Pei Hua Lu

Glutamine synthetase down-regulation reduces astrocyte protection against glutamate excitotoxicity to neurons.

Although the role of astrocyte glutamate transporters in glutamate clearance is well illustrated, the role of glutamine synthetase (GS) that influences this process remains to be elucidated. We examined whether GS affected the uptake of glutamate in astrocytes in vitro. The glutamate uptake was assessed by measuring the concentration of glutamate and glutamine in culture medium in the presence or absence of glutamate. We demonstrated that inhibition of GS in astrocytes by MSO significantly impaired glutamate uptake and glutamine release. Conversely, induction of GS expression in astrocytes by gene transfer significantly enhanced the glutamate uptake and glutamine release. When an inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) was applied to the cultures, it significantly reduced GS expression and inhibited glutamate-induced GS activation resulting in increased excitotoxicity to neurons. These results suggest that GS in astrocytes may represent a novel target for neuroprotection against neuronal dysfunction and death that occur in many neurological disorders.

A novel role of phospholipase A2 in mediating spinal cord secondary injury

To investigate whether phospholipase A2 (PLA2) plays a role in the pathogenesis of spinal cord injury (SCI).

Chondroitin sulfate proteoglycans regulate the growth, differentiation and migration of multipotent neural precursor cells through the integrin signaling pathway.

BackgroundNeural precursor cells (NPCs) are defined by their ability to proliferate, self-renew, and retain the potential to differentiate into neurons and glia. Deciphering the factors that regulate their behaviors will greatly aid in their use as potential therapeutic agents or targets. Chondroitin sulfate proteoglycans (CSPGs) are prominent components of the extracellular matrix (ECM) in the central nervous system (CNS) and are assumed to play important roles in controlling neuronal differentiation and development.ResultsIn the present study, we demonstrated that CSPGs were constitutively expressed on the NPCs isolated from the E16 rat embryonic brain. When chondroitinase ABC was used to abolish the function of endogenous CSPGs on NPCs, it induced a series of biological responses including the proliferation, differentiation and migration of NPCs, indicating that CSPGs may play a critical role in NPC development and differentiation. Finally, we provided evidence suggesting that integrin signaling pathway may be involved in the effects of CSPGs on NPCs.ConclusionThe present study investigating the influence and mechanisms of CSPGs on the differentiation and migration of NPCs should help us to understand the basic biology of NPCs during CNS development and provide new insights into developing new strategies for the treatment of the neurological disorders in the CNS.

Differential gene expression in neural stem cells and oligodendrocyte precursor cells: a cDNA microarray analysis.

The use of neural stem cells (NSCs) or their progeny oligodendrocyte precursor cells (OPCs) represents a promising repair strategy for many neurological disorders. However, the molecular events and biological features during the transition from NSCs to OPCs remain unclear. In the present study, we isolated NSCs from the embryonic rat forebrain and induced them into OPCs by using B104 conditioned medium (B104CM) in vitro. We then employed cDNA array technology to compare changes in gene expression between the two cell populations. Among 1,176 genes examined, 40 were differentially expressed, and some of them may be involved in OPC differentiation from NSCs. Our findings thus provide new insights into the molecular basis of differentiation of OPCs from NSCs.

Immunization with recombinant Nogo-66 receptor (NgR) promotes axonal regeneration and recovery of function after spinal cord injury in rats.

Nogo-66 receptor (NgR), a common receptor for the three known myelin-associated inhibitors, i.e., Nogo-A, myelin-associated glycoprotein (MAG), and oligodendrocyte myelin glycoprotein (OMgp), plays a key role in the failure of axonal regeneration in the adult mammalian central nervous system (CNS). Here we report a novel vaccine approach that stimulates the production of anti-NgR antibody to overcome NgR-mediated growth inhibition after spinal cord injury (SCI). We showed that adult rats immunized with recombinant NgR produced high titers of the anti-NgR antibody and that antisera obtained from the immunized rats promoted neurite outgrowth of rat cerebellar neurons on the inhibitory MAG substrate in vitro. In a spinal cord dorsal hemisection model, NgR immunization promoted regeneration of lesioned corticospinal tract (CST) axons, anterogradely labeled with biotin dextran amine (BDA), beyond the lesion site. In a contusive SCI model, NgR immunization markedly reduced the total lesion volume and improved Basso, Beattie, and Bresnahan (BBB) locomotor rating scale and grid walking performance. Thus, the NgR vaccine approach may represent a promising repair strategy to promote structural and functional recovery following SCI.

Methods for isolating highly-enriched embryonic spinal cord neurons: A comparison between enzymatic and mechanical dissociations

Spinal cord neuronal culture is a useful system to study normal and abnormal functions of the spinal cord. For many bioassays, obtaining large quantities of highly purified spinal cord neurons is required. However, technical difficulties exist in obtaining these cells reliably and consistently. By comparing two dissociation methods, mechanical and enzymatic dissociations, we found that the enzymatic dissociation of embryonic day 14-15 spinal cords resulted in significantly higher cell yield than the mechanical dissociation (25.40 +/- 5.41 x 10(6) versus 3.43 +/- 0.52 x 10(6) cells per 12 embryos; n = 6/group; p < 0.01). Furthermore, cell viability was significantly higher after the enzymatic than the mechanical dissociation (83.40 +/- 3.08% versus 32.81 +/- 3.49%, n = 4/group; p < 0.01). In both methods, highly purified populations of primary neurons were obtained (mechanical: 85.17 +/- 2.84%; enzymatic: 87.67 +/- 2.52%; n = 3/group). Critical measures that affect culture outcomes include, but not limited to, the age of embryo, cell seeding density, dissociation time, and elimination of non-neuronal cells. Thus, the present study has identified the enzymatic dissociation method to be a preferred method for obtaining large quantity of highly-enriched embryonic spinal cord neurons.

