Jiangkai Lin

Curcumin attenuates acute inflammatory injury by inhibiting the TLR4/MyD88/NF-κB signaling pathway in experimental traumatic brain injury

BackgroundTraumatic brain injury (TBI) initiates a neuroinflammatory cascade that contributes to substantial neuronal damage and behavioral impairment, and Toll-like receptor 4 (TLR4) is an important mediator of thiscascade. In the current study, we tested the hypothesis that curcumin, a phytochemical compound with potent anti-inflammatory properties that is extracted from the rhizome Curcuma longa, alleviates acute inflammatory injury mediated by TLR4 following TBI.MethodsNeurological function, brain water content and cytokine levels were tested in TLR4-/- mice subjected to weight-drop contusion injury. Wild-type (WT) mice were injected intraperitoneally with different concentrations of curcumin or vehicle 15 minutes after TBI. At 24 hours post-injury, the activation of microglia/macrophages and TLR4 was detected by immunohistochemistry; neuronal apoptosis was measured by FJB and TUNEL staining; cytokines were assayed by ELISA; and TLR4, MyD88 and NF-κB levels were measured by Western blotting. In vitro, a co-culture system comprised of microglia and neurons was treated with curcumin following lipopolysaccharide (LPS) stimulation. TLR4 expression and morphological activation in microglia and morphological damage to neurons were detected by immunohistochemistry 24 hours post-stimulation.ResultsThe protein expression of TLR4 in pericontusional tissue reached a maximum at 24 hours post-TBI. Compared with WT mice, TLR4-/- mice showed attenuated functional impairment, brain edema and cytokine release post-TBI. In addition to improvement in the above aspects, 100 mg/kg curcumin treatment post-TBI significantly reduced the number of TLR4-positive microglia/macrophages as well as inflammatory mediator release and neuronal apoptosis in WT mice. Furthermore, Western blot analysis indicated that the levels of TLR4 and its known downstream effectors (MyD88, and NF-κB) were also decreased after curcumin treatment. Similar outcomes were observed in the microglia and neuron co-culture following treatment with curcumin after LPS stimulation. LPS increased TLR4 immunoreactivity and morphological activation in microglia and increased neuronal apoptosis, whereas curcumin normalized this upregulation. The increased protein levels of TLR4, MyD88 and NF-κB in microglia were attenuated by curcumin treatment.ConclusionsOur results suggest that post-injury, curcumin administration may improve patient outcome by reducing acute activation of microglia/macrophages and neuronal apoptosis through a mechanism involving the TLR4/MyD88/NF-κB signaling pathway in microglia/macrophages in TBI.

Functional recovery in acute traumatic spinal cord injury after transplantation of human umbilical cord mesenchymal stem cells

Objective:Spinal cord injury results in loss of neurons, degeneration of axons, formation of glial scar, and severe functional impairment. Human umbilical cord mesenchymal stem cells can be induced to form neural cells in vitro. Thus, these cells have a potential therapeutic role for treating spinal cord injury. Design and Setting:Rats were randomly divided into three groups: sham operation group, control group, and human umbilical cord mesenchymal stem cell group. All groups were subjected to spinal cord injury by weight drop device except for sham group. Subjects:Thirty-six female Sprague-Dawley rats. Interventions:The control group received Dulbeccos modified essential media/nutrient mixture F-12 injections, whereas the human umbilical cord mesenchymal stem cell group undertook cells transplantation at the dorsal spinal cord 2 mm rostrally and 2 mm caudally to the injury site at 24 hrs after spinal cord injury. Measurements:Rats from each group were examined for neurologic function and contents of brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, and neurotrophin-3. Survival, migration, and differentiation of human umbilical cord mesenchymal stem cells, regeneration of axons, and formation of glial scar were also explored by using immunohistochemistry and immunofluorescence. Main Results:Recovery of hindlimb locomotor function was significantly enhanced in the human umbilical cord mesenchymal stem cells grafted animals at 5 wks after transplantation. This recovery was accompanied by increased length of neurofilament-positive fibers and increased numbers of growth cone-like structures around the lesion site. Transplanted human umbilical cord-mesenchymal stem cells survived, migrated over short distances, and produced large amounts of glial cell line-derived neurotrophic factor and neurotrophin-3 in the host spinal cord. There were fewer reactive astrocytes in both the rostral and caudal stumps of the spinal cord in the human umbilical cord-mesenchymal stem cell group than in the control group. Conclusions:Treatment with human umbilical cord mesenchymal stem cells can facilitate functional recovery after traumatic spinal cord injury and may prove to be a useful therapeutic strategy to repair the injured spinal cord.

