Xiao-hong Li

Sirt1 Promotes Axonogenesis by Deacetylation of Akt and Inactivation of GSK3

Accumulating evidence shows that Sirt1 regulates a variety of neurological functions through the deacetylation of many proteins besides histone; however, the literature on the relationship between Sirt1 and axonal outgrowth is limited. Here, we first demonstrated that Sirt1 was located in the axon, especially in the growth cone. Then, we found that genetic inhibition of Sirt1 retarded axonal development in embryonic hippocampal neurons, whereas genetic and pharmacologic upregulation of Sirt1 promoted not only the formation but also the elongation of axons. Sirt1 can deacetylate and thus activate Akt, and inhibition of Akt significantly reversed the axonogenesis induced by Sirt1 overexpression. We also found that Sirt1 inhibited the activity of glycogen synthase kinase 3 (GSK3), whereas activation of GSK3 could abolish the effect of Sirt1. These results suggest that Sirt1 promotes axonogenesis by deacetylating Akt and thereby activates the Akt/GSK3 pathway, which could be a promising therapeutic target for axonopathy.

Aβ-AGE aggravates cognitive deficit in rats via RAGE pathway

β-Amyloid (Aβ) accumulation has been proved to be responsible for the pathogenesis of Alzheimers disease (AD). However, it is not yet clear what makes Aβ accumulate and become toxic in the AD brains. Our previous studies demonstrated that glycated Aβ (Aβ-AGE) could be formed, and it exacerbated the authentic Aβ-mediated neurotoxicity in vitro, but we did not show the role of Aβ-AGE in vivo and the underlying mechanism. In the current study, we synthesized Aβ-AGE by incubating Aβ with methylglyoxal in vitro, and then stereotactically injected Aβ-AGE into lateral ventricle of Sprague-Dawley (SD) rats. We found that Aβ-AGE aggravated Aβ-induced cognitive impairment, which was characterized by higher speed of deterioration of long-term potentiation (LTP), more decrease of dendritic spines density and more down-regulation of synaptic proteins. We also observed the overexpression of receptor for advanced glycation endproducts receptor for AGEs (RAGE) and the activation of downstream molecular (GSK3, NF-κB, p38) in RAGE-mediated pathways. On the other hand, simultaneous application of RAGE antibody or GSK3 inhibitor LiCl reversed the cognitive decline amplified by Aβ-AGE. Our data revealed that in vivo the Aβ-AGE is more toxic than Aβ, and Aβ-AGE could lead to the aggravation of AD-like pathology though the RAGE pathway, suggesting that Aβ-AGE and RAGE may be new therapeutic targets for AD.

Combination of temperature-sensitive stem cells and mild hypothermia: a new potential therapy for severe traumatic brain injury.

Stem cell transplantation holds great potential for the treatment of traumatic brain injury (TBI). However, the micro-environment of reduced oxygen and accumulated toxins leads to low survival rates of grafted cells, which dramatically limits their clinical application. Mild hypothermia has been demonstrated to improve the micro-environment after severe TBI. Thus, we speculate that combinational therapy of mild hypothermia may promote survival of grafted cells, especially temperature-sensitive stem cells, which show the most activity in mild temperatures. In this study, we first isolated mesenchymal stem cells from umbilical cord (UCSMCs) and generated the temperature-sensitive UCSMCs (tsUCSMCs) by infection with a retrovirus carrying the temperature-sensitive tsA58 SV40 LT antigen gene. We demonstrated that tsUCSMCs grew and proliferated with more activity at 33°C than at 37°C by counting cell numbers with a hematocytometer, measuring the cell cycle with flow cytometry, and detecting proliferating cell nuclear antigen (PCNA) with immunofluorescence staining. Thereafter, we established the rat severe TBI model by fluid percussion, and injected PBS, UCSMCs, or tsUCSMCs into the injured region, and subject the animals to normothermia or mild hypothermia (33°C). We found that, compared with UCSMC or tsUCSMC treatment alone, their combination with hypothermia could significantly improve motor and cognitive function with more survival of the grafted cells. Furthermore, we observed that combined therapy with hypothermia and tsUCSMCs exerted the most protective effect on the recovery of neurological function of all the tested treatments, with the highest survival and proliferation rates, and the lowest apoptosis rate. Thus this may represent a new therapeutic strategy for the treatment of severe TBI.

FoxQ1 promotes glioma cells proliferation and migration by regulating NRXN3 expression.

