Cuprizone-induced Demyelination in Mouse Brain is not due to Depletion of Copper.

The demyelinating effects of CPZ are not due to Cu deficiency but are instead consistent with acute toxicity of a CPZ + Cu complex.

Label-free assessment of myelin status using birefringence microscopy.

Background: Label-free methods for quantifying myelination can reduce expense, time, and variability in results when examining tissue white matter pathology.

New Method: We sought to determine whether the optical birefringent properties of myelin could be exploited to determine myelination status of white matter in tissue sections. Sections of forebrains of mice (normal, and treated with cuprizone to cause demyelination) were examined by birefringence using a birefringence imaging system (Thorlabs LCC7201), and results compared with sections stained using Luxol Fast Blue.

Nile Red fluorescence spectroscopy reports early physicochemical changes in myelin with high sensitivity.

The molecular composition of myelin membranes determines their structure and function. Even minute changes to the biochemical balance can have profound consequences for axonal conduction and the synchronicity of neural networks. Hypothesizing that the earliest indication of myelin injury involves changes in the composition and/or polarity of its constituent lipids, we developed a sensitive spectroscopic technique for defining the chemical polarity of myelin lipids in fixed frozen tissue sections from rodent and human.

Ferroptosis Mediates Cuprizone-Induced Loss of Oligodendrocytes and Demyelination.

Multiple sclerosis (MS) is a chronic demyelinating disease of the CNS. Cuprizone (CZ), a copper chelator, is widely used to study demyelination and remyelination in the CNS, in the context of MS. However, the mechanisms underlying oligodendrocyte (OL) cell loss and demyelination are not known.

Spectral Characterization of Stem Cell-Derived Myelination within the Injured Adult PNS Using the Solvatochromic Dye Nile Red.

Background: Myelin is an essential component of the peripheral and central nervous system, enabling fast axonal conduction and supporting axonal integrity; limited tools exist for analysis of myelin composition in-vivo.

Objective: To demonstrate that the photophysical properties of myelin-incorporated solvatochromic dyes can be exploited to probe the biochemical composition of living peripheral nerve myelin at high spatial resolution.

Methods: Using the myelin-incorporated fluorescent dye Nile Red we sequentially analyzed the spectral characteristics of remyelinating myelin membranes both in-vitro and in-vivo, including in living rats.

Deficient Surveillance and Phagocytic Activity of Myeloid Cells Within Demyelinated Lesions in Aging Mice Visualized by Live Multiphoton Imaging.

Aging impairs regenerative processes including remyelination, the synthesis of a new myelin sheath. Microglia and other infiltrating myeloid cells such as macrophages are essential for remyelination through mechanisms that include the clearance of inhibitory molecules within the lesion. Prior studies have shown that the quantity of myeloid cells and the clearance of inhibitory myelin debris are deficient in aging, contributing to the decline in remyelination efficiency with senescence.

A novel approach to 32-channel peripheral nervous system myelin imaging in vivo, with single axon resolution.

OBJECTIVE Intravital spectral imaging of the large, deeply situated nerves in the rat peripheral nervous system (PNS) has not been well described. Here, the authors have developed a highly stable platform for performing imaging of the tibial nerve in live rodents, thus allowing the capture of high-resolution, high-magnification spectral images requiring long acquisition times. By further exploiting the qualities of the topically applied myelin dye Nile red, this technique is capable of visualizing the detailed microenvironment of peripheral nerve demyelination injury and recovery, while allowing us to obtain images of exogenous Schwann cell myelination in a living animal.

Mechanisms of lysophosphatidylcholine-induced demyelination: A primary lipid disrupting myelinopathy.

For decades lysophosphatidylcholine (LPC, lysolecithin) has been used to induce demyelination, without a clear understanding of its mechanisms. LPC is an endogenous lysophospholipid so it may cause demyelination in certain diseases. We investigated whether known receptor systems, inflammation or nonspecific lipid disruption mediates LPC-demyelination in mice.

Toll-like receptor 2-mediated alternative activation of microglia is protective after spinal cord injury.

Improving neurological outcome after spinal cord injury is a major clinical challenge because axons, once severed, do not regenerate but 'dieback' from the lesion site. Although microglia, the immunocompetent cells of the brain and spinal cord respond rapidly to spinal cord injury, their role in subsequent injury or repair remains unclear. To assess the role of microglia in spinal cord white matter injury we used time-lapse two-photon and spectral confocal imaging of green fluorescent protein-labelled microglia, yellow fluorescent protein-labelled axons, and Nile Red-labelled myelin of living murine spinal cord and revealed dynamic changes in white matter elements after laser-induced spinal cord injury in real time.

Feline immunodeficiency virus-based lentiviral vectors.

Feline immunodeficiency virus (FIV)-based lentiviral vectors are useful for introducing integrated transgenes into nondividing human cells. This article describes the production and use of advanced generation FIV vectors. Key properties are discussed in comparison to other lentiviral vectors.

Titration of feline immunodeficiency virus-based lentiviral vector preparations.

Feline immunodeficiency virus (FIV)-based lentiviral vectors are useful for introducing integrated transgenes into nondividing human cells. This protocol describes methods for measuring and calculating vector titers in transducing units (TU)/mL. Alternate methods are provided for green fluorescent protein (GFP) vectors and for β-galactosidase vectors.

Production and harvest of feline immunodeficiency virus-based lentiviral vector from cells grown in T75 tissue-culture flasks.

Feline immunodeficiency virus (FIV)-based lentiviral vectors are useful for introducing integrated transgenes into nondividing human cells. This protocol describes the production and harvesting of vector from cells grown in T75 tissue-culture flasks. The methods are for production for basic science laboratory use and in vivo experimentation.