Glial scar and neuroregeneration: histological, functional, and magnetic resonance imaging analysis in chronic spinal cord injury.

OBJECT A glial scar is thought to be responsible for halting neuroregeneration following spinal cord injury (SCI). However, little quantitative evidence has been provided to show the relationship of a glial scar and axonal regrowth after injury. METHODS In this study performed in rats and dogs, a traumatic SCI model was made using a weight-drop injury device, and tissue sections were stained with H & E for immunohistochemical analysis. The function and behavior of model animals were tested using electrophysiological recording and the Basso-Beattie-Bresnahan Locomotor Rating Scale, respectively. The cavity in the spinal cord after SCI in dogs was observed using MR imaging. RESULTS The morphological results showed that the formation of an astroglial scar was defined at 4 weeks after SCI. While regenerative axons reached the vicinity of the lesion site, the glial scar blocked the extension of regrown axons. In agreement with these findings, the electrophysiological, behavioral, and in vivo MR imaging tests showed that functional recovery reached a plateau at 4 weeks after SCI. The thickness of the glial scars in the injured rat spinal cords was also measured. The mean thickness of the glial scar rostral and caudal to the lesion cavity was 107.00 +/- 20.12 microm; laterally it was 69.92 +/- 15.12 microm. CONCLUSIONS These results provide comprehensive evidence indicating that the formation of a glial scar inhibits axonal regeneration at 4 weeks after SCI. This study reveals a critical time window of postinjury recovery and a detailed spatial orientation of glial scar, which would provide an important basis for the development of therapeutic strategy for glial scar ablation.

Immediate splenectomy decreases mortality and improves cognitive function of rats after severe traumatic brain injury.

BACKGROUND Traumatic brain injury (TBI) is a major health problem all over the world. It frequently causes a considerable social burden because of its high incidence of death and long-term disability, especially in the case of severe TBI. Recent studies revealed that the spleen might contribute to secondary brain injury after ischemia or intracerebral hemorrhage. The purpose of this study was to evaluate the significance of the spleen in traumatic brain edema after severe TBI. METHODS We established a severe TBI model with rats and performed splenectomy to observe the mortality, brain water content, cognitive function (water maze), and cytokines levels, including interleukin (IL)-1β, tumor necrosis factor-α (TNF-α), IL-6, and IL-10, in the blood plasma (enzyme-linked immunosorbent assay) and their mRNA expression levels in injured brain tissue (quantitative reverse transcriptase-polymerase chain reaction). RESULTS The immediate splenectomy after TBI significantly decreased the death rate from 35.42% to 14.89% and eliminated the brain water content of the injured brain, especially at days 2 and 3. The Morris water maze assessment showed an improved spatial reference memory in rats that underwent both TBI and splenectomy when compared with those in the TBI group, 4 weeks later. Splenectomy reduced the IL-1β, TNF-α, and IL-6 contents in the blood serum after TBI, and the mRNA expression levels of IL-1β, TNF-α, and IL-6 in the ipsilateral brain tissue also decreased. CONCLUSIONS Our study demonstrates that splenectomy has a protective effect on rats with severe TBI by inhibiting proinflammatory cytokines, including IL-1β, TNF-α, and IL-6, both systematically and locally in the injured brain, hence leading to a decreased mortality and improved cognitive function.

In Vitro Labeling of Human Umbilical Cord Mesenchymal Stem Cells With Superparamagnetic Iron Oxide Nanoparticles

Human umbilical cord mesenchymal stem cells (hUC‐MSCs) transplantation has been shown to promote regeneration and neuroprotection in central nervous system (CNS) injuries and neurodegenerative diseases. To develop this approach into a clinical setting it is important to be able to follow the fates of transplanted cells by noninvasive imaging. Neural precursor cells and hematopoietic stem cells can be efficiently labeled by superparamagnetic iron oxide (SPIO) nanoparticle. The purpose of our study was to prospectively evaluate the influence of SPIO on hUC‐MSCs and the feasibility of tracking for hUC‐MSCs by noninvasive imaging. In vitro studies demonstrated that magnetic resonance imaging (MRI) can efficiently detect low numbers of SPIO‐labeled hUC‐MSCs and that the intensity of the signal was proportional to the number of labeled cells. After transplantation into focal areas in adult rat spinal cord transplanted SPIO‐labeled hUC‐MSCs produced a hypointense signal using T2‐weighted MRI in rats that persisted for up to 2 weeks. This study demonstrated the feasibility of noninvasive imaging of transplanted hUC‐MSCs. J. Cell. Biochem. 108: 529–535, 2009.