Background Forkhead box Q1 (FoxQ1) is a member of the forkhead transcription factor family, and it has recently been found to participate in cancer development. However, whether FoxQ1 expression contributes to glioma development and progression is not known. We investigate FoxQ1 expression in gliomas and the role of FoxQ1 during tumorgenesis. Methods Reverse transcription quantitative real-time PCR (RT-qPCR) and Western blot were used to determine the FoxQ1 and Neurexins 3 (NRXN3) expression in gliomas. Chromatin immunoprecipitation (ChIP) and luciferase assays were used to determine the regulation between FoxQ1 and NRXN3. We established depleted FoxQ1 stable clones in U-87MG cells and overexpressed FoxQ1 stable clones in SW1088 cells. MTT and transwell were used to evaluate the ability of proliferation and migration, respectively. Results FoxQ1 mRNA and protein were up-regulated in gliomas and negatively related to the NRXN3 expression (r = −0.373, P = 0.042). FoxQ1 directly binds to NRXN3 promoter region and suppresses the promoter activity. Furthermore, the ability of proliferation and migration is reduced in depleted FoxQ1 cells. Conclusion FoxQ1 promotes glioma cell proliferation and migration by down-regulation of NRXN3 expression.

Mesenchymal stem cells maintain the microenvironment of central nervous system by regulating the polarization of macrophages/microglia after traumatic brain injury.

ABSTRACT Mesenchymal stem cells (MSCs), which are regarded as promising candidates for cell replacement therapies, are able to regulate immune responses after traumatic brain injury (TBI). Secondary immune response following the mechanical injury is the essential factor leading to the necrosis and apoptosis of neural cells during and after the cerebral edema has subsided and there is lack of efficient agent that can mitigate such neuroinflammation in the clinical application. By means of three molecular pathways (prostaglandin E2 (PGE2), tumor-necrosis-factor-inducible gene 6 protein (TSG-6), and progesterone receptor (PR) and glucocorticoid receptors (GR)), MSCs induce the activation of macrophages/microglia and drive them polarize into the M2 phenotypes, which inhibits the release of pro-inflammatory cytokines and promotes tissue repair and nerve regeneration. The regulation of MSCs and the polarization of macrophages/microglia are dynamically changing based on the inflammatory environment. Under the stimulation of platelet lysate (PL), MSCs also promote the release of pro-inflammatory cytokines. Meanwhile, the statue of macrophages/microglia exerts significant effects on the survival, proliferation, differentiation and activation of MSCs by changing the niche of cells. They form positive feedback loops in maintaining the homeostasis after TBI to relieving the secondary injury and promoting tissue repair. MSC therapies have obtained great achievements in several central nervous system disease clinical trials, which will accelerate the application of MSCs in TBI treatment.

Mild hypothermia facilitates the long-term survival of newborn cells in the dentate gyrus after traumatic brain injury by diminishing a pro-apoptotic microenvironment.

Although previous research has demonstrated that traumatic brain injury (TBI) accelerates the proliferation of neural stem cells in dentate gyrus of the hippocampus, most of these newborn cells undergo apoptosis in a traumatic microenvironment. Thus, promoting the long-term survival of newborn cells during neurogenesis is a compelling goal for the treatment of TBI. In this study, we investigated whether mild hypothermia (MHT) therapy, which mitigates the multiple secondary injury cascades of TBI, enhances the survival of newborn cells. SD rats were subjected to unilateral fluid percussion injury and received MHT therapy for 4h (33.5°C). Bromodeoxyuridine (BrdU) was administered to label the mitotic cells. Spatial learning and memory were evaluated with the Morris water maze test. Brain sections were immunostained with antibodies against BrdU, DCX (a neuroblast marker) or NeuN (a mature neuron marker). The apoptosis levels in the dentate gyrus were examined with antibodies against the apoptotic proteins FAS, FASL, Bcl-2 and cleaved caspase 3. The results indicated that MHT could significantly prevent TBI-induced cognitive impairments. At 1week after injury, the density of BrdU-immunoreactive cells significantly increased in both TBI and TBI+MHT rats. At 4weeks after injury, the density of BrdU-positive cells further increased in TBI+MHT rats, whereas the density declined in the TBI rats. The density of DCX-positive cells in SGZ of the hippocampus at 1week after injury in the TBI+MHT rats was significantly greater than in the TBI rats. Moreover, the density of NeuN-positive cells in the subgranular zone at 4weeks after injury and in the granule cell layer at 7weeks after injury was significantly increased in the TBI+MHT rats. The TBI+MHT rats displayed a lower level of apoptosis in the dentate gyrus compared with the TBI rats. These data indicate that TBI could only facilitate a burst of proliferation and short-term survival of newborn cells, whereas TBI+MHT could facilitate long-term survival and maturation of newborn cells through diminishing pro-apoptotic microenvironment. These results suggest that MHT-mediated neurogenesis may have an important therapeutic potential for the endogenous repair of TBI.

