Shengli Hu

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.

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.

Hyperbaric oxygen preconditioning protects against traumatic brain injury at high altitude.

BACKGROUND Recent studies have shown that preconditioning with hyperbaric oxygen (HBO) can reduce ischemic and hemorrhagic brain injury. We investigated effects of HBO preconditioning on traumatic brain injury (TBI) at high altitude and examined the role of matrix metalloproteinase-9 (MMP-9) in such protection. METHODS Rats were randomly divided into 3 groups: HBO preconditioning group (HBOP; n = 13), high-altitude group (HA; n = 13), and high-altitude sham operation group (HASO; n = 13). All groups were subjected to head trauma by weight-drop device, except for HASO group. HBOP rats received 5 sessions of HBO preconditioning (2.5 ATA, 100% oxygen, 1 h daily) and then were kept in hypobaric chamber at 0.6 ATA (to simulate pressure at 4000m altitude) for 3 days before operation. HA rats received control pretreatment (1 ATA, room air, 1 h daily), then followed the same procedures as HBOP group. HASO rats were subjected to skull opening only without brain injury. Twenty-four hours after TBI, 7 rats from each group were examined for neurological function and brain water content; 6 rats from each group were killed for analysis by H&E staining and immunohistochemistry. RESULTS Neurological outcome in HBOP group (0.71 +/- 0.49) was better than HA group (1.57 +/- 0.53; p < 0.05). Preconditioning with HBO significantly reduced percentage of brain water content (86.24 +/- 0.52 vs. 84.60 +/- 0.37; p < 0.01). Brain morphology and structure seen by light microscopy was diminished in HA group, while fewer pathological injuries occurred in HBOP group. Compared to HA group, pretreatment with HBO significantly reduced the number of MMP-9-positive cells (92.25 +/- 8.85 vs. 74.42 +/- 6.27; p < 0.01). CONCLUSIONS HBO preconditioning attenuates TBI in rats at high altitude. Decline in MMP-9 expression may contribute to HBO preconditioning-induced protection of brain tissue against TBI.

Deferoxamine alleviates chronic hydrocephalus after intraventricular hemorrhage through iron chelation and Wnt1/Wnt3a inhibition

Post-hemorrhagic chronic hydrocephalus (PHCH) is a common complication after intraventricular hemorrhage (IVH). The mechanism of PHCH is not fully understood, and its treatment is relatively difficult. In the present study, a rat model of PHCH was used to elucidate the role of iron in the pathogenesis of PHCH. The action of deferoxamine (DFX) in IVH-induced PHCH, the expression of brain ferritin, the concentration of iron in cerebrospinal fluid (CSF), and changes in Wnt1/Wnt3a gene expression were determined. Results indicate that iron plays an important role in the occurrence of hydrocephalus after IVH. The iron chelator, DFX, can decrease the concentrations of iron and ferritin after cerebral hemorrhage and can thereby decrease the incidence of hydrocephalus. In addition, after IVH, the gene expression of Wnt1 and Wnt3a was enhanced, with protein expression also upregulated; DFX was able to suppress both gene and protein expression of Wnt1 and Wnt3a in brain tissue. This indicates that iron may be the key stimulus that activates the Wnt signaling pathway and regulates subarachnoid fibrosis after cerebral hemorrhage, and that DFX may be a candidate for preventing PHCH in patients with IVH.

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.

Interleukin-1 receptor associated kinases-1/4 inhibition protects against acute hypoxia/ischemia-induced neuronal injury in vivo and in vitro

Neuronal Toll-like receptors (TLRs)-2 and -4 have been shown to play a pivotal role in ischemic brain injury, and the interleukin-1 receptor associated kinases (IRAKs) are considered to be the key signaling molecules involved downstream of TLRs. Here, we investigated the expression levels of IRAK-1 and -4 and the effects of IRAK-1/4 inhibition on brain ischemic insult and neuronal hypoxia-induced injury. Male Sprague-Dawley (SD) rats and the rat neuroblastoma B35 cell line were used in these experiments. Permanent middle cerebral artery occlusion (MCAO) was induced by the intraluminal filament technique, and B35 cells were stimulated with the hypoxia-mimetic, cobalt chloride (CoCl(2)). Following induction of hypoxia/ischemia (H/I), B35 cells and cerebral cortical neurons expressed higher levels of IRAK-1 and -4. Furthermore, IRAK-1/4 inhibition decreased the mortality rate, functional deficits, and ischemic infarct volume by 7 days after MCAO. Similarly, IRAK-1/4 inhibition attenuated CoCl(2)-induced cytotoxicity and apoptosis in B35 cells in vitro. Our results show that IRAK-1/4 inhibition decreased the nuclear translocation of the nuclear factor-kappaB (NF-κB) p65 subunit, the levels of activated (phosphorylated) c-jun N-terminal kinase (JNK) and cleaved caspase-3, and the secretion of TNF-α and IL-6 in B35 cells at 6 h after CoCl(2) treatment. These data suggest that IRAK-1/4 inhibition plays a neuroprotective role in H/I-induced brain injury.

