3D printing collagen/chitosan scaffold ameliorated axon regeneration and neurological recovery after spinal cord injury.

Spinal cord injury (SCI) is a disaster that can cause severe motor, sensory, and functional disorders. Implanting biomaterials have been regarded as hopeful strategies to restore neurological function. However, no optimized scaffold has been available. In this study, a novel 3D printing technology was used to fabricate the scaffold with designed structure. The composite biomaterials of collagen and chitosan were also adopted to balance both compatibility and strength. Female Sprague-Dawley rats were subjected to a T8 complete-transection SCI model. Scaffolds of C/C (collagen/chitosan scaffold with freeze-drying technology) or 3D-C/C (collagen/chitosan scaffold with 3D printing technology) were implanted into the lesion. Compared with SCI or C/C group, 3D-C/C implants significantly promoted locomotor function with the elevation in Basso-Beattie-Bresnahan (BBB) score and angle of inclined plane. Decreased latency and increased amplitude were observed both in motor-evoked potential and somatosensory-evoked potential in 3D-C/C group compared with SCI or C/C group, which further demonstrated the improvement of neurological recovery. Fiber tracking of diffusion tensor imaging (DTI) showed the most fibers traversing the lesion in 3D-C/C group. Meanwhile, we observed that the correlations between the locomotor (BBB score or angle of inclined plane) and the DTI parameters (fractional anisotropy values) were positive. Although C/C implants markedly enhanced biotin dextran amine (BDA)-positive neural profiles compared with SCI group, rats implanted with 3D-C/C scaffold displayed the largest degree of BDA profiles regeneration. Collectively, our 3D-C/C scaffolds demonstrated significant therapeutic effects on rat complete-transected spinal cord model, which provides a promising and innovative therapeutic approach for SCI. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1898-1908, 2019.

Mechanism of interaction between ocular and nasal neurogenic inflammation in allergic rhinoconjunctivitis.

PURPOSE: The mechanisms of naso-ocular interaction in allergic rhinoconjunctivitis are not well understood. Neurogenic inflammation affects both eyes and nose via the same neurogenic factors. The purpose of this study was to investigate the effects of neurogenic inflammation on conjunctival inflammation following nasal allergen provocation. METHODS: Sensitized rats were exposed to ovalbumin (OVA) via the nose. Parts of the nasal mucosa and conjunctivae were sliced and used for hematoxylin-eosin staining, immunohistochemical analysis, western blotting, and real-time polymerase chain reaction. The slides were observed under a light microscope, and the acquired images were analyzed. The levels of substance P (SP), vasoactive intestinal peptide (VIP), and nerve growth factor (NGF) were detected. RESULTS: The levels of SP, VIP, and NGF were increased in both nasal mucosa and conjunctivae 1 h and 24 h after OVA administration (p < 0.05). Higher levels of SP, VIP, and NGF expression were observed in the nasal mucosa and conjunctivae 24 h after OVA administration (p < 0.05). Following damage of the nasal sensory nerves by capsaicin, the protein and mRNA levels of SP, VIP, and NGF were reduced. CONCLUSION: In conclusion, the increased levels of VIP, SP, and NGF might be responsible for the ocular reaction following nasal challenge with allergen in rats.

Optical Depolarization of DCX-Expressing Cells Promoted Cognitive Recovery and Maturation of Newborn Neurons via the Wnt/ß-Catenin Pathway.

Electrical excitability by membrane depolarization is crucial for survival and maturation of newborn cells in the dentate gyrus of the hippocampus. However, traditional technology for membrane depolarization lacks temporal and spatial precision. Optogenetics can be used to activate channelrhodopsin-2 (ChR2), allowing cationic current to depolarize genetically targeted cells. In this study, we used ChR2-EGFP driven by doublecortin (DCX) to promote survival and maturation of newborn cells in the dentate gyrus after traumatic brain injury (TBI). C57BL/6 mice underwent lateral fluid percussion TBI. TBI mice were transfected with a lentivirus carrying the DCX-ChR2-EGFP gene. We observed that not only immature neurons but also type-2b intermediate progenitor (IPs) and neuroblasts expressed DCX-EGFP, indicating that DCX-expressing newborn cells could provide a long time window for electrical activity regulation. Quantitative results showed that the number of EGFP-expressing cells began to rise at 3 days after TBI and peaked at 9 days after TBI. By optical depolarization of DCX-EGFP-expressing cells between 3 and 12 days, we observed significantly improved cognitive deficits after TBI with enhanced survival and maturation of newborn cells in the dentate gyrus. We also investigated the role of optical depolarization in neural stem cells transfected with a lentivirus carrying the ChR2-DCX-EGFP gene in vitro. By administrating verapamil to block L-type calcium channels, we verified that the up-regulation of MAP2, NeuN, Neurog2, NeuroD1 and GluR2 in newborn cells was mediated by ChR2-elicted depolarization. By using ß-catenin inhibitor Dkk1, we demonstrated that optical depolarization of DCX-EGFP-expressing cells facilitated survival and maturation probably through the Wnt/ß-catenin signaling cascade.

