Early application of tail nerve electrical stimulation-induced walking training promotes locomotor recovery in rats with spinal cord injury.

STUDY DESIGN: This is a randomized controlled prospective trial with two parallel groups. OBJECTIVES: The objective of this study was to determine whether early application of tail nerve electrical stimulation (TANES)-induced walking training can improve the locomotor function. SETTING: This study was conducted in SCS Research Center in Colorado, USA. METHODS: A contusion injury to spinal cord T10 was produced using the New York University impactor device with a 25 -mm height setting in female, adult Long-Evans rats. Injured rats were randomly divided into two groups (n=12 per group). One group was subjected to TANES-induced walking training 2 weeks post injury, and the other group, as control, received no TANES-induced walking training. Restorations of behavior and conduction were assessed using the Basso, Beattie and Bresnahan open-field rating scale, horizontal ladder rung walking test and electrophysiological test (Hoffmann reflex). RESULTS: Early application of TANES-induced walking training significantly improved the recovery of locomotor function and benefited the restoration of Hoffmann reflex. CONCLUSION: TANES-induced walking training is a useful method to promote locomotor recovery in rats with spinal cord injury.

X-irradiation reduces lesion scarring at the contusion site of adult rat spinal cord.

Spinal cord injury (SCI) results in cell death and tissue destruction, and ultimately cavitation followed by the formation of lesion scars at the injury site. The lesion scars include an astrocytic component (glial scar) and a fibroblastic component (connective tissue scar). The purpose of the present study is to determine if X-irradiation could minimize the formation of lesion scars and reduce the levels of chondroitin sulfate proteoglycans (CSPGs) in the contusion SCI model of the adult rat. Two weeks after SCI, a connective tissue scar formed at the injury site consisting primarily of fibroblasts and exhibits strong CSPG immunoreactivity. The fibroblasts might originate from the connective tissue of pia mater or arachnoid mater. At the same time, reactive astrocytes in the spared tissue accumulate surrounding the lesion cavity to form a thick glial scar with significant enhancement of glial fibrillary acidic protein (GFAP) and CSPG immunoreactivity. After X-irradiation (40 Gy) of the injury site 2 days post-injury, that results in an attenuated dose to the lesion, the connective tissue scar was not observed, and accordingly, almost no CSPG immunoreactivity was detected at this area. Meanwhile, the glial scar and its CSPG immunoreactivity were prominently reduced. X-irradiation did not show significant improvement in locomotor recovery, but resulted in a slight delay of body weight recovery following injury. This preparative treatment could be used to reduce secondary scarring in the lesion resulting in an enriched site for further treatment such as growth related transplantation.

A horseradish peroxidase-light and electron microscopic study of immunoliposomes utilized for intracellular delivery to the rat striatum.

Liposomes can deliver plasmid DNA, viruses, antisense oligonucleotides, and pharmacological agents to the central nervous system. Conjugation of antibodies to liposomes increases delivery specificity. Immunoliposomes created with Thy 1.1 antibody have previously been shown to be effective for neuronal delivery. The intracellular delivery of these immunoliposomes is evaluated by light and electron microscopy. Thy 1.1 conjugated liposomes were loaded with horseradish peroxidase and stereotactically injected into rat striatum. On light microscopy, immunoliposomes were concentrated within 0.2 mm of the injection site 8 h following delivery but, 24 h post-operatively, had diffused more than 0.5 mm from the injection site. With transmission electron microscopy, immunoliposomes were observed entering numerous neurons and some astrocytes in a process distinct from the clathrin-coated pit mechanism. These findings suggest that Thy 1.1 immunoliposomes are effective for intracellular delivery in vivo and their endocytosis occurs independently of a coated pit process. The research has helped to elucidate alternative mechanisms for immunoliposomal delivery. A more fundamental understanding of these attributes is needed to achieve the therapeutic potential of immunoliposomes.

Transfecting neurons and glia in the rat using pH-sensitive immunoliposomes.

Immunoliposomes were constructed using antibody 5-113 (directed to an antigen on the external surface rat glial cells), the antibody Thy 1.1, and a non-immune antibody. The antibodies were conjugated to N-gluytaryl-phosphatidylethanolamine. Liposomes were constructed with these conjugated antibodies, other lipids and a beta-galactosidase plasmid under the control of the cytomegalovirus promoter. When immunoliposomes decorated with one of three different antibodies were injected into the brain or spinal cord of adult rats, the X-gal reaction product was observed in neurons, astrocytes and vascular elements. There was an increase in neuronal labeling when animals were injected with Thy 1.1 conjugated liposomes and there was an increase in glial labeling in animals injected with 5-113 liposomes. In spinal cords, the immunoliposomes appear to penetrate a substantial distance, transfecting neurons several centimeters from the site of delivery. These data suggest that immunoliposomes may provide an effective transfection system for gene delivery in the CNS.

