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L Guth

Publications and source records attributed to L Guth.

At least 19 recordsLinked to original sources
PubMed1988

Quantitative evaluation of axonal regeneration by immunochemical assay for neurofilament protein.

In experiments on nerve regeneration requiring assessment of the rate and extent of axonal outgrowth, the availability of a simple and accurate method of quantification would be extremely useful. We approached this issue by modifying the conventional ELISA procedure so as to provide a sensitive, specific, and quantitative biochemical assay of the phosphorylated neurofilament content of homogenates or sections of nerve tissue. The technique involves four sequential steps: (i) adhesion of fixed or fresh homogenates or tissue sections to wells of microtiter plates, (ii) binding of a monoclonal antibody against phosphorylated neurofilament to the tissue, (iii) secondary binding to the anti-phosphorylated neurofilament of a phosphatase-labeled second antibody (antimouse IgG), and (iv) enzymatic assay of alkaline phosphatase activity using a fluorescent substrate (4-methylumbelliferyl phosphate). The technique is sufficiently sensitive to measure the phosphorylated neurofilament content of a 1:100,000 (w/v) homogenate of brain, spinal cord, or peripheral nerve and of single 10-microns paraffin sections of Bouin-fixed rat spinal cord. To validate the applicability of the procedure to the study of nerve regeneration, the sciatic nerve of adult rats was either crushed (to permit regeneration) or transected and ligated (to preclude regeneration). The animals were autopsied 1 to 16 weeks later, when four segments 3-mm in length taken from regions proximal and distal to the lesion site were assayed for phosphorylated filament content. The temporal course of its disappearance during degeneration and its reappearance during regeneration coincided with the known histologic changes in crushed and transected nerves. These findings demonstrate the validity of using the immunochemical assay for PNF in studies of nerve regeneration in the peripheral nervous system and the potential applicability of this procedure to studies on regeneration in the central nervous system.

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PubMed1987

Distribution of carbonic anhydrase activity in neurons of the rat.

Certain neurons of dorsal root ganglia (DRG) and some fibers of the sciatic nerve contain histochemically demonstrable carbonic anhydrase activity. Since the distribution of this enzyme throughout the nervous system has not yet been evaluated systematically, we conducted a comprehensive histochemical survey focusing particularly on structures derived from the neural crest and nonneural crest ectoderm. In the peripheral nervous system, we observed carbonic anhydrase activity in some, but not all, neurons of dorsal root, trigeminal, celiac, and myenteric ganglia as well as in glial cells throughout the CNS. Some neurons of the nodose ganglion also showed carbonic anhydrase activity. In all first order sensory ganglia that were studied, the enzyme was found only in large (50 micron or above) and medium (20-50 micron) size neurons; in the case of spinal ganglia, the reactive neurons constituted approximately 30% of the total neuronal population. Of these reactive neurons, 56% were heavily stained and 44% were moderately stained. Several possible roles for neuronal carbonic anhydrase are considered.

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PubMed1985

Essentiality of a specific cellular terrain for growth of axons into a spinal cord lesion.

To date, there are no reports of growth of significant numbers of axons into or across a lesion of the mammalian spinal cord. However, recent studies