Biomechanical Comparison of Different Surgical Strategies for Skip-level Cervical Degenerative Disc Disease: A Finite Element Study.

STUDY DESIGN: We constructed finite element (FE) models of the cervical spine consisting of C2-C7 and predicted the biomechanical effects of different surgical procedures and instruments on adjacent segments, internal fixation systems, and the overall cervical spine through FE analysis. OBJECTIVE: To compare the biomechanical effects between zero-profile device and cage-plate device in skip-level multistage anterior cervical discectomy and fusion (ACDF). SUMMARY OF BACKGROUND DATA: ACDF is often considered as the standard treatment for degenerative cervical spondylosis. However, the selection of surgical methods and instruments in cases of skip-level cervical degenerative disc disease is still controversial. METHODS: Three FE models were constructed, which used noncontiguous 2-level Zero-P (NCZP) devices for C3/4 and C5/6, a noncontiguous 2-level cage-plate (NCCP) for C3/4 and C5/6, and a contiguous 3-level cage-plate (CCP) for C3/6. Simulate daily activities in ABAQUS. The range of motion (ROM), von Mises stress distribution of the endplate and internal fixation system, and intervertebral disc pressure (IDP) of each model were recorded and compared. RESULTS: Similar to the stress of the cortical bone, the maximum stress of the Zero-P device was higher than that of the CP device for most activities. The ROM increments of the superior, inferior, and intermediate segments of the NCZP model were lower than those of the NCCP and CCP models in many actions. In terms of the IDP, the increment value of stress for the NCZP model was the smallest, whereas those of the NCCP and CCP models were larger. Similarly, the increment value of stress on the endplate also shows the minimum in the NCZP model. CONCLUSIONS: Noncontiguous ACDF with zero-profile can reduce the stress on adjacent intervertebral discs and endplates, resulting in a reduced risk of adjacent segment disease development. However, the high cortical bone stress caused by the Zero-P device may influence the risk of fractures.

Finite element biomechanical analysis of 3D printed intervertebral fusion cage in osteoporotic population.

OBJECTIVE: To study the biomechanical characteristics of each tissue structure when using different 3D printing Cage in osteoporotic patients undergoing interbody fusion. METHODS: A finite element model of the lumbar spine was reconstructed and validated with regarding a range of motion and intervertebral disc pressure from previous in vitro studies. Cage and pedicle screws were implanted and part of the lamina, spinous process, and facet joints were removed in the L4/5 segment of the validated mode to simulate interbody fusion. A 280 N follower load and 7.5 N·m moment were applied to different postoperative models and intact osteoporotic model to simulate lumbar motion. The biomechanical characteristics of different models were evaluated by calculating and analyzing the range of motion of the fixed and cephalic adjacent segment, the stress of the screw-rod system, the stress at the interface between cage and L5 endplate, and intervertebral disc pressure of the adjacent segment. RESULTS: After rigid fixation, the range of motion of the fixed segment of model A-C decreased significantly, which was much smaller than that of the osteoporotic model. And with the increase of the axial area of the interbody fusion cages, the fixed segment of model A-C tended to be more stable. The range of motion and intradiscal pressure of the spinal models with different interbody fusion cages were higher than those of the complete osteoporosis model, but there was no significant difference between the postoperative models. On the other hand, the L5 upper endplate stress and screw-rod system stress of model A-C show a decreasing trend in different directions of motion. The stress of the endplate is the highest during flexion, which can reach 40.5 MPa (model A). The difference in endplate stress between models A-C was the largest during lateral bending. The endplate stress of models A and B was 150.5% and 140.9% of that of model C, respectively. The stress of the screw-rod system was the highest during lateral bending (model A, 102.0 MPa), which was 108.4%, 102.4%, 110.4%, 114.2% of model B and 158.5%, 110.1%, 115.8%, 125.4% of model C in flexion, extension, lateral bending, and rotation, respectively. CONCLUSIONS: For people with osteoporosis, no matter what type of cage is used, good immediate stability can be achieved after surgery. Larger cage sizes provide better fixation without significantly increasing ROM and IDP in adjacent segments, which may contribute to the development of ASD. In addition, larger cage sizes can disperse endplate stress and reduce stress concentration, which is of positive significance in preventing cage subsidence after operation. The cage and screw rod system establish a stress conduction pathway on the spine, and a larger cage greatly enhances the stress-bearing capacity of the front column, which can better distribute the stress of the posterior spine structure and the stress borne by the posterior screw rod system, reduce the stress concentration phenomenon of the nail rod system, and avoid exceeding the yield strength of the material, resulting in the risk of future instrument failure.

