Evaluating the toxic mechanism of 1,2-diacetylbenzene in neural cells/tissues: The favorable impact of silibinin.

1,2-diacetylbenzene (1,2-DAB) is a neurotoxic component of aromatic solvents commonly used in industrial applications that induces neuropathological changes in animals. This study unraveled the toxic impact of 1,2-DAB in nerve tissues, explant cultures, and neuron-glial cultures, and explored whether herbal products can mitigate its toxicity. The effects of DAB on axonal transport were studied in retinal explant cultures grown in a micro-patterned dish. The mitochondrial movement in the axons was captured using time-lapse video recordings. The results showed that 1,2-DAB, but not 1,3-DAB inhibited axonal outgrowth and mitochondrial movement in a dose-dependent manner. The toxicity of 1,2-DAB was further studied in spinal cord tissues and cultures. 1,2-DAB selectively induced modifications of microtubules and neurofilaments in spinal cord tissues. 1,2-DAB also potently induced cell damage in both neuronal and glial cultures. Further, 1,2-DAB-induced cellular ATP depletion precedes cell damage in glial cells. Interestingly, treatment with the herbal products silibinin or silymarin effectively mitigated 1,2-DAB-induced toxicity in spinal cord tissues and neuronal/glial cultures. Collectively, the molecular toxicity of 1,2-DAB in neural tissues involves protein modification, ATP depletion, and axonal transport defects, leading to cell death. Silibinin and silymarin show promising neuroprotective effects against 2-DAB-induced toxicity.

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.

Improving the regenerative potential of olfactory ensheathing cells by overexpressing prostacyclin synthetase and its application in spinal cord repair.

BACKGROUND: Olfactory ensheathing cells (OEC), specialized glia that ensheathe bundles of olfactory nerves, have been reported as a favorable substrate for axonal regeneration. Grafting OEC to injured spinal cord appears to facilitate axonal regeneration although the functional recovery is limited. In an attempt to improve the growth-promoting properties of OEC, we transduced prostacyclin synthase (PGIS) to OEC via adenoviral (Ad) gene transfer and examined the effect of OEC with enhanced prostacyclin synthesis in co-culture and in vivo. Prostacyclin is a vasodilator, platelet anti-aggregatory and cytoprotective agent. RESULTS: Cultured OEC expressed high level of cyclooxygneases, but not PGIS. Infection of AdPGIS to OEC could selectively augument prostacyclin synthesis. When cocultured with either OEC or AdPGIS-OEC, neuronal cells were resistant to OGD-induced damage. The resulted OEC were further transplanted to the transected cavity of thoracic spinal cord injured (SCI) rats. By 6 weeks post-surgery, significant functional recovery in hind limbs occurred in OEC or AdPGIS-OEC transplanted SCI rats compared with nontreated SCI rats. At 10-12 weeks postgraft, AdPGIS-OEC transplanted SCI rats showed significantly better motor restoration than OEC transplanted SCI rats. Futhermore, regenerating fiber tracts in the distal spinal cord stump were found in 40-60% of AdPGIS-OEC transplanted SCI rats. CONCLUSIONS: Enhanced synthesis of prostacyclin in grafted OEC improved fiber tract regeneration and functional restoration in spinal cord injured rats. These results suggest an important potential of prostacyclin in stimulating OEC therapeutic properties that are relevant for neural transplant therapies.

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-1α 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.

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-1ß 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-1ß 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-1ß-positive astrocytes and the development of allodynia. Our treatment significantly decreased IL-1ß-positive astrocytes, thus preventing the occurrence of neuropathic pain following multiple cervical root injuries.

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

BACKGROUND: 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. METHODS: We constructed a recombinant adeno-associated virus (AAV) vector to express human aFGF and evaluated aFGF expression and function in AAV-aFGF-infected PC12 cells. We analyzed AAV-green fluorescent protein (GFP) tropism and AAV-mediated aFGF expression in contused spinal cords. Animals received behavioural testing to evaluate the functional recovery. RESULTS: Overexpression of aFGF was shown in AAV-aFGF-infected PC12 cells in a dose-dependent manner. Concurrently, neurite extension and cell number were significantly increased in AAV-aFGF infected cells. AAV-mediated GFP expression persisted for at least 5 weeks in contused spinal cords, and the most prominently transduced cells were neurones. Contusive injury reduced endogenous aFGF expression in spinal cords. Overexpression of aFGF was demonstrated in AAV-aFGF transduced spinal cords compared to AAV-GFP transduced spinal cords at 3 and 14 days post-injury. Evaluation of motor function revealed that the improvement of AAV-aFGF-treated rats was prominent. Both AAV-aFGF- and recombinant human aFGF-treated rats revealed significantly better recovery at 5 weeks post-injury, compared to vehicle- and AAV-GFP-treated rats. CONCLUSIONS: These data suggest that supplement of aFGF improve the functional recovery of spinal cord-contused rats and that AAV-aFGF-mediated gene transfer could be a clinically feasible therapeutic approach for patients after nervous system injuries.