In vivo magnetic resonance imaging tracking of SPIO‐labeled human umbilical cord mesenchymal stem cells

Human umbilical cord mesenchymal stem cells (hUC‐MSCs) can be efficiently labeled by superparamagnetic iron oxide (SPIO) nanoparticles, which produces low signal intensity on magnetic resonance imaging (MRI) in vitro. This study was to evaluate the feasibility of in vivo tracking for hUC‐MSCs labeled by SPIO with noninvasive MRI. SPIO was added to cultures at concentrations equivalent to 0, 7, 14, 28, and 56 µg Fe/ml (diluted with DMEM/F12) and incubated for 16 h. Prussian Blue staining was used to determinate the labeling efficiency. Rats were randomly divided into three groups, control group, hUC‐MSCs group, and SPIO‐labeled hUC‐MSCs group. All groups were subjected to spinal cord injury (SCI) by weight drop device. Rats were examined for neurological function. In vivo MRI was used to track SPIO‐labeled hUC‐MSCs transplanted in rats spinal cord. Survival and migration of hUC‐MSCs were also explored using immunofluorescence. Significant improvements in locomotion were observed in the hUC‐MSCs groups. There was statistical significance compared with control group. In vivo MRI 1 and 3 weeks after injection showed a large reduction in signal intensity in the region transplanted with SPIO‐labeled hUC‐MSCs. The images from unlabeled hUC‐MSCs showed a smaller reduction in signal intensity. Transplanted hUC‐MSCs engrafted within the injured rats spinal cord and survived for at least 8 weeks. In conclusion, hUC‐MSCs can survive and migrate in the host spinal cord after transplantation, which promote functional recovery after SCI. Noninvasive imaging of transplanted SPIO‐labeled hUC‐MSCs is feasible. J. Cell. Biochem. 113: 1005–1012, 2012.

Deferoxamine attenuates iron-induced long-term neurotoxicity in rats with traumatic brain injury

This study investigated whether deferoxamine (DFO), an iron chelator attenuates iron-induced toxicity in rats with traumatic brain injury. In this study, three groups of Sprague–Dawley rats (sham, injury and DFO groups) were examined. Rats were killed on day 28 after Morris water maze testing and brains perfused for either non-heme brain binding or hemosiderin staining. Western blotting was used to measure protein levels of ferritin, transferrin and transient receptor potential canonical channel 6 (TRPC6). In TBI rats, there was a significant increase in brain iron on day 28, ferritin L, ferritin H, transferrin and TRPC6 levels were all significantly elevated post-TB1. There were also deficits in spatial learning and memory; however, DFO administration attenuated these effects in TBI rats supporting the notion that DFO may reduce brain injury accentuated by iron overload.

Immediate splenectomy down-regulates the MAPK-NF-κB signaling pathway in rat brain after severe traumatic brain injury.

BACKGROUND The treatment of severe traumatic brain injury (TBI) remains a difficult process. One key to improving treatment efficacy is to reduce secondary brain injury. Local and systemic inflammatory responses play an important role in secondary injury after TBI, which if unchecked can lead to fatal cerebral edema. Previous studies focused mainly on local brain tissue, whereas little is known about the contribution of peripheral organs in the pathogenesis of TBI. We previously showed that immediate splenectomy decreases mortality and improves cognitive function in rats after severe TBI by inhibiting the release of proinflammatory cytokines both systematically and locally in the injured brain. In this study, we further investigated the molecular mechanisms responsible for the effect of the spleen on local brain inflammation after TBI. METHODS We established a severe TBI model with rats and performed splenectomy to study the effect of the spleen on mitogen-activated protein kinase (MAPK)–NF-&kgr;B activation in the brain tissue. The expression of p38 MAPK, extracellular regulated protein kinases (ERK), and NF-&kgr;B protein in the trauma region was examined by Western blotting. The neuron-like PC-12 cell line and microglia-like BV-2 cell line were used for in vitro experiments to test the effects of spleen supernatant after TBI. Cell apoptosis (annexin V/propidium iodide staining), NF-&kgr;B nuclear translocation (immunofluorescence microscopy), and MAPK signaling (phosphorylation of p-p38 and p-ERK) were examined. RESULTS We found that TBI significantly up-regulated MAPK signaling in the injured brain region, whereas immediate splenectomy suppressed MAPK activation. In vitro, the spleen supernatant from rats after TBI also resulted in increased MAPK activation and NF-&kgr;B nuclear translocation in microglia-like BV-2 cells, whereas the application of interleukin (IL)-1R antagonist (IL-1Ra) significantly reduced the expression of p-p38 and p-ERK as well as NF-&kgr;B nuclear translocation. In addition, spleen supernatant after TBI induced apoptosis in neuron-like PC-12 cells, and IL-1Ra could effectively reduce apoptosis. CONCLUSION Our study demonstrates that immediate splenectomy down-regulates the MAPK–NF-&kgr;B signaling pathway in rat brain after severe TBI. We also provide experimental evidence for the potential use of IL-1Ra to alleviate brain inflammation after TBI.