Magnetic resonance imaging-three-dimensional printing technology fabricates customized scaffolds for brain tissue engineering

Conventional fabrication methods lack the ability to control both macro- and micro-structures of generated scaffolds. Three-dimensional printing is a solid free-form fabrication method that provides novel ways to create customized scaffolds with high precision and accuracy. In this study, an electrically controlled cortical impactor was used to induce randomized brain tissue defects. The overall shape of scaffolds was designed using rat-specific anatomical data obtained from magnetic resonance imaging, and the internal structure was created by computer-aided design. As the result of limitations arising from insufficient resolution of the manufacturing process, we magnified the size of the cavity model prototype five-fold to successfully fabricate customized collagen-chitosan scaffolds using three-dimensional printing. Results demonstrated that scaffolds have three-dimensional porous structures, high porosity, highly specific surface areas, pore connectivity and good internal characteristics. Neural stem cells co-cultured with scaffolds showed good viability, indicating good biocompatibility and biodegradability. This technique may be a promising new strategy for regenerating complex damaged brain tissues, and helps pave the way toward personalized medicine.

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.

Collagen/heparin sulfate scaffolds fabricated by a 3D bioprinter improved mechanical properties and neurological function after spinal cord injury in rats

Effective treatments promoting axonal regeneration and functional recovery for spinal cord injury (SCI) are still in the early stages of development. Most approaches have been focused on providing supportive substrates for guiding neurons and overcoming the physical and chemical barriers to healing that arise after SCI. Although collagen has become a promising natural substrate with good compatibility, its low mechanical properties restrict its potential applications. The mechanical properties mainly rely on the composition and pore structure of scaffolds. For the composition of a scaffold, we used heparin sulfate to react with collagen by crosslinking. For the structure, we adopted a three-dimensional (3D) printing technology to fabricate a scaffold with a uniform pore distributions. We observed that the internal structure of the scaffold printed with a 3D bioprinter was regular and porous. We also found that both the compression modulus and strengths of the scaffold were significantly enhanced by the collagen/heparin sulfate composition compared to a collagen scaffold. Meanwhile, the collagen/heparin sulfate scaffold presented good biocompatibility when it was co-cultured with neural stem cells in vitro. We also demonstrated that heparin sulfate modification significantly improved bFGF immobilization and absorption to the collagen by examining the release kinetics of bFGF from scaffolds. Two months after implantating the scaffold into transection lesions in T10 of the spinal cord in rats, the collagen/heparin sulfate group demonstrated significant recovery of locomotor function and according to electrophysiological examinations. Parallel to functional recovery, collagen/heparin sulfate treatment further ameliorated the pathological process and markedly increased the number of neurofilament (NF) positive cells compared to collagen treatment alone. These data suggested that a collagen/heparin sulfate scaffold fabricated by a 3D bioprinter could enhance the mechanical properties of collagen and provide continuous guidance channels for axons, which would improve the neurological function after SCI.

Acupuncture Improved Neurological Recovery after Traumatic Brain Injury by Activating BDNF/TrkB Pathway

How to promote neural repair following traumatic brain injury (TBI) has long been an intractable problem. Although acupuncture has been demonstrated to facilitate the neurological recovery, the underlying mechanism is elusive. Brain-derived neurotrophic factor (BDNF) exerts substantial protective effects for neurological disorders. In this study, we found that the level of BDNF and tropomyosin receptor kinase B (TrkB) was elevated spontaneously after TBI and reached up to the peak at 12 h. Nevertheless, this enhancement is quickly declined to the normal at 48 h. After combined stimulation at the acupoints of Baihui, Renzhong, Hegu, and Zusanli, we found that BDNF and TrkB were still significantly elevated at 168 h. We also observed that the downstream molecular p-Akt and p-Erk1/2 were significantly increased, suggesting that acupuncture could persistently activate the BDNF/TrkB pathway. To further verify that acupuncture improved recovery through activating BDNF/TrkB pathway, K252a (specific inhibitor of TrkB) was treated by injection stereotaxically into lateral ventricle. We observed that K252a could significantly prevent the acupuncture-induced amelioration of motor, sensation, cognition, and synaptic plasticity. These data indicated that acupuncture promoted the recovery of neurological impairment after TBI by activating BDNF/TrkB signaling pathway, providing new molecular mechanism for understanding traditional therapy of acupuncture.