Production, harvest, and concentration of feline immunodeficiency virus-based lentiviral vector from cells grown in CF10 or CF2 devices.

Feline immunodeficiency virus (FIV)-based lentiviral vectors are useful for introducing integrated transgenes into nondividing human cells. This protocol describes the production of FIV-based lentiviral vectors using cells grown in CF10 or CF2 devices. It also details the harvesting and concentration of these vectors.

Prolonged transgene expression with lentiviral vectors in the aqueous humor outflow pathway of nonhuman primates.

We injected lentiviral vectors into the eyes of live nonhuman primates to assess potential for glaucoma gene therapy. Anterior chambers of five cynomolgus monkeys were injected with green fluorescent protein (GFP)-encoding feline immunodeficiency viral vectors. The monkeys were monitored for in vivo transgene expression and clinical parameters.

Durable, safe, multi-gene lentiviral vector expression in feline trabecular meshwork.

Multiple disease-specific considerations have led to interest in the potential of gene therapy to permanently correct elevated intraocular pressure (IOP), the main causal risk factor for primary open angle glaucoma (POAG). Since IOP elevation results from abnormal resistance to aqueous humor outflow from the eye through the trabecular meshwork (TM), a means to genetically modify this specialized outflow organ permanently and safely is a prioritized goal. Here we tested different lentiviral vector designs and doses for long-term transgene expression in a large animal model, and investigated whether exogenously introduced myocilin proteins influenced IOP.

An essential role for LEDGF/p75 in HIV integration.

Chromosomal integration enables human immunodeficiency virus (HIV) to establish a permanent reservoir that can be therapeutically suppressed but not eradicated. Participation of cellular proteins in this obligate replication step is poorly understood. We used intensified RNA interference and dominant-negative protein approaches to show that the cellular transcriptional coactivator lens epithelium-derived growth factor (LEDGF)/p75 (p75) is an essential HIV integration cofactor.

Perfusion of his-tagged eukaryotic myocilin increases outflow resistance in human anterior segments in the presence of aqueous humor.

Purpose: A previous study by the authors has shown that recombinant myocilin purified from a prokaryotic expression system increases outflow resistance in cultured human anterior segments. The present study was performed to determine whether full-length myocilin purified from a human trabecular meshwork cell expression system alters outflow resistance after infusion into human anterior segments.

Methods: A feline immunodeficiency virus vector encoding both full-length myocilin (amino acids 1-503 fused to C-terminal V5 and six-histidine epitopes) and puromycin resistance was used to transduce a transformed trabecular meshwork cell line (TM5).

Restriction of feline immunodeficiency virus by Ref1, Lv1, and primate TRIM5alpha proteins.

The Ref1 and Lv1 postentry restrictions in human and monkey cells have been analyzed for lentiviruses in the primate and ungulate groups, but no data exist for the third (feline) group. We compared feline immunodeficiency virus (FIV) to other restricted (human immunodeficiency virus type 1 [HIV-1], equine infectious anemia virus [EIAV]) and unrestricted (NB-tropic murine leukemia virus [NB-MLV]) retroviruses across wide ranges of viral inputs in cells from multiple primate and nonprimate species. We also characterized restrictions conferred to permissive feline and canine cells engineered to express rhesus and human TRIM5alpha proteins and performed RNA interference (RNAi) against endogenous TRIM5alpha.

Long-term, targeted genetic modification of the aqueous humor outflow tract coupled with noninvasive imaging of gene expression in vivo.

Purpose: To address a problem impeding research into glaucoma-associated genetic mutations and glaucoma gene therapy and achieve permanent, targeted transgene expression in the trabecular meshwork (TM). Lentiviral vectors are known to transduce human donor eye TM ex vivo, but efficacy in vivo has not been shown. More generally in the field of gene therapy, the authors hypothesized that distinctive properties of the intraocular aqueous circulation could facilitate solving problems of accessibility, targeting, and scale that have hindered realization of gene therapy in other settings.

Long-term retinal transgene expression with FIV versus adenoviral vectors.

Purpose: Gene therapy for chronic retinal diseases will require long-term expression of therapeutic transgenes. Lentiviral and adenoviral (Ad) vectors are gene delivery systems with markedly different properties. Lentiviral vectors require integration into the host genome, which facilitates long-term expression, while Ad vectors remain episomal.

Comparison of wild-type and class I integrase mutant-FIV vectors in retina demonstrates sustained expression of integrated transgenes in retinal pigment epithelium.

Background: In neonatal and adult rodent retina, substantial lentiviral vector expression has been detected primarily in retinal pigment epithelium (RPE), except in very young animals (2-5 days post-natal). In non-retinal tissues, studies of lentiviral vectors have utilized various controls. Among the most stringent are class I integrase mutants, which selectively block the integration reaction while leaving all other gag/pol-encoded functions intact.

Preservation of aqueous outflow facility after second-generation FIV vector-mediated expression of marker genes in anterior segments of human eyes.

Purpose: Feline immunodeficiency virus (FIV)-based lentiviral vectors produce effective genetic modification of the trabecular meshwork (TM) of human eyes in organ-perfusion culture, resulting in high-level expression of a beta-galactosidase marker gene (lacZ) without loss of TM cellularity or architecture. However, effects on aqueous outflow physiology have not been determined, and the ability to monitor FIV vector transgene expression in living TM in situ has not been established. In the current study, transgene expression and outflow facility were evaluated in perfused human anterior segments after FIV vector transduction of lacZ or of a marker gene that can be monitored noninvasively, enhanced green fluorescent protein (eGFP).