Effects of autoimmunity on recovery of function in adult rats following spinal cord injury

The central nervous system (CNS) is considered to be an immune-privileged site. For a long time, autoimmunity-induced inflammation has been viewed as an important mediator of secondary damage in the CNS following injury. However, other studies also suggest that autoimmunity is protective and beneficial. To investigate whether protective autoimmunity is present following spinal cord injury (SCI), we employed neonatally thymectomized (Tx) rats which contain few T lymphocytes in their peripheral blood, and passively immunized them with T lymphocytes activated by myelin basic protein (MBP) or spinal cord homogenate (SCH). Here we report that, among Tx, sham-Tx (sTx) and normal rats that received a contusive SCI, no significant histological and behavioral differences were found, suggesting that the endogenous T lymphocytes had no significant influence on the pathogenesis of secondary SCI. In rats passively immunized with MBP- or SCH-activated T cells (MBP-T or SCH-T, respectively), similar numbers of CD4(+) T cells were found to infiltrate into the injured spinal cords. However, only the MBP-T immunization showed neuroprotection, evidenced by the reduction of post-traumatic neuronal losses and improvement of functional recovery. These results collectively suggest that not all T lymphocytes against CNS antigens are neuroprotective and that a subpopulation of them, such as those of MBP-T cells, could be beneficial for SCI repair.

DNA vaccine against NgR promotes functional recovery after spinal cord injury in adult rats.

NgR is a common receptor for three myelin-associated inhibitors and mediates their inhibitory activities on neurite outgrowth. In the present study, we investigated whether a DNA vaccine targeting NgR could play a beneficial role in improving recovery from spinal cord injury (SCI). We demonstrated that a DNA vaccine against NgR was successfully constructed and expressed efficiently in vitro and in vivo. After immunization with anti-NgR DNA vaccine, a low level of antibody response and a T cell-mediated immune response were induced in the vaccinated rats. And the antisera taken from the anti-NgR DNA vaccinated rats could partly reverse the inhibition of MAG on neurite outgrowth. When the rats were subjected to a contusive SCI, the vaccinated rats showed much better functional recovery than the controls. In those vaccinated rats that induced a T cell response and generated antibodies against NgR, functional improvements were even better. Histological assessments by three-dimensional reconstruction further demonstrated that the total lesion volume in the vaccinated rats was reduced by 30.8% compared to the controls. These results collectively suggest that DNA vaccine against NgR can significantly improve functional recovery in rats that received contusive SCI and that the vaccination approach may provide a promising strategy for promoting SCI repair.

Expression and regulation of versican in neural precursor cells and their lineages

AbstractAim:To have a better understanding of the expression and regulation of versican isoforms in neural precursor cells (NPC) and oligodendrogliogenesis.Methods:By immunocytochemistry, RT-PCR, and real-time PCR, we examined the temporal expression of versican in NPC isolated from embryonic d 16 rats as well as in oligodendrocyte (OL) lineage cells induced to differentiate from NPC, which mimicked the oligodendrogliogenesis in vivo.Results:We found that versican was constitutively expressed in NPC and their lineage cells, including neurons, astrocytes, and OL. In addition, 2 versican isoforms, V1/V0 and V2, were found to express at low levels in NPC, but at significantly higher levels in OL lineage cells. The peak expression of versican V2 was found at the oligo-dendrocyte precursor cell stage. Furthermore, the treatment of 2 pro-inflammatory cytokines, TNF-α and IFN-γ, enhanced the transcription of versican V2 in NPC in a dose-dependent manner, but showed no effect on V1/V0 expression.Conclusion:Taken together, our results demonstrate that versican, particularly the inhibitory V2 isoform, is increasingly expressed in OL lineage cells induced to differentiate from NPC. An increase in versican V2 expression after cytokine stimulation implies the interplay between the injury-induced upregulation of inflammatory cytokines and chondroitin sulfate proteoglycan-mediated inhibition of axonal regeneration after central nervous system injury.

Expression and localization of p80 interleukin-1 receptor protein in the rat spinal cord

The biological effects of interleukin (IL)-1 are mediated by two distinct receptors, the p80 or type I (IL-1RI) and p68 or type II (IL-1RII) receptors. Because IL-1RII has a short, 29-amino acid cytoplasmic domain which may not be sufficient for signaling, there is considerable evidence indicating that IL-1 may signal exclusively through the IL-1RI receptor. Here, we report the expression, distribution, and cellular localization of the IL-1RI protein in the adult rat spinal cord in vivo and embryonic spinal cord in vitro. We found that IL-1RI was expressed in both the gray and white matter throughout the entire length of the spinal cord and was localized in neurons of the anterior horn, astrocytes, oligodendrocytes, and central canal ependymal cells. Interestingly, resting microglia were negative for IL-1RI. In primary cultures obtained from the embryonic day (E) 15 rats, IL-1RI was expressed in eeurons, astrocytes, and oligodendrocytes as well as microglia. These data provide both in vivo and in vitro evidence that neurons and glial cells express the IL-1RI proteins. The differential expression of IL-1RI in the developing, but not mature, microglia may indicate the difference of these cells in response to IL-1 stimuli during maturation. The distribution and cellular localization of IL-1RI proteins in the spinal cord provide a molecular basis for understanding the reciprocal interaction between the immune and the central nervous systems.