Meng-Jen Lee

Acid fibroblast growth factor and peripheral nerve grafts regulate Th2 cytokine expression, macrophage activation, polyamine synthesis, and neurotrophin expression in transected rat spinal cords.

Spinal cord injury elicits an inflammatory response that recruits macrophages to the injured spinal cord. Quantitative real-time PCR results have shown that a repair strategy combining peripheral nerve grafts with acidic fibroblast growth factor (aFGF) induced higher interleukin-4 (IL-4), IL-10, and IL-13 levels in the graft areas of rat spinal cords compared with transected spinal cords at 10 and 14 d. This led to higher arginase I-positive alternatively activated macrophage (M2 macrophage) responses. The gene expression of several enzymes involved in polyamine biosynthesis pathways was also upregulated in the graft areas of repaired spinal cords. The treatment induced a twofold upregulation of polyamine levels at 14 d, as confirmed by HPLC. Polyamines are important for the repair process, as demonstrated by the observation that treatment with inhibitors of arginase I and ornithine decarboxylase attenuates the functional recoveries of repaired rats. After 14 d, the treatment also induced the expression of neurotrophin nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), as well as M2 macrophages within grafted nerves expressing BDNF. IL-4 was upregulated in the injury sites of transected rats that received aFGF alone compared with those that received nerve grafts alone at 10 d. Conversely, nerve graft treatment induced NGF and BDNF expression at 14 d. Macrophages expressing polyamines and BDNF may benefit axonal regeneration at 14 d. These results indicate that aFGF and nerve grafts regulate different macrophage responses, and M2 macrophages may play an important role in axonal regeneration after spinal cord injury in rats.

Combined treatment using peripheral nerve graft and FGF-1: Changes to the glial environment and differential macrophage reaction in a complete transected spinal cord

We used a complete spinal cord transection model in which the T8 spinal segment was removed to study the effect of combined treatment of peripheral nerve graft and application of FGF-1 on the glial environment. The combined treatment resulted in reduced astrocytic glial scarring, reactive macrophage gliosis, and inhibitory proteoglycan in the back-degenerated white matter tract. While the macrophage activities in the back-degenerative tract were down-regulated, those in the grafted peripheral nerves and in the distal Wallerian degenerative tracts were not. We concluded that the combined treatment changed the glial environment in the back-degenerative tract, and differentially regulated the macrophage activities in the system, in favor of CNS regeneration.

Effect of Enhanced Prostacyclin Synthesis by Adenovirus‐Mediated Transfer on Lipopolysaccharide Stimulation in Neuron‐Glia Cultures

Abstract: Prostacyclin (PGI2) is known as a short‐lived, potent vasodilator and platelet anti‐aggregatory eicosanoid. This work attempts to selectively augment PGI2 synthesis in neuron‐glia cultures by adenoviral (Ad) gene transfer of PGI synthase (PGIS) or bicistronic cyclooxygenase 1 (COX‐1)/PGIS and examines whether PGI2 confers protection against lipopolysaccharide (LPS) stimulation. Cultures released low levels of eicosanoids. Upon Ad‐PGIS or Ad‐COX‐1/PGIS infection, cultures selectively increased prostacyclin release. Both PGIS‐ and COX‐1/PGIS‐overexpressed cultures contained fewer microglial numbers. Further, they significantly attenuated LPS‐induced iNOS expression and lactate, nitric oxide, and TNF‐α production. Taken together, enhanced prostacyclin synthesis in neuron‐glial cultures reduced microglia number and suppressed LPS stimulation.

