Neuroprotocols最新文献

A method for the quantitative analysis of the molecular species of glycerolipids present In biological samples has been described. 1,2-Diacyl-sn-glycerol, either isolated from biological samples or enzymatically generated from phosphoglycerides, is benzoylated at the sn-3 position and then subjected to reverse-phase (C₁₈-silica) HPLC to separate the molecular species of different hydrophobicitles. An internal standard (1,2-distearoyl-sn-glycerol) is used to identify and quantify the various species eluted from the reverse-phase column. Examples are given for the quantitative analysis of molecular species and precursor-product relationships of glycerolipids generated In SK-N-SH neuroblastoma cells after stimulation of the cell-surface muscarinic acetylcholine receptors.

The ability of lithium to interfere with signal transduction pathways that involve neurotransmitter receptor activation of phosphoinositide turnover has been proposed as a potential mechanistic explanation of the therapeutic actions of lithium in manic-depressive illness. Noncompetitive inhibition of inositol monophosphatase by submillimolar concentrations of lithium deprives active neurons of endogenously generated myo-inositol. If this deficit cannot be compensated for by uptake of extracellular myo-inositol, then the ability of the cell to synthesize and maintain inositol phospholipid pools will be compromised. Here we describe methods for the investigation of the phosphoinositide cycle, with particular emphasis on methods that have been used to highlight the complex actions of lithium to disrupt activation of this important signal transduction pathway by neurotransmitters.

Glycosyl-phosphoinositide molecules have both structural and functional roles in mammalian cells. These glycophospholipids can serve as membrane anchors for cell surface proteins or as precursors for the generation of second messengers in hormone action. Methodology for analysis of the synthesis and metabolism of these molecules is outlined. Tissue culture cells are used for experiments involving labeling with radioactive precursors. After exposure to hormones, glycosyl-phosphoinositides and their metabolites can be analyzed by a combination of thin-layer and high-performance liquid chromatography.

Experimental modeling of spinal cord injury is based mostly on mechanical effects, such as the impact of a weight dropped on the exposed spinal cord. The development of the resultant lesion is influenced by many interactive factors, e.g., the efficiencies of momentum and energy transfer to the cord and their profiles in time, and consequently the histopathologic reproducibility of the lesion is often inconsistent. We describe here a recoilless method that avoids these complications (as well as laminectomy) inherently. The vascular endothelium is injured photochemically, yielding chiefly small-vessel thrombosis and associated vasogenic edema sufficient to generate spinal cord necrosis to predetermined, reproducible degrees. This model is thus intended to simulate the secondary response of the vasculature to mechanical injury In the absence of hemorrhage. The most efficient version of this technique utilizes argon-dye laser excitation of the photosensitizing dye rose bengal at its 562-nm absorption maximum in tissue. With the laser beam focused in the shape of a thin (0.3-mm) line transverse to the spinal column at T8, a narrow zone of necrosis is initially produced. Within 1 week this initial zone expands in volume (length, 6-7 mm) to create a space that, when cleared of cellular debris, is suitable for cell Implantation. In cross section, striking features are the sharp horizontal demarcation between necrosed and viable tissue and the uniform progression of lesion depth as a function of Irradiation time. The necrotic region is bordered dorsally and laterally by a thin rim of viable tissue except at the beam focus; starting at 5 days there is evidence of demyelination in this peripheral region. By 14 days, myelination by oligodendrocytes and Schwann cells begins near this rim; large numbers of Schwann cells enter the dorsal cord at the epicenter, and myelinated axons occupy previously degenerated areas. By 2 months, the initial necrotic area begins to diminish, flattening laterally into clefts. Large, empty cavities develop secondarily with luminal surfaces that are smoothly contoured, as seen by electron microscopy, in contrast to the relatively irregular border of the initial necrotic lesion. Many of these morphological attributes mirror those observed after contusion injury. We anticipate that the uniformity and reproducibility of the photochemical lesion will prove useful in conducting statistically efficient tests of strategies, such as the administration of drugs, trophic factors, or transplanted cells, which are proposed to improve outcome after spinal cord injury.