Biomechanical behaviour of a novel bone cement screw in the minimally invasive treatment of Kummell's disease: a finite element study.

OBJECTIVE: To investigate and evaluate the biomechanical behaviour of a novel bone cement screw in the minimally invasive treatment of Kummell's disease (KD) by finite element (FE) analysis. METHODS: A validated finite element model of healthy adult thoracolumbar vertebrae T12-L2 was given the osteoporotic material properties and the part of the middle bone tissue of the L1 vertebral body was removed to make it wedge-shaped. Based on these, FE model of KD was established. The FE model of KD was repaired and treated with three options: pure percutaneous vertebroplasty (Model A), novel unilateral cement screw placement (Model B), novel bilateral cement screw placement (Model C). Range of motion (ROM), maximum Von-Mises stress of T12 inferior endplate and bone cement, relative displacement of bone cement, and stress distribution of bone cement screws of three postoperative models and intact model in flexion and extension, as well as lateral bending and rotation were analyzed and compared. RESULTS: The relative displacements of bone cement of Model B and C were similar in all actions studied, and both were smaller than that of Model A. The minimum value of relative displacement of bone cement is 0.0733 mm in the right axial rotation of Model B. The maximum Von-Mises stress in T12 lower endplate and bone cement was in Model C. The maximum Von-Mises stress of bone cement screws in Model C was less than that in Model B, and it was the most substantial in right axial rotation, which is 34%. There was no substantial difference in ROM of the three models. CONCLUSION: The novel bone cement screw can effectively reduce the relative displacement of bone cement by improving the stability of local cement. Among them, novel unilateral cement screw placement can obtain better fixation effect, and the impact on the biomechanical environment of vertebral body is less than that of novel bilateral cement screw placement, which provides a reference for minimally invasive treatment of KD in clinical practice.

Optimization of in-brace corrective force in adolescents with Lenke type 5 curve using finite element model.

BACKGROUND: Pelvic parameters have been taken into consideration for the evaluation of the outcomes of bracing in AIS. To discuss the stress required to correct the pelvic deformity related to Lenke5 adolescent idiopathic scoliosis (AIS) by finite element analysis, and provide a reference for the shaping of the pelvic region of the brace. METHODS: An three-dimensional (3D) corrective force on the pelvic area was defined. Computed tomography images were used to reconstruct a 3D model of Lenke5 AIS. Computer-aided engineering software Abaqus was used to implement finite element analysis. By adjusting the magnitude and position of corrective forces, coronal pelvic coronal plane rotation (PCPR) and Cobb angle (CA) of lumbar curve in the coronal plane, horizontal pelvic axial plane rotation, and apical vertebra rotation (AVR) were minimized to achieve the best effect on the spine and pelvic deformity correction. The proposed corrective conditions were divided into three groups: (1) forces applied on X-axis; (2) forces applied both in the X- and Y-axis; and (3) forces applied along the X-, Y-, and Z-axis at the same time. RESULTS: In three groups, CA correction reduced by 31.5%, 42.5%, and 59.8%, and the PCPR changed to 12°, 13°, and 1° from 6.5°, respectively. The best groups of correction forces should simultaneously locate on the sagittal, transverse, and coronal planes of the pelvis. CONCLUSIONS: For Lenke5 AIS, 3D correction forces can sufficiently reduce scoliosis and pelvic asymmetrical state. Force applied along the Z-axis is vital to correct the pelvic coronal pelvic tilt associated with Lenke5 AIS.