Hypothermia-Modulating Matrix Elasticity of Injured Brain Promoted Neural Lineage Specification of Mesenchymal Stem Cells.

Both chemical and physical microenvironments appear to be important for lineage specification of umbilical cord mesenchymal stem cells (UCMSCs). However, physical factors such as the elastic modulus in traumatic brain injury (TBI) are seldom studied. Intracranial hypertension and cerebral edema after TBI may change the brain's physical microenvironment, which inhibits neural lineage specification of transplanted UCMSCs. The purpose of this study is to investigate the potential regulatory effect of mild hypothermia on the elastic modulus of the injured brain. First, we found that more UCMSCs grown on gels mimicking the elastic modulus of the brain (0.5 kPa) differentiated into neural cells, which were verified with the formation of branched cells and the expression of neural markers. Then, UCMSCs were transplanted into TBI rats, and we observed that mild hypothermia resulted in the differentiation of more neurons and astrocytes from transplanted UCMSCs. To demonstrate that more neural specification of UCMSCs was due to the regulation of the elastic modulus, we monitored intracranial pressure and cerebral edema. The results showed that mild hypothermia significantly reduced intracranial pressure and brain water content, indicating modulation of the elastic modulus by mild hypothermia. An examination with atomic force microscopy (AFM) in a cell injury model in vitro further verified hypothermia-regulated elastic modulus. In this study, we found a novel role of mild hypothermia in modulating the elastic modulus of the injured brain, resulting in the promotion of neural lineage specification of UCMSCs, which suggested that the combination of mild hypothermia had more advantages in cell-based therapy after TBI.

The Distribution of Transplanted Umbilical Cord Mesenchymal Stem Cells in Large Blood Vessel of Experimental Design With Traumatic Brain Injury.

The authors aim to track the distribution of human umbilical cord mesenchymal stem cells (MSCs) in large blood vessel of traumatic brain injury -rats through immunohistochemical method and small animal imaging system. After green fluorescent protein (GFP) gene was transfected into 293T cell, virus was packaged and MSCs were transfected. Mesenchymal stem cells containing GFP were transplanted into brain ventricle of rats when the infection rate reaches 95%. The immunohistochemical and small animal imaging system was used to detect the distribution of MSCs in large blood vessels of rats. Mesenchymal stem cells could be observed in large vessels with positive GFP expression 10 days after transplantation, while control groups (normal group and traumatic brain injury group) have negative GFP expression. The vascular endothelial growth factor in transplantation group was higher than that in control groups. The in vivo imaging showed obvious distribution of MSCs in the blood vessels of rats, while no MSCs could be seen in control groups. The intravascular migration and homing of MSCs could be seen in rats received MSCs transplantation, and new angiogenesis could be seen in MSCs-transplanted blood vessels.

The research progress of mesenchymal stem cells in the treatment of Traumatic brain injury.

Traumatic brain injury (TBI) is the leading cause of mortality and morbidity in children and adults throughout the world. It is urgent to ameliorate TBI damage and reduce the disability and case-fatality rate. Stem cell therapy is another medical revolution after drug and surgical medication. Mesenchymal stem cells (MSCs) are a class of cells with significant self-renewal and multi-lineage differentiation properties and be favorable for the treatment of various diseases and injuries, it could be envisioned that MSCs transplantation may be a promising treatment for TBI. Currently, stem cell therapy has shown promising effects in the treatment of many diseases. In this article, we will review the characteristics of MSCs, MSCs for neuronal function restoration, the properties of immune-modulatory of MSCs, and the anti-apoptotic effects of MSCs, the angiogenesis effect of MSCs,and the safety issues in MSCs therapy in TBI.

Establishment of an ideal time window model in hypothermic-targeted temperature management after traumatic brain injury in rats.

Although hypothermic-targeted temperature management (HTTM) holds great potential for the treatment of traumatic brain injury (TBI), translation of the efficacy of hypothermia from animal models to TBI patientshas no entire consistency. This study aimed to find an ideal time window model in experimental rats which was more in accordance with clinical practice through the delayed HTTM intervention. Sprague-Dawley rats were subjected to unilateral cortical contusion injury and received therapeutic hypothermia at 15mins, 2 h, 4 h respectively after TBI. The neurological function was evaluated with the modified neurological severity score and Morris water maze test. The brain edema and morphological changes were measured with the water content and H&E staining. Brain sections were immunostained with antibodies against DCX (a neuroblast marker) and GFAP (an astrocyte marker). The apoptosis levels in the ipsilateral hippocampi and cortex were examined with antibodies against the apoptotic proteins Bcl-2, Bax, and cleaved caspase-3 by the immunofluorescence and western blotting. The results indicated that each hypothermia therapy group could improve neurobehavioral and cognitive function, alleviate brain edema and reduce inflammation. Furthermore, we observed that therapeutic hypothermia increased DCX expression, decreased GFAP expression, upregulated Bcl-2 expression and downregulated Bax and cleaved Caspase-3 expression. The above results suggested that HTTM at 2h or even at 4h post-injury revealed beneficial brain protection similarly, despite the best effect at 15min post-injury. These findings may provide relatively ideal time window models, further making the following experimental results more credible and persuasive.