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.

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 H(2)O(2) 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.

Functional recovery after the repair of transected cervical roots in the chronic stage of injury.

The treatment of root injury is typically performed at the more chronic stages post injury, by which time a substantial number of neurons have died. Therefore, before being applied in the clinical setting, a treatment strategy for these lesions should prove to be as effective in the chronic stages of injury as it is in the acute stage. In this study, we simulated the most severe clinical scenarios to establish an optimal time window for repair at a chronic stage. The sixth to eighth cervical roots on the left side of female SD rats were cut at their junction with the spinal cord. One or three weeks later, the wound was reopened and these roots were repaired with intercostal nerve grafts, with subsequent application of aFGF and fibrin glue. In the control group, the wound was closed after re-exploration without further repair procedures. Sensory and motor functions were measured after the surgery. Spinal cord morphology, neuron survival, and nerve fiber regeneration were traced by CTB-HRP. Results showed that both the sensory and motor functions had significant recovery in the 1-week repair group, but not in the 3-week repair group. By CTB-HRP tracing, we found that the architecture of the spinal cords was relatively preserved in the 1-week repair group, while those of the control group showed significant atrophic change. There were regenerating nerve fibers in the dorsal horn and more motor neuron survival in the 1-week repair group compared to that of the 3-week group. It was concluded that treating transected cervical roots at a chronic stage with microsurgical nerve grafting and application of aFGF and fibrin glue can lead to significant functional recovery, as long as the repair is done before too many neurons die.

PAL31 may play an important role as inflammatory modulator in the repair process of the spinal cord injury rat.

Functional regeneration in a complete T8 transection model Cheng et al. (1996) and most recently, acidic fibroblast growth factor (aFGF; also known as FGF-1) involved in the repair process of the spinal cord injury (SCI) rat Tsai et al. (2008) have been reported. To further reveal the mechanism of the repair process of SCI, in additionally, we have identified a 30 kDa specific protein kinase A substrate induced at 6 days after SCI. However, the induction of the transducing signal was reduced in samples treated with aFGF. The 30 kDa protein was purified and identified by mass spectrometry as a novel protein, PAL31. The results of immunohistochemical study showed that PAL31 is abundantly expressed in the epicenter of the injured spinal cord and colocalizes with ED1-positive cells (macrophages) and CD8 T lymphocytes. Over-expression of PAL31 in RAW 264.7 cells resulted in the down-regulation of macrophage chemoattractant protein 1, inducible nitric oxide synthase, and signal transducer and activator of transcription-1. However, knockdown of PAL31 by small interfering RNA seems to lead to apoptosis when the cells were treated with inflammatory inducers. These experimental results suggest that PAL31 may involve in the modulation of the inflammatory response and, at the same time, prevent apoptosis process of macrophage after SCI.

Involvement of acidic fibroblast growth factor in spinal cord injury repair processes revealed by a proteomics approach.

Acidic fibroblast growth factor (aFGF; also known as FGF-1) is a potent neurotrophic factor that affects neuronal survival in the injured spinal cord. However, the pathological changes that occur with spinal cord injury (SCI) and the attribution to aFGF of a neuroprotective effect during SCI are still elusive. In this study, we demonstrated that rat SCI, when treated with aFGF, showed significant functional recovery as indicated by the Basso, Beattie, and Bresnahan locomotor rating scale and the combined behavior score (p < 0.01-0.001). Furthermore proteomics and bioinformatics approaches were adapted to investigate changes in the global protein profile of the damaged spinal cord tissue when experimental rats were treated either with or without aFGF at 24 h after injury. We found that 51 protein spots, resolvable by two-dimensional PAGE, had significant differential expression. Using hierarchical clustering analysis, these proteins were categorized into five major expression patterns. Noticeably proteins involved in the process of secondary injury, such as astrocyte activation (glial fibrillary acidic protein), inflammation (S100B), and scar formation (keratan sulfate proteoglycan lumican), which lead to the blocking of injured spinal cord regeneration, were down-regulated in the contusive spinal cord after treatment with aFGF. We propose that aFGF might initiate a series of biological processes to prevent or attenuate secondary injury and that this, in turn, leads to an improvement in functional recovery. Moreover the quantitative expression level of these proteins was verified by quantitative real time PCR. Furthermore we identified various potential neuroprotective protein factors that are induced by aFGF and may be involved in the spinal cord repair processes of SCI rats. Thus, our results could have a remarkable impact on clinical developments in the area of spinal cord injury therapy.

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.