Biomechanical evaluation of different sizes of 3D printed cage in lumbar interbody fusion-a finite element analysis.

OBJECTIVE: To study the biomechanical characteristics of various tissue structures of different sizes of 3D printed Cage in lumbar interbody fusion. METHODS: A finite element model of normal spine was reconstructed and verified. Pedicle screws and Cage of different sizes were implanted in the L4/5 segment to simulate lumbar interbody fusion. The range of motion of the fixed and cephalic adjacent segment, the stress of the screw-rod system, the stress at the interface between cage and L5 endplate, and intervertebral disc pressure of the adjacent segment were calculated and analyzed. RESULTS: The range of motion and intervertebral disc pressure of the adjacent segment of each postoperative model were larger than those of the intact model, but there was not much difference between them. The stress of cage-endplate interface was also larger than that of the intact model. However, the difference is that the stress of the endplate and the screw-rod system has a tendency to decrease with the increase of the axial area of cage. CONCLUSIONS: Cage with larger axial area in lumbar interbody fusion can reduce the stress of internal fixation system and endplate, but will not increase the range of motion and intervertebral disc pressure of adjacent segment. It has a certain effect in preventing the cage subsidence, internal fixation system failure and screw rod fracture.

Biomechanical comparison of spinal column shortening - a finite element study.

BACKGROUND: At present, research on spinal shortening is mainly focused on the safe distance of spinal shortening and the mechanism of spinal cord injury, but there is no research on the biomechanical characteristics of different shortening distances. The purpose of this study was to study the biomechanical characteristics of spine and internal fixation instruments at different shortening distances by the finite element (FE) method. METHODS: An FE model of lumbar L1-S was established and referred to the previous in vitro experiments to verify the rationality of the model by verifying the Intradiscal pressure (IDP) and the range of motion (ROM) of the motion segment. Five element models of spinal shortening were designed under the safe distance of spinal shortening, and the entire L3 vertebra and both the upper and lower intervertebral discs were resected. Model A was not shortened, while models B-E were shortened by 10%, 20%, 30% and 50% of the vertebral body, respectively. Constraining the ROM of the sacrum in all directions, a 7.5 N ·m moment and 280 N follower load were applied on the L1 vertebra to simulate the motion of the lumbar vertebrae in three planes. The ROM of the operated segments, the Von Mises stress (VMS) of the screw-rod system, the VMS of the upper endplate at the interface between the titanium cage and the L4 vertebral body, and the ROM and the IDP of the adjacent segment (L5/S) were recorded and analysed. RESULTS: All surgical models showed good stability at the operated segments (L1-5), with the greatest constraint in posterior extension (99.3-99.7%), followed by left-right bending (97.9-98.7%), and the least constraint in left-right rotation (84.9-86.3%) compared with the intact model. The VMS of the screw-rod system and the ROM and IDP of the distal adjacent segments of models A-E showed an increasing trend, in which the VMS of the screw-rod system of model E was the highest under flexion (172.5 MPa). The VMS of the endplate at the interface between the cage and L4 upper endplate of models A-E decreased gradually, and these trend were the most obvious in flexion, which were 3.03, 2.95, 2.83, 2.78, and 2.61 times that of the intact model, respectively. CONCLUSION: When performing total vertebrae resection and correcting the spinal deformity, if the corrected spine has met our needs, the distance of spinal shortening should be minimized to prevent spinal cord injury, fracture of internal fixations and adjacent segment disease (ASD).

Changes in cervical alignment of Zero-profile device versus conventional cage-plate construct after anterior cervical discectomy and fusion: a meta-analysis.