Local Delivery of High-Dose Chondroitinase ABC in the Sub-Acute Stage Promotes Axonal Outgrowth and Functional Recovery after Complete Spinal Cord Transection

Chondroitin sulfate proteoglycans (CSPGs) are glial scar-associated molecules considered axonal regeneration inhibitors and can be digested by chondroitinase ABC (ChABC) to promote axonal regeneration after spinal cord injury (SCI). We previously demonstrated that intrathecal delivery of low-dose ChABC (1 U) in the acute stage of SCI promoted axonal regrowth and functional recovery. In this study, high-dose ChABC (50 U) introduced via intrathecal delivery induced subarachnoid hemorrhage and death within 48 h. However, most SCI patients are treated in the sub-acute or chronic stages, when the dense glial scar has formed and is minimally digested by intrathecal delivery of ChABC at the injury site. The present study investigated whether intraparenchymal delivery of ChABC in the sub-acute stage of complete spinal cord transection would promote axonal outgrowth and improve functional recovery. We observed no functional recovery following the low-dose ChABC (1 U or 5 U) treatments. Furthermore, animals treated with high-dose ChABC (50 U or 100 U) showed decreased CSPGs levels. The extent and area of the lesion were also dramatically decreased after ChABC treatment. The outgrowth of the regenerating axons was significantly increased, and some partially crossed the lesion site in the ChABC-treated groups. In addition, retrograde Fluoro-Gold (FG) labeling showed that the outgrowing axons could cross the lesion site and reach several brain stem nuclei involved in sensory and motor functions. The Basso, Beattie and Bresnahan (BBB) open field locomotor scores revealed that the ChABC treatment significantly improved functional recovery compared to the control group at eight weeks after treatment. Our study demonstrates that high-dose ChABC treatment in the sub-acute stage of SCI effectively improves glial scar digestion by reducing the lesion size and increasing axonal regrowth to the related functional nuclei, which promotes locomotor recovery. Thus, our results will aid in the treatment of spinal cord injury.

Regulation of chondroitin sulphate proteoglycan and reactive gliosis after spinal cord transection: effects of peripheral nerve graft and fibroblast growth factor 1.

M‐J. Lee, C. J. Chen, W‐C. Huang, M‐C. Huang, W‐C. Chang, H‐S. Kuo, M‐J. Tsai, Y‐L. Lin and H. Cheng (2011) Neuropathology and Applied Neurobiology37, 585–599

Adeno-associated virus-mediated human acidic fibroblast growth factor expression promotes functional recovery of spinal cord–contused rats

Following spinal cord injury, the delivery of neurotrophic factors to the injured spinal cord has been shown to promote axonal regeneration and functional recovery. In previous studies, we showed that acidic fibroblast growth factor (aFGF) is a potent neurotrophic factor that promotes the regeneration of axotomized spinal cord or dorsal root ganglion neurones.

Enhanced expression of glycine N-methyltransferase by adenovirus-mediated gene transfer in CNS culture is neuroprotective

Glycine N‐methyltransferase (GNMT) is the most abundant hepatic methyltransferase and plays important roles in regulating methyl group metabolism. In the central nervous system, GNMT expression is low and its function has not been revealed. The present study examines the effect of GNMT overexpression by adenovirus‐mediated transfer in cortical mixed neuron‐glial cultures. Infection of adenovirus encoding green fluorescence protein to cultures demonstrates high preference for non‐neuronal cells. Optimal GNMT overexpression in cultures by adenoviral GNMT (Ad‐GNMT) infection not only induces protein kinase C phosphorylation, but also increases neuronal/oligodendroglial survival. Furthermore, these Ad‐GNMT‐infected cultures are significantly resistant to H2O2 toxicity and lipopolysaccharide stimulation. Conditioned media from Ad‐GNMT‐infected microglia also significantly enhance neuronal survival. Taken together, enhanced GNMT expression in mixed neuronal‐glial cultures is neuroprotective, most likely mediated through non‐neuronal cells.

Treatment with nerve grafts and aFGF attenuates allodynia caused by cervical root transection injuries.