Superoxide plays critical roles in electrotaxis of fibrosarcoma cells via activation of ERK and reorganization of the cytoskeleton.

Direct-current electrical field (DCEF) induces directional migration in many cell types by activating intracellular signaling pathways. However, the mechanisms coupling the extracellular electric stimulation to the intracellular signals remain largely unknown. In this study, we show that DCEF directs migration of HT-1080 fibrosarcoma cells to the cathode, stimulates generation of hydrogen peroxide and superoxide through the activation of NADPH oxidase, induces anode-facing cytoskeleton polarization, and activates ERK signaling. Subsequent studies demonstrate that the electrotaxis of HT-1080 fibrosarcoma cells is abolished by NADPH oxidase inhibitor and overexpression of manganese superoxide dismutase (MnSOD), an enzyme that hydrolyzes superoxide. In contrast, overexpression of catalases, which hydrolyze hydrogen peroxide, does not affect electrotaxis. MnSOD overexpression also eliminates cytoskeleton polarization as well as the activation of AKT, ERKs, and p38. In contrast, under catalase overexpression, the cytoskeleton still polarizes and p38 activation is affected. Finally, we show that inhibition of ERK activation also abolishes DCEF-induced directional migration and cytoskeleton polarization. Collectively, our results indicate that superoxide plays critical roles in DCEF-induced directional migration of fibrosarcoma cells, possibly by regulating the activation of ERKs. This study provides novel insights into the current understanding of DCEF-mediated cancer cell directional migration and metastasis.

G-protein coupled estrogen receptor 1 mediated estrogenic neuroprotection against spinal cord injury.

Objective:What underlies the protection of estrogen against spinal cord injury remains largely unclear. Here, we investigated the expression pattern of a new estrogen receptor, G-protein coupled estrogen receptor 1 in the spinal cord and its role in estrogenic protection against spinal cord injury. Design and Settings:Department of Neurosurgery and Key Laboratory of Neurotrauma, Southwest Hospital. Subjects:Male Sprague–Dawley rats. Interventions:The animals subjected to spinal cord injury were divided into six groups and given vehicle solution, 17&bgr;-estradiol, or G-protein coupled estrogen receptor 1 agonist G-1 at 15 mins and 24 hrs postinjury, or given nuclear estrogen receptor antagonist ICI 182,780 at 1 hr before spinal cord injury followed by 17&bgr;-estradiol administration at 15 mins and 24 hrs postinjury, or given G-protein coupled estrogen receptor 1 specific antisense or random control oligonucleotide at 4 days before spinal cord injury followed by 17&bgr;-estradiol administration at 15 mins and 24 hrs postinjury. Measurements:Male Sprague–Dawley rats were subjected to spinal cord injury using a weight-drop injury approach. Immunohistochemical assays were used to observe the distribution and cell-type expression pattern of G-protein coupled estrogen receptor 1. The terminal deoxynucleotidyl transferase dUTP nick-end labeling-staining assay and behavior tests were employed to assess the role of G-protein coupled estrogen receptor 1 in mediating estrogenic protection against spinal cord injury. Main Results:We show that G-protein coupled estrogen receptor 1 is mainly distributed in the ventral horn and white matter of the spinal cord, which is totally different from nuclear estrogen receptors. We also show that G-protein coupled estrogen receptor 1 is specifically expressed by neurons, oligodendrocytes, and microglial cells, but not astrocytes. Furthermore, estrogen treatment prevents spinal cord injury-induced apoptotic cell death and enhances functional recovery after spinal cord injury, which can be mimicked by the specific G-protein coupled estrogen receptor 1 agonist G-1 and inhibited by specific knockdown of G-protein coupled estrogen receptor 1 expression, but not pure nuclear ER antagonist ICI 182,780. Finally, we show that estrogen or G-1 up-regulates the protein expression level of G-protein coupled estrogen receptor 1 to intensify estrogenic effects during spinal cord injury. Conclusions:These results reveal that G-protein coupled estrogen receptor 1 may mediate estrogenic neuroprotection against spinal cord injury, and underline the promising potential of estrogen with its new target G-protein coupled estrogen receptor 1 for the treatment of spinal cord injury patients.