BACKGROUND: Anterior cervical diskectomy and fusion (ACDF) has been widely accepted as a gold standard for patients with cervical spondylotic myelopathy (CSM). However, there was insufficient evidence to compare the changes in the cervical alignment with different fusion devices in a long follow-up period. This meta-analysis was performed to compare the radiologic outcomes and loss of correction (LOC) in cervical alignment of Zero-profile (ZP) device versus cage-plate (CP) construct for the treatment of CSM. METHODS: Retrospective and prospective studies directly comparing the outcomes between the ZP device and CP construct in ACDF were included. Data extraction was conducted and study quality was assessed independently. A meta-analysis was carried out by using fixed effects and random effects models to calculate the odds ratio and mean difference in the ZP group and the CP group. RESULTS: Fourteen trials with a total of 1067 participants were identified. ZP group had a lower rate of postoperative dysphagia at the 2- or 3-month and 6-month follow-up than CP group, and ZP group was associated with a decreased ASD rate at the last follow-up when compared with the CP group. The pooled data of radiologic outcomes revealed that there was no significant difference in postoperative and last follow-up IDH. However, postoperative and last follow-up cervical Cobb angle was significantly smaller in the ZP group when compared with the CP group. In subgroup analyses, when the length of the last follow-up was less than 3 years, there was no difference between two groups. However, as the last follow-up time increased, cervical Cobb angle was significantly lower in the ZP group when compared with the CP group. CONCLUSION: Based on the results of our analysis, the application of ZP device in ACDF had a lower rate of postoperative dysphagia and ASD than CP construct. Both devices were safe in anterior cervical surgeries, and they had similar efficacy in correcting radiologic outcomes. However, as the last follow-up time increased, ZP group showed greater changes cervical alignment. In order to clarify the specific significance of LOC, additional large clinical studies with longer follow-up period are required.

Biomechanical comparison of different prosthetic reconstructions in total en bloc spondylectomy: a finite element study.

OBJECTIVE: To analyse and compare the biomechanical differences between 3D-printed prostheses, titanium mesh cages and poorly matched titanium mesh cages in total en bloc spondylectomy (TES). METHODS: The finite element model of T10-L2 for healthy adults was modified to make three models after T12 total spondylectomy. These models were a 3D-printed prosthesis, titanium mesh cage and prosthesis-endplate mismatched titanium mesh cage for reconstruction. The range of motion (ROM), stress distribution of the endplate and internal fixation system of three models in flexion and extension, lateral bending and axial rotation were simulated and analysed by ABAQUS. RESULT: In flexion, due to the support of the anterior prosthesis, the fixation system showed the maximum fixation strength. The fixation strength of the 3D-printed prosthesis model was 26.73 N·m /°, that of the TMC support model was 27.20 N·m /°, and that of the poorly matched TMC model was 24.16 N·m /°. In flexion, the L1 upper endplate stress of the poorly matched TMC model was 35.5% and 49.6% higher than that of the TMC and 3D-printed prosthesis, respectively. It was 17% and 28.1% higher in extension, 39.3% and 42.5% higher in lateral bending, and 82.9% and 91.2% higher in axial rotation, respectively. The lower endplate of T11 showed a similar trend, but the magnitude of the stress change was reduced. In the stress analysis of the 3D-printed prosthesis and TMC, it was found that the maximum stress was in flexion and axial rotation, followed by left and right bending, and the least stress was in extension. However, the mismatched TMC withstood the maximum von Mises stress of 418.7 MPa (almost twice as much as the buckling state) in rotation, 3 times and 5.83 times in extension, and 1.29 and 2.85 times in lateral bending, respectively. CONCLUSION: Different prostheses with good endplate matching after total spondylectomy can obtain effective postoperative stable support, and the reduction in contact area caused by mismatch will affect the biomechanical properties and increase the probability of internal fixation failure.

Biomechanical behaviour of tension-band-reconstruction titanium plate in open-door laminoplasty: a study based on finite element analysis.