PURPOSE Nerve root traction injuries induce spinal cord inflammation and lead to neuronal death within days. In the present study, we examined the inflammatory response one week after multiple cervical root transections. METHODS In the transection group, the left cervical roots (C6-8) of rats were cut at the spinal cord junction. In the repair group, transected roots were repaired with nerve grafts and the subsequent application of aFGF and fibrin glue. A sham group had nerve roots exposed without transection. Mechanical allodynia and spinal glial responses were evaluated. RESULTS Allodynia did not differ between the treatment groups on day 2. Rats with transected spinal nerve roots had significantly more allodynia by 7 days, which was associated with IL-1β expression in dorsal and ventral horn astrocytes, and microglia activation. Repair of nerve roots with autologous intercostal nerve grafts and FGF in fibrin glue attenuated the allodynia, reduced IL-1β expression in astroctyes and reduced microglia activation, along with a significant increase in arginase I expression. CONCLUSION This study demonstrated a correlation between an increased number of IL-1β-positive astrocytes and the development of allodynia. Our treatment significantly decreased IL-1β-positive astrocytes, thus preventing the occurrence of neuropathic pain following multiple cervical root injuries.

Evaluation of the antiangiogenic effect of Kringle 1-5 in a rat glioma model.

BACKGROUND: Kringle 1-5 (K1-5) is a potent antiangiogenesis factor for treating breast cancer and hepatocellular carcinoma. However, its use in treating brain tumors has not been studied. OBJECTIVE: To evaluate whether K1-5 is effective at treating gliomas. METHODS: The effects of K1-5 on cell morphology and cytotoxicity with or without lipopolysaccharide were tested in primary mixed neuronal-glial cultures. The antiglioma activity of K1-5 was evaluated by intra-arterial administration of K1-5 at 4 days after implantation of C6 glioma cells into the rat hippocampus. In 1 group of animals, tumor size, tumor vasculature, and tumor histology were evaluated on day 12. Animal survival was assessed in the other group. RESULTS: In vitro studies showed that K1-5 did not induce cytotoxicity in neurons and glia. In vivo studies demonstrated that K1-5 reduced vessel length and vessel density and inhibited perivascular tumor invasion. In addition, K1-5 normalized vessel morphology, decreased expression of hypoxia-inducible factor-1&agr; and vascular endothelial growth factor, decreased tumor hypoxia, and decreased pseudopalisading necrosis. The average tumor volume was smaller in the treated than in the untreated group. Furthermore, animals treated with K1-5 survived significantly longer. CONCLUSION: Kringle 1-5 effectively reduces the growth of malignant gliomas in the rat. Although still far from translation in humans, K1-5 might be a possible future alternative treatment option for patients with gliomas.

Local inhibition of matrix metalloproteinases reduced M2 macrophage activity and impeded recovery in spinal cord transected rats after treatment with fibroblast growth factor-1 and nerve grafts

Alternatively activated macrophages (M2 macrophages) promote central nervous system regeneration. Our previous study demonstrated that treatment with peripheral nerve grafts and fibroblast growth factor-1 recruited more M2 macrophages and improved partial functional recovery in spinal cord transected rats. The migration of macrophages is matrix metalloproteinase (MMP) dependent. We used a general inhibitor of MMPs to influence macrophage migration, and we examined the migration of macrophage populations and changes in spinal function. Rat spinal cords were completely transected at T8, and 5 mm of spinal cord was removed (group T). In group R, spinal cord-transected rats received treatment with fibroblast growth factor-1 and peripheral nerve grafts. In group RG, rats received the same treatment as group R with the addition of 200 μM GM6001 (an MMP inhibitor) to the fibrin mix. We found that MMP-9, but not MMP-2, was upregulated in the graft area of rats in group R. Local application of the MMP inhibitor resulted in a reduction in the ratio of arginase-1 (M2 macrophage subset)/inducible nitric oxide synthase-postive cells. When the MMP inhibitor was applied at 8 weeks postoperation, the partial functional recovery observed in group R was lost. This effect was accompanied by a decrease in brain-derived neurotrophic factor levels in the nerve graft. These results suggested that the arginase-1 positive population in spinal cord transected rats is a migratory cell population rather than the phenotypic conversion of early iNOS+ cells and that the migration of the arginase-1+ population could be regulated locally. Simultaneous application of MMP inhibitors or promotion of MMP activity for spinal cord injury needs to be considered if the coadministered treatment involves M2 recruitment.