Curcumin attenuates ischemia-like injury induced IL-1ß elevation in brain microvascular endothelial cells via inhibiting MAPK pathways and nuclear factor-κB activation.

Inflammatory reactions play a key role in the cerebral injury after stroke or other ischemic brain diseases. Curcumin, which is extracted from herb turmeric, has been reported to have anti-inflammatory effects. The present study was aimed to investigate the anti-inflammatory effects of curcumin on oxygen-glucose deprivation (OGD) injured brain microvascular endothelial cells (BMECs). Rat BMECs were used and the results showed that OGD induced a significant elevation of the leakage of lactate dehydrogenase and the secretion of the proinflammation cytokine, IL-1ß. Activation of p38, JNK MAPKs, and NF-κB in BMECs was also observed after OGD. The treatment of curcumin (20 µM) inhibited the increased production of IL-1ß both at the protein and mRNA levels. The increased phosphorylation of p38 and JNK induced by OGD was decreased under the treatment of curcumin, whereas the p38 inhibitor, SB203580, significantly inhibited OGD-induced IL-1ß production, but the JNK inhibitor, SP600125, failed to do so. These results suggest that the inhibition of IL-1ß by curcumin may dependent on the p38 signaling pathway. The OGD-induced IL-1ß production was also inhibited by the NF-κB inhibitor, and curcumin suppressed OGD-induced NF-κB activation. Furthermore, the NF-κB activation was attenuated by the SB203580, indicating that NF-κB activation was dependent on p38 signaling pathway. The present study suggests that curcumin displays an anti-inflammatory effect on OGD-injured BMECs via down-regulating of MAPK and NF-κB signaling pathways and might have therapeutic potential for the ischemic brain diseases.

[L-arginine suppresses ischemia/reperfusion induced up-regulation of endothelin-1 production in a rat model of acute myocardial injury].

OBJECTIVE: To examine the level of endothelin-1 (ET-1) in serum and its expression in myocardium tissue during the development of acute myocardial ischemia/reperfusion (I/R) injury in rat and the effects of L-arginine (L-Arg) administration on these indexes. METHODS: One hundred and ten Wistar rats were randomly divided into nine groups to receive: (1) sham surgery, (2)ischemia (I, by ligation of anterior descending coronary artery for 30 minutes), (3) I+reperfusion (R, by the removal of the ligature) for 0.5 hour, (4) I+R for 1 hour, (5) I+R for 2 hours; group (6) ~ (9) also received I/R treatment as in group 2 ~ 5 respectively but with L-Arg pretreatment. Blood and myocardium tissue samples were collected by the end of the experiment for the analysis of: serum level of creatine kinase (CK), lactate dehydrogenase [LDH, by enzyme linked immunosorbent assay (ELISA)], ET-1 (radioimmunoassay), and the tissue content of ET-1 mRNA/peptide [by reverse-transcription polymerase chain-reaction (RT-PCR) and Western blotting]. RESULTS: In comparison with the sham treated control animals, the serum levels of CK, LDH, and ET-1 were all significantly higher in the groups treated with I/R (particularly those exposed to reperfusion). The myocardial tissue content of ET-1 mRNA/peptide were also significantly increased in I/R treated groups (particularly the I+R 2 hours group) as compared to control (ET-1 mRNA: 0.775 ± 0.029 vs. 0.310 ± 0.076; ET-1 peptide: 0.773 ± 0.055 vs. 0.340 ± 0.099, both P < 0.05). The i.v. administration of L-Arg significantly suppressed the up-regulation of tissue content of ET-1 mRNA /peptide in I/R treated animals (ET-1 mRNA: 0.340 ± 0.049 vs. 0.775 ± 0.029; ET-1 peptide: 0.390 ± 0.094 vs. 0.773 ± 0.055, both P < 0.05). CONCLUSION: L-Arg may be tested during certain stage of I/R injury as a therapeutic intervention for the suppression of ET-1 up-regulation.

Improved method of acute myocardial ischemic rat model / 法医学杂志

OBJECTIVE@#To improve the successful rate of operation and the livability in establishing acute myocardial ischemic rat model.@*METHODS@#The successful rate of animal experiment is compared between traditional method and improved method.@*RESULTS@#The successful rate of improved method was 90%, which was much higher than 30% in successful rate of traditional method.@*CONCLUSION@#The improved method may reduce the difficulty of operation remarkably and cut down the experiment expenditure, which is superior to traditional method.