OBJECTIVE: To investigate and evaluate the biomechanical behaviour of tension-band-reconstruction (TBR) and ordinary titanium plates in open-door laminoplasty by finite element (FE) analysis. METHODS: TBR titanium plate and ordinary titanium plate were implanted into a validated finite element model of healthy adult cervical vertebrae. Among them, 5 ordinary titanium plate were used in model A, 2 TBR titanium plates and 3 ordinary titanium plates were used in model B, and 5 TBR titanium plates were used in model C. The same loading conditions was applied identically to all models. Range of motion (ROM) of the vertebral body, stress distribution of the titanium plate and intradiscal pressure (IDP) were compared in flexion, extension, lateral bending and rotation. RESULTS: The ROM of model B and C was similar in flexion and extension, and both were smaller than that of model A. The highest von Mises stress in the titanium plate appears is in model C. The IDP in C2/3 was significantly higher than that in other segments in flexion. There was no significant difference in IDP among three models in left lateral bending and left axial rotation. CONCLUSION: Application of TBR titanium plate in open-door laminoplasty can reduced ROM in flexion, extension and axial rotation of the cervical vertebrae. But the increase of stress in TBR titanium plate could lead to higher risk of adverse events such as titanium plate deformation. Moreover, compared with complete TBR titanium plate, the combination of TBR titanium plate for C3 and C7 with ordinary titanium plate for the other vertebrae largely reduce the stress of the titanium plates by ensuring stability. The proposed FE model (C2-T1) exhibits a great potential in evaluating biomechanical behaviour of TBR titanium plate for open-door laminoplasty.

Application of dual-trajectory screws in revision surgery for lumbar adjacent segment disease: a finite element study.

BACKGROUND: Advancements in medicine and the popularity of lumbar fusion surgery have made lumbar adjacent segment disease (ASDz) increasingly common, but there is no mature plan for guiding its surgical treatment. Therefore, in this study, four different finite element (FE) ASDz models were designed and their biomechanical characteristics were analysed to provide a theoretical basis for clinical workers to choose the most appropriate revision scheme for ASDz. METHODS: According to whether internal fixation was retained, different FE models were created to simulate ASDz revision surgery, and flexion, extension, axial rotation and lateral bending were simulated by loading. The biomechanical characteristics of the adjacent segments of the intervertebral disc and the internal fixation system and the range of motion (ROM) of the lumbar vertebrae were analysed. RESULTS: The difference in the ROM of the fixed segment between FE models that did or did not retain the original internal fixation was less than 0.1°, and the difference was not significant. However, the stress of the screw-rod system when the original internal fixation was retained and prolonged fixation was performed with dual-trajectory screws was less than that when the original internal fixation was removed and prolonged fixation was performed with a long bar. Especially in axial rotation, the difference between models A and B is the largest, and the difference in peak stress reached 30 MPa. However, for the ASDz revision surgery segment, the endplate stress between the two models was the lowest, and the intradiscal pressure (IDP) of the adjacent segment was not significantly different between different models. CONCLUSION: Although ASDz revision surgery by retaining the original internal fixation and prolonging fixation with dual-trajectory screws led to an increase in stress in the fusion segment endplate, it provides stability similar to ASDz revision surgery by removing the original internal fixation and prolonging fixation with a long bar and does not lead to a significant change in the IDP of the adjacent segment while avoiding a greater risk of rod fracture.

The mouse cortico-basal ganglia-thalamic network.

The cortico-basal ganglia-thalamo-cortical loop is one of the fundamental network motifs in the brain. Revealing its structural and functional organization is critical to understanding cognition, sensorimotor behaviour, and the natural history of many neurological and neuropsychiatric disorders. Classically, this network is conceptualized to contain three information channels: motor, limbic and associative1-4. Yet this three-channel view cannot explain the myriad functions of the basal ganglia. We previously subdivided the dorsal striatum into 29 functional domains on the basis of the topography of inputs from the entire cortex5. Here we map the multi-synaptic output pathways of these striatal domains through the globus pallidus external part (GPe), substantia nigra reticular part (SNr), thalamic nuclei and cortex. Accordingly, we identify 14 SNr and 36 GPe domains and a direct cortico-SNr projection. The striatonigral direct pathway displays a greater convergence of striatal inputs than the more parallel striatopallidal indirect pathway, although direct and indirect pathways originating from the same striatal domain ultimately converge onto the same postsynaptic SNr neurons. Following the SNr outputs, we delineate six domains in the parafascicular and ventromedial thalamic nuclei. Subsequently, we identify six parallel cortico-basal ganglia-thalamic subnetworks that sequentially transduce specific subsets of cortical information through every elemental node of the cortico-basal ganglia-thalamic loop. Thalamic domains relay this output back to the originating corticostriatal neurons of each subnetwork in a bona fide closed loop.