Sensory and motor recovery after repairing transected cervical roots.

BACKGROUND: Adult mammal sensory axons avulsed through spinal dorsal root traction injuries, especially of the brachial plexus or cauda equina, cannot normally regenerate through axonal outgrowth from the DRG into the spinal cord, thus causing clinical conditions that require neuronal regeneration for sensory recovery and for which no successful treatment has yet been reported. METHODS: To evaluate the sensory recovery of the forelimb after transection of their left cervical dorsal and ventral roots (C6-C8) at their spinal cord junctions, 22 SD rats were randomly assigned to 3 groups: transection only (control 1); transection followed by repair using intercostal nerve grafts and fibrin glue (control 2); transection, repair, and application of aFGF and fibrin glue (experimental group). The following tests were reperformed after retransecting the repaired nerve roots to discount collateral innervation from adjacent nerve roots: motor function (grasping power), mechanical sensitivity to pain and touch (foot-withdrawal response to mechanical stimuli), temperature sensitivity (foot-withdrawal response to cold stimulus), and electrophysiologic sensory responses (measurement of cortical SEP). RESULTS: After transection and repair, the experimental group rats showed recovery in both motor (grasping power) and sensory (touch, pain, and temperature sensation) nerve functions. Neuronal regeneration was confirmed by the reappearance of cortical SEP and by its disappearance after retransection of the repaired cervical nerve roots. CONCLUSION: Using our strategy for repairing transected cervical nerve roots, motor and sensory recovery was achieved in adult rats. The success of our study highlights possible treatment options for humans with avulsion injuries of the dorsal roots from the spinal cord.

Dual effect of adenovirus-mediated transfer of BMP7 in mixed neuron-glial cultures: neuroprotection and cellular differentiation.

Bone morphogenetic proteins (BMPs), members of the TGF-beta superfamily, have been implicated in nervous system development and in response to injury. Previous studies have shown that recombinant BMP7 can enhance dendritic growth and protect cultured neurons from oxidative stress. Because of the presence of extracellular BMP antagonists, BMP7 seems to act locally. Therefore, the present study uses BMP7 overexpression using adenovirus (Ad)-mediated gene transfer to examine its effect in mixed neuronal cultures. Enhanced BMP7 expression selectively induces neuronal CGRP expression in a time-dependent manner. BMP7 overexpression not only significantly protects cultures from H2O2 toxicity but reduces lipopolysaccharide (LPS) stimulation. Concurrently, it profoundly reduces microglial numbers, but increases oligodendroglial and endothelial cells. Together, low-dose and continuously expressed BMP7 is both neuroprotective and differentiation-inductive.

The combination of peripheral nerve grafts and acidic fibroblast growth factor enhances arginase I and polyamine spermine expression in transected rat spinal cords.

Treatment with a combination of peripheral nerve grafts and acidic fibroblast growth factor improves hind limb locomotor function after spinal cord transection. This study examined the effect of treatment on expression of arginase I (Arg I) and polyamines. Arg I expression was low in the spinal cords of normal rats but increased following spinal injury. Only fully repaired spinal cords expressed higher Arg I levels 6-14 days following repair. In 10-day repaired spinal cords, high Arg I immunoreactivity was detected in motoneurons and alternatively activated macrophages in the graft area and graft-stump edges, and high levels of the polyamine spermine were expressed by macrophages within the intercostal nerve graft. Thus, in addition to enhancing the expression of Arg I and spermine in repaired spinal cords, our treatment may recruit activated macrophages and create a more favorable environment for axonal regrowth.

Effect of enhanced prostacyclin synthesis by adenovirus-mediated transfer on lipopolysaccharide stimulation in neuron-glia cultures.

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-alpha production. Taken together, enhanced prostacyclin synthesis in neuron-glial cultures reduced microglia number and suppressed LPS stimulation.

Gene transfer into human keloid tissue with adeno-associated virus vector.

BACKGROUND Gene transfer is a new territory for clinicians. Intractable disorders might be approached in such a way. Adeno-associated virus (AAV) vector has been transfected successfully into a variety of tissues including skin. We evaluated the ability of this vector to transfer and cause expression of the reporter gene in human keloid tissue. METHODS Human keloid specimens were injected with an AAV vector encoding beta-galactosidase and incubated for 4 weeks after injection. The presence of mRNA and beta-galactosidase enzymatic activity were assayed by reverse-transcriptase polymerase chain reaction and the X-gal technique. RESULTS Gene expression shown by reverse-transcriptase polymerase chain reaction was observed in keloid tissue 4 weeks after injection, and so was the positive X-gal staining. CONCLUSION Our results showed that AAV vector could transduce human keloid tissue effectively. Replacement of the reporter gene with a functioning gene might be feasible for keloid treatment.