DNA synthesis is induced in adult neurons after expression of E2F1 and E1A.

Differentiated neurons in several brain regions express the retinoblastoma (Rb) tumor suppressor gene. Changing the tumor suppressor function of Rb by expressing transcription factor E2F1 and viral oncoprotein E1A in cerebellar granular neurons in vitro and in cerebral cortical neurons in vivo results in the induction of DNA synthesis in these neurons. Immunoliposome-mediated transfection of E1A and E2F1 cDNAs into the adult cortical neurons of rats in vivo results in initiation of DNA synthesis in 5-15% of the transfected neurons. These results indicate that expression of Rb may be necessary to prevent induction of differentiated neurons to proliferate since many mitogenic growth factors are expressed in the brain.

Delivery of plasmid DNA to glial cells using pH-sensitive immunoliposomes.

Immunoliposomes were constructed with an antibody specific to glial cells. They were used to examine the specificity and efficacy of cell type plasmid transfection. Liposomes contained a beta-galactosidase gene under control of an SV-40 promotor. Two different monoclonal antibodies of a different subclass, IgM and IgG, were examined for their targeting ability using immunoliposomes. Cultured C6 glioma (specific target cell type) and NIH 3T3 (control cell type, fibroblast) cells were transfected using these immunoliposomes. Results indicate a three-fold increase in transfection by the glial specific immunoliposomes, "gliasomes", in glial cell culture over control liposomes. Gliasomes were exposed to NIH 3T3 cells and showed no enhanced transfection over control liposomes. Gliasomes were tested for their specificity by the addition of excess antibody to the cell culture in order to saturate specific receptors on C6 glioma cells. Results indicate a reduced transfection, nearly three-fold, in cells that were saturated with excess antibody prior to exposure to the immunoliposomes.

Proton transport and Na+/H+ exchange in vesicles isolated from sockeye salmon (Oncorhynchus nerka) kidneys during migration from salt to fresh water.

Renal epithelial function, proton flux and sodium stimulated proton flux, was observed in vesicles isolated from the brush border of the proximal tubule of Sockeye Salmon (Oncorhynchus nerka) during migration. Brush border membrane vesicles (BBMV) were isolated from the body kidney of Sockeye Salmon using aggregation/differential centrifugation techniques. Vesicle purity was tested using a series of epithelial and basal lateral markers including alkaline phosphatase, maltase, gamma-glutamyl transferase (GGTP), Mg(2+)-activated ATP-ase, Na(+)+K(+)-activated ATPase, and 5'-nucleotidase and the lysosomal marker acid phosphatase. An enrichment/depletion factor for each marker was determined by comparison of purified BBMV with kidney homogenate. Vesicles exhibit an enrichment factor for alkaline phosphatase, GGTP, maltase, Mg(2+)-activated ATP-ase, Na(+)+K(+)-activated ATPase, and 5'-nucleotidase. A depletion factor was observed for acid phosphatase. Vesicle integrity was tested by measuring the time course of proton flux in the presence of a pH gradient. Amiloride sensitive sodium stimulated proton flux was observed in these vesicles. The presence of sodium caused a saturable increase in the rate of proton flux, indicating the activity of a sodium/proton antiport protein in BBMV.

Mechanism of acridine orange interaction with phospholipids and proteins in renal microvillus vesicles.

The mechanism of interaction of acridine orange (AO), a fluorescent, weak base, with rabbit kidney brush border membrane vesicles (BBMV) has been studied by absorption, and steady-state and time-resolved fluorescence spectroscopy. Equilibrium binding experiments indicate that AO binds to an apparent single class of sites on BBMV with a dissociation constant of 90 microM and site stoichiometry of 810 nmol/mg protein. The absorption spectra AO indicate that BBMV induces aggregation of AO; experiments with lipid vesicles show that the aggregation requires BBMV membrane proteins. Fluorescence stopped-flow experiments in which 0.15 mg/ml BBMV is mixed with increasing concentrations of AO result in a time course of fluorescence enhancement for [AO] less than 1.5 microM, and of fluorescence quenching for [AO] greater than 1.5 microM. Similar stopped-flow experiments with phosphatidylcholine lipid vesicles result only in a fluorescence enhancement time course. These results indicate the presence of two parallel pathways for AO binding to BBMV: one for AO binding to BBMV lipid, the other for AO binding to BBMV protein. Nanosecond lifetime measurements and fluorescence titration experiments confirm the presence of two environments for AO in BBMV. Fluorescence stopped-flow experiments indicate that AO responds to the imposition of an outwardly directed proton gradient by a rapid (less than 0.5 s) decrease in fluorescence, corresponding to re-equilibration of AO into the acidic intravesicular compartment, followed by an increase in fluorescence, corresponding to proton flux across the membrane. These findings have been incorporated into a stepwise mechanism for AO interaction with BBMV which have direct implications for the use of AO as a pH indicator in biological systems.