Biomechanics of artificial pedicle fixation in a 3D-printed prosthesis after total en bloc spondylectomy: a finite element analysis.

BACKGROUND: This study compared the biomechanics of artificial pedicle fixation in spine reconstruction with a 3-dimensional (3D)-printed prosthesis after total en bloc spondylectomy (TES) by finite element analysis. METHODS: A thoracolumbar (T10-L2) finite element model was developed and validated. Two models of T12 TES were established in combination with different fixation methods: Model A consisted of long-segment posterior fixation (T10/11, L1/2) + 3D-printed prosthesis; and Model B consisted of Model A + two artificial pedicle fixation screws. The models were evaluated with an applied of 7.5 N·m and axial force of 200 N. We recorded and analyzed the following: (1) stiffness of the two fixation systems, (2) hardware stress in the two fixation systems, and (3) stress on the endplate adjacent to the 3D-printed prosthesis. RESULTS: The fixation strength of Model B was enhanced by the screws in the artificial pedicle, which was mainly manifested as an improvement in rotational stability. The stress transmission of the artificial pedicle fixation screws reduced the stress on the posterior rods and endplate adjacent to the 3D-printed prosthesis in all directions of motion, especially in rotation. CONCLUSIONS: After TES, the posterior long-segment fixation combined with the anterior 3D printed prosthesis could maintain postoperative spinal stability, but adding artificial pedicle fixation increased the stability of the fixation system and reduced the risk of prosthesis subsidence and instrumentation failure.

Biomechanical finite element analysis of vertebral column resection and posterior unilateral vertebral resection and reconstruction osteotomy.

BACKGROUND: Regarding the repair of vertebral compression fractures, there is a lack of adequate biomechanical verification as to whether only half of the vertebral body and the upper and lower intervertebral discs affect spinal biomechanics; there also remains debate as to the appropriate length of fixation. METHODS: A model of old vertebral compression fractures with kyphosis was established based on CT data. Vertebral column resection (VCR) and posterior unilateral vertebral resection and reconstruction (PUVCR) were performed at T12; long- and short-segment fixation methods were applied, and we analyzed biomechanical changes after surgery. RESULTS: Range of motion (ROM) decreased in all fixed models, with lumbar VCR decreasing the most and short posterior unilateral vertebral resection and reconstruction (SPUVCR) decreasing the least; in the long posterior unilateral vertebral resection and reconstruction (LPUVCR) model, the internal fixation system produced the maximum VMS stress of 213.25 mPa in a lateral bending motion and minimum stress of 40.22 mPa in a lateral bending motion in the SVCR. CONCLUSION: There was little difference in thoracolumbar ROM between PUVCR and VCR models, while thoracolumbar ROM was smaller in long-segment fixation than in short-segment fixation. In all models, the VMS was most significant at the screw-rod junction and greatest at the ribcage-vertebral body interface, partly explaining the high probability of internal fixation failure and prosthesis migration in these two positions.

Precise segmentation of densely interweaving neuron clusters using G-Cut.

Characterizing the precise three-dimensional morphology and anatomical context of neurons is crucial for neuronal cell type classification and circuitry mapping. Recent advances in tissue clearing techniques and microscopy make it possible to obtain image stacks of intact, interweaving neuron clusters in brain tissues. As most current 3D neuronal morphology reconstruction methods are only applicable to single neurons, it remains challenging to reconstruct these clusters digitally. To advance the state of the art beyond these challenges, we propose a fast and robust method named G-Cut that is able to automatically segment individual neurons from an interweaving neuron cluster. Across various densely interconnected neuron clusters, G-Cut achieves significantly higher accuracies than other state-of-the-art algorithms. G-Cut is intended as a robust component in a high throughput informatics pipeline for large-scale brain mapping projects.