Amelioration of rCBF and PbtO2 following TBI at high altitude by hyperbaric oxygen pre-conditioning.

Abstract Objectives: Hypobaric hypoxia at high altitude can lead to brain damage and pre-conditioning with hyperbaric oxygen (HBO) can reduce ischemic/hypoxic brain injury. This study investigates the effects of high altitude on traumatic brain injury (TBI) and examines the neuroprotection provided by HBO pre-conditioning against TBI. Methods: Rats were randomly divided into four groups: HBO pre-conditioning group (HBOP, n = 10), high altitude group (HA, n = 10), plain control group (PC, n = 10) and plain sham operation group (sham, n = 10). All groups were subjected to head trauma by weight drop device except for the sham group. Rats from each group were examined for neurological function, regional cerebral blood flow (rCBF) and brain tissue oxygen pressure (PbtO2) and were killed for analysis by transmission electron microscope. Results: The score of neurological deficits in the HA group was highest, followed by the HBOP group and the PC group, respectively. Both rCBF and PbtO2 were the lowest in the HA group. Brain morphology and structure seen via the transmission electron microscope was diminished in the HA group, while fewer pathological injuries occurred in the HBOP and PC groups. Conclusions: High altitude aggravates TBI significantly and HBO pre-conditioning can attenuate TBI in rats at high altitude by improvement of rCBF and PbtO2. Pre-treatment with HBO might be beneficial for people traveling to high altitude locations.

Superoxide Mediates Direct Current Electric Field-Induced Directional Migration of Glioma Cells through the Activation of AKT and ERK

Direct current electric fields (DCEFs) can induce directional migration for many cell types through activation of intracellular signaling pathways. However, the mechanisms that bridge extracellular electrical stimulation with intracellular signaling remain largely unknown. In the current study, we found that a DCEF can induce the directional migration of U87, C6 and U251 glioma cells to the cathode and stimulate the production of hydrogen peroxide and superoxide. Subsequent studies demonstrated that the electrotaxis of glioma cells were abolished by the superoxide inhibitor N-acetyl-l-cysteine (NAC) or overexpression of mitochondrial superoxide dismutase (MnSOD), but was not affected by inhibition of hydrogen peroxide through the overexpression of catalase. Furthermore, we found that the presence of NAC, as well as the overexpression of MnSOD, could almost completely abolish the activation of Akt, extracellular-signal-regulated kinase (Erk)1/2, c-Jun N-terminal kinase (JNK), and p38, although only JNK and p38 were affected by overexpression of catalase. The presenting of specific inhibitors can decrease the activation of Erk1/2 or Akt as well as the directional migration of glioma cells. Collectively, our data demonstrate that superoxide may play a critical role in DCEF-induced directional migration of glioma cells through the regulation of Akt and Erk1/2 activation. This study provides novel evidence that the superoxide is at least one of the “bridges” coupling the extracellular electric stimulation to the intracellular signals during DCEF-mediated cell directional migration.

Antisense vimentin cDNA combined with chondroitinase ABC reduces glial scar and cystic cavity formation following spinal cord injury in rats.

The formation of glial scar and cystic cavities restricts axon regeneration after spinal cord injury. Chondroitin sulphate proteoglycans (CSPGs) are regarded as the prominent inhibitory molecules in the glial scar, and their inhibitory effects may be abolished in part by chondroitinase ABC (ChABC), which can digest CSPGs. CSPGs are secreted mostly by reactive astrocytes, which form dense scar tissues. The intermediate filament protein vimentin underpins the cytoskeleton of reactive astrocytes. Previously we have shown that retroviruses carrying full-length antisense vimentin cDNA reduce reactive gliosis. Here we administered both antisense vimentin cDNA and ChABC to hemisected rat spinal cords. Using RT-PCR, Western blotting and immunohistochemistry, we found that the combined treatment reduced the formation of glial scar and cystic cavities through degrading CSPGs molecules and inhibiting intermediate filament proteins. The modified intra- and extra-cellular architecture may alter the physical and biochemical characteristics of the scar, and the combined therapy might be used to inhibit glial scar formation.