Serum vascular endothelial growth factor affects tissue fluid accumulation and is associated with deteriorating tissue perfusion and oxygenation in severe sepsis: a prospective observational study.

BACKGROUND: Positive fluid balance and tissue fluid accumulation are associated with adverse outcomes in sepsis. Vascular endothelial growth factor (VEGF) increases in sepsis, promotes vascular permeability, and may affect tissue fluid accumulation and oxygenation. We used near-infrared spectroscopy (NIRS) to estimate tissue hemoglobin (Hb) oxygenation and water (H2O) levels to investigate their relationship with serum VEGF levels. MATERIAL AND METHODS: New-onset severe sepsis patients admitted to the intensive care unit were enrolled. Relative tissue concentrations of oxy-Hb ([HbO2]), deoxy-Hb ([HbR]), total Hb ([HbT]), and H2O ([H2O]) were estimated by near-infrared spectroscopy (NIRS) for three consecutive days and serum VEGF levels were measured. Comparisons between oliguric and non-oliguric patients were conducted and the correlations between variables were analyzed. RESULTS: Among 75 eligible patients, compared with non-oliguric patients, oliguric patients were administrated more intravascular fluids (median [IQR], 1926.00 [1348.50-3092.00] mL/day vs. 1069.00 [722.00-1486.75] mL/day, p < 0.001) and had more positive daily net intake and output (mean [SD], 1,235.06 [1303.14] mL/day vs. 313.17 [744.75] mL/day, p = 0.012), lower [HbO2] and [HbT] over the three-day measurement (analyzed by GEE p = 0.01 and 0.043, respectively) and significantly higher [H2O] on the third day than on the first two days (analyzed by GEE p = 0.034 and 0.018, respectively). Overall, serum VEGF levels were significantly negatively correlated with [HbO2] and [HbT] (rho = - 0.246 and - 0.266, p = 0.042 and 0.027, respectively) but positively correlated with [H2O] (rho = 0.449, p < 0.001). Subgroup analysis revealed a significant correlation between serum VEGF and [H2O] in oliguric patients (rho = 0.532, p = 0.003). Multiple regression analysis determined the independent effect of serum VEGF on [H2O] (standardized coefficient = 0.281, p = 0.038). CONCLUSIONS: In severe sepsis, oliguria relates to higher positive fluid balance, lower tissue perfusion and oxygenation, and progressive tissue fluid accumulation. Elevated serum VEGF is associated with worsening tissue perfusion and oxygenation and independently affects tissue fluid accumulation.

Lactobacillus plantarum PS128 alleviates neurodegenerative progression in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced mouse models of Parkinson's disease.

Evidence suggests that the Parkinson's disease (PD) pathogenesis is strongly associated with bidirectional pathways in the microbiota-gut-brain axis (MGBA), and psychobiotics may inhibit PD progression. We previously reported that the novel psychobiotic strain, Lactobacillus plantarum PS128 (PS128), ameliorated abnormal behaviors and modulated neurotransmissions in dopaminergic pathways in rodent models. Here, we report that orally administering PS128 for 4 weeks significantly alleviated the motor deficits, elevation in corticosterone, nigrostriatal dopaminergic neuronal death, and striatal dopamine reduction in 1-methyl-4-phenyl-1,2,3,6-tetrathydropyridine (MPTP)-induced PD mouse models. PS128 ingestion suppressed glial cell hyperactivation and increased norepinephrine and neurotrophic factors in the striatum of the PD-model mice. PS128 administration also attenuated MPTP-induced oxidative stress and neuroinflammation in the nigrostriatal pathway. Fecal analysis showed that PS128 modulated the gut microbiota. L. plantarum abundance was significantly increased along with methionine biosynthesis-related microbial modules. PS128 also suppressed the increased family Enterobacteriaceae and lipopolysaccharide and peptidoglycan biosynthesis-related microbial modules caused by MPTP. In conclude, PS128 ingestion alleviated MPTP-induced motor deficits and neurotoxicity.PS128 supplementation inhibited neurodegenerative processes in PD-model mice and may help prevent PD.

New nerve regeneration strategy combining laminin-coated chitosan conduits and stem cell therapy.

Nerve regeneration remains a difficult challenge due to the lack of safe and efficient matrix support. We designed a laminin (LN)-modified chitosan multi-walled nerve conduit combined with bone marrow stem cell (BMSC) grating to bridge a 10 mm long gap in the sciatic nerve of Sprague-Dawley rats. The repair outcome was monitored during 16 weeks after surgery. Successful grafting of LN onto the chitosan film, confirmed by immunolocalization, significantly improved cell adhesion. In vivo study showed that newly formed nerve cells covered the interior of the conduit to connect the nerve gap successfully in all groups. The rats implanted with the conduit combined with BMSCs showed the best results, in terms of nerve regrowth, muscle mass of gastrocnemius, function recovery and tract tracing. Neuroanatomical horseradish peroxidase tracer analysis of motor neurons in the lumbar spinal cord indicated that the amount and signal intensity were significantly improved. Furthermore, BMSCs suppressed neuronal cell death and promoted regeneration by suppressing the inflammatory and fibrotic response induced by chitosan after long-term implantation. In summary, this study suggests that LN-modified chitosan multi-walled nerve conduit combined with BMSCs is an efficient and safe conduit matrix for nerve regeneration.

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.

Effects of laminin-coated carbon nanotube/chitosan fibers on guided neurite growth.

This study assesses the ability and potential of carbon nanotube (CNT)/chitosan to guide axon re-growth after nerve injuries. The CNT/chitosan fibers were produced via the coagulation and hydrodynamic focusing method. Fiber width and morphology were adjusted using such parameters as syringe pumping rate and the coagulant used. The CNT/chitosan fiber diameters were 50-300 µm for syringe pumping rates of 6-48 mL/h. Polyethylene glycol/NaOH (25%, w/w) solution was a suitable coagulant for forming fibers with small diameters. Physical property tests demonstrate that the CNT/chitosan composites had superior tensile strength and electrical conductivity compared with those of chitosan alone. The MTT and LDH tests reveal that CNT/chitosan composites were not cytotoxic. To improve the neural cell affinity of CNT/chitosan fibers, laminin was incorporated onto fiber surfaces via the oxygen plasma technique; cell adhesion ratio increased significantly from 3.5% to 72.2% with this surface modification. Immunofluorescence staining and SEM imaging indicate that PC12 cells adhered successfully and grew on the laminin (LN)-coated CNT/chitosan films and fibers. Experimental results show that PC12 grown on LN-coated CNT/chitosan fibers in vitro extend longitudinally oriented neurites in a manner similar to that of native peripheral nerves. With the inherent electrical properties of CNTs, oriented CNT/chitosan fibers have a potential for use as nerve conduits in nerve tissue engineering.

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.

Gait analysis of spinal cord injured rats after delivery of chondroitinase ABC and adult olfactory mucosa progenitor cell transplantation.

Chondroitin sulfate proteoglycan (CSPG) is a major component of glial scar to restrict axonal regeneration in the lesion site after spinal cord injury (SCI). Chondroitinase ABC (ChABC), a bacteria enzyme, which has been demonstrated to digest the glycosaminoglycan (GAG) side chain of CSPG to promote axonal re-growth across the injured site. Our previous study suggested that long-term delivery of ChABC (1U/ml, injection volume 0.6 microl for one animal) via intrathecal catheter could decrease the inhibitory effect of limiting axonal re-growth after SCI. The functional behavior has been shown to improve following ChABC treatment. Little axons re-grow across the lesion site of the spinal cord but not enough to support axon innervations to targets. In this article, we show that ChABC administration combining olfactory mucosa progenitor cell (OMPC) transplantation can promote axonal re-growth across the lesion site and enhance the consistency of stepping in spinally transected rats. These OMPCs generated NG2(+) cell lineages after transplanting into the spinal cord parenchyma, and OMPCs were found to spread and migrate toward the lesion region of spinal cord. Moreover, the spatial and temporal characteristics of the step cycle in rats that receive a complete spinal cord transaction following continuous ChABC supply and OMPC transplantation. The gait characteristics of treated rats on a treadmill were consistent and approached that of intact rats. In future, the mechanism of restoring the injured spinal cord will be further investigated.

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.

Preparation and characterization of an immunogenic meningococcal group A conjugate vaccine for use in Africa.

Periodic epidemics of group A meningococcal (Mn A) meningitis continue to occur in sub-Saharan Africa. For its prevention, a Mn A polysaccharide (PS)-tetanus toxoid (TT) conjugate vaccine was developed using reductive amination of polysaccharide aldehydes and toxoid hydrazides. In mouse immunization studies, a schedule of three bi-weekly s.c. immunizations of 0.1 or 1mug of the conjugate (PS content) without an adjuvant induced serum antibody levels of >10,000units/mL measured by enzyme-linked immunosorbent assay (ELISA) as compared to approximately 100units/mL in PS control mice. The elicited antibodies were active in bactericidal assays using either baby rabbit or human complement (titers >1500 compared to approximately 200 for the PS control group). The synthesis process is reproducible and scalable, and has been successfully used for manufacturing a Mn A PS-TT conjugate vaccine based on a paradigm of shared manufacturing with transfer of new technology [Jodar L, LaForce FM, Ceccarini C, Aguado T, Granoff DM. Meningococcal conjugate vaccine for Africa: a model for development of new vaccine for the poorest countries. Lancet 2003, 361:1092-4]. A phase 1 clinical trial of the manufactured Men A-TT conjugate vaccine has been successfully carried out in adults in India, and a phase 2 clinical trial in young children is currently underway in Africa.

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.

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.

Chondroitinase ABC promotes axonal re-growth and behavior recovery in spinal cord injury.

In spinal cord injury, the injury could trigger some inhibitory signal cascades to promote chondroitin sulfate proteoglycans (CSPGs), the structures of scar tissues, formation. CSPGs could limit axonal regeneration mainly through the glycosaminoglycan (GAG) chain in the lesion site were suggested. We hypothesized that the digestion of CSPGs by chondroitinase ABC (ChABC) might decrease the inhibitory effects of limiting axonal re-growth after spinal cord injury. We compared the digesting products of CSPGs such as 2B6 by ChABC with the untreated control group and found no immunostaining of 2B6 in control group. The smaller size scars of ChABC-treatment were observed via CS-56, a type of CSPGs, 8 weeks after transection by immunohistochemistry. The inhibitory effects of CSPGs withdraw GAGs following ChABC-treatment would reduce, and immunopositive GAP-43 newly outgrown fibers were identified. In the animal trials, ChABC-treatment could improve motor function through BBB locomotor's test and reduce limiting ability of scar tissues to promote axonal regeneration via changing the structure of CSPGs by immunohistochemistry with GAP-43.