A cortical astrocyte subpopulation inhibits axon growth in vitro and in vivo.

Astrocytes are the most heterogeneous and predominant glial cell type in the central nervous system. However, the functional significance of this heterogeneity remains to be elucidated. Following injury, damaged astrocytes inhibit axonal regeneration in vivo and in vitro. Cultured primary astrocytes are commonly considered good supportive substrates for neuron attachment and axon regeneration. However, it is not known whether different populations of cells in the heterogeneous astrocyte culture affect neuron behavior in the same way. In the present study, the effect of astrocyte heterogeneity on neuronal attachment and neurite outgrowth was examined using an in vitro and in vivo coculture system. In vitro, neonatal cortical astrocytes were co-cultured with purified dorsal root ganglia (DRG) neurons and astrocyte growth morphology, neuron attachment and neurite growth were evaluated. The results demonstrated that the heterogeneous astrocyte cells showed two different types of growth pattern, typical and atypical. Typical astrocytes were supportive to neuron attachment and neurite growth, which was consistent with previous studies, whereas atypical astrocytes inhibited neuron attachment and neurite growth. These inhibitory astrocytes exhibited a special growth pattern with various shapes and sizes, a high cell density, few oligodendrocytes on the top layer and occupied a smaller growth area compared with typical astrocytes. Neurites extended freely on typical supportive astrocyte populations, however, moved away when they reached atypical astrocyte growth pattern. Neurons growing on the atypical astrocyte pattern demonstrated minimal neurite outgrowth and these neurites had a dystrophic appearance, however, neuronal survival was unaffected. Immunocytochemistry studies demonstrated that these atypical inhibitory astrocytes were glial fibrillary acidic protein (GFAP) positive cells. The existence of inhibitory astrocyte subpopulations in normal astrocytes reflects the complexity of the function of astrocyte populations. In vivo, DRG neurons in grey matter did not show neurite growth, while DRG neurons survived and showed robust axon outgrowth along the corpus callosum. In conclusion, further studies on this new type of inhibitory astrocyte subpopulation may deepen our understanding of the complex biology of astrocytes.

An efficient method for dorsal root ganglia neurons purification with a one-time anti-mitotic reagent treatment.

BACKGROUND: The dorsal root ganglia (DRG) neuron is an invaluable tool in axon growth, growth factor regulation, myelin formation and myelin-relevant researches. The purification of DRG neurons is a key step in these studies. Traditionally, purified DRG neurons were obtained in two weeks after exposure to several rounds of anti-mitotic reagent. METHODS AND RESULTS: In this report, a novel, simple and efficient method for DRG purification is presented. DRG cultures were treated once with a high-dose anti-mitotic reagent cocktail for 72 hours. Using this new method, DRG neurons were obtained with 99% purification within 1 week. We confirmed that the neurite growth and the viability of the purified DRG neurons have no difference from the DRG neurons purified by traditional method. Furthermore, P0 and MBP expression was observed in myelin by immunocytochemistry in the DRG/SC co-culture system. The formation of mature node of Ranvier in DRG-Schwann cell co-culture system was observed using anti-Nav 1.6 and anti-caspr antibody. CONCLUSION AND SIGNIFICANCE: The results indicate that this high dose single treatment did not compromise the capacity of DRG neurons for myelin formation in the DRG/SC co-culture system. In conclusion, a convenient approach for purifying DRG neurons was developed which is time-saving and high-efficiency.

Extraocular muscle characteristics related to myasthenia gravis susceptibility.

BACKGROUND: The pathogenesis of extraocular muscle (EOM) weakness in myasthenia gravis might involve a mechanism specific to the EOM. The aim of this study was to investigate characteristics of the EOM related to its susceptibility to myasthenia gravis. METHODS: Female F344 rats and female Sprague-Dawley rats were assigned to experimental and control groups. The experimental group received injection with Ringer solution containing monoclonal antibody against the acetylcholine receptor (AChR), mAb35 (0.25 mg/kg), to induce experimental autoimmune myasthenia gravis, and the control group received injection with Ringer solution alone. Three muscles were analyzed: EOM, diaphragm, and tibialis anterior. Tissues were examined by light microscopy, fluorescence histochemistry, and transmission electron microscopy. Western blot analysis was used to assess marker expression and ELISA analysis was used to quantify creatine kinase levels. Microarray assay was conducted to detect differentially expressed genes. RESULTS: In the experimental group, the EOM showed a simpler neuromuscular junction (NMJ) structure compared to the other muscles; the NMJ had fewer synaptic folds, showed a lesser amount of AChR, and the endplate was wider compared to the other muscles. Results of microarray assay showed differential expression of 54 genes in the EOM between the experimental and control groups. CONCLUSION: Various EOM characteristics appear to be related to the increased susceptibility of the EOM and the mechanism of EOM weakness in myasthenia gravis.

Study glial cell heterogeneity influence on axon growth using a new coculture method.

In the central nervous system of all mammals, severed axons after injury are unable to regenerate to their original targets and functional recovery is very poor. The failure of axon regeneration is a combined result of several factors including the hostile glial cell environment, inhibitory myelin related molecules and decreased intrinsic neuron regenerative capacity. Astrocytes are the most predominant glial cell type in central nervous system and play important role in axon functions under physiology and pathology conditions. Contrast to the homologous oligodendrocytes, astrocytes are a heterogeneous cell population composed by different astrocyte subpopulations with diverse morphologies and gene expression. The functional significance of this heterogeneity, such as their influences on axon growth, is largely unknown. To study the glial cell, especially the function of astrocyte heterogeneity in neuron behavior, we established a new method by co-culturing high purified dorsal root ganglia neurons with glial cells obtained from the rat cortex. By this technique, we were able to directly compare neuron adhesion and axon growth on different astrocytes subpopulations under the same condition. In this report, we give the detailed protocol of this method for astrocytes isolation and culture, dorsal root ganglia neurons isolation and purification, and the co-culture of DRG neurons with astrocytes. This method could also be extended to other brain regions to study cellular or regional specific interaction between neurons and glial cells.

Femtosecond laser-assisted microinjection into living neurons.

A Ti:sapphire femtosecond laser micro-manipulation system was used to perforate vital cells. Propidium iodide was successfully transferred into both astrocytes and PC12 cells via the channel of a femtosecond laser, which induced transiently recoverable opening of the cell membrane. The transfection efficiency reached almost 100%. This method also had the advantage of contact-free, non-disruptive and stable transfection.

[Isolation, cultivation and identification of stem cells from cerebral cortex of mouse embryo].

OBJECTIVE: To isolate, cultivate and identify the neural stem cells from the cortex of embryo mouse in vitro for the further related research. METHODS: Cerebral hemisphere of mouse fetuses were taken out. Suspension of cerebral cells was made. Neural stem cells were identified by immunofluorescent cytochemistry to the primary culture. Neural stem cells were isolated and cultured in special culture medium using single-cell clone culture technique. After the stem cells were stably cultured successively for 20 generations, nestin, glial fibrillary acidic protein, beta-tublin, and galactosidase (Galc) were detected by indirect immunofluorescence cytochemistry. RESULTS: The stem cells isolated from the cerebral hemisphere of mouse embryo with positive expression of nestin could be passaged in vitro for at least 20 generations. Inoculated on the coverglass with poly-1-ornithine and cultured in medium containing serum, the stem cells were induced to differentiate. The cells differentiated from these stem cells showed green fluorescence by GFAP fluorescent staining, red fluorescence by beta-tublin fluorescent staining, or green fluorescence by Galc fluorescent staining. CONCLUSION: The stem cells from cortex of mouse embryo can be successfully isolated and cultured in vitro. The stem cells can be passaged in series and possessed the potential of multi-directional differentiation and express the specific antigens of neuron, astrocytes, and oligodendrocytes. Thus they can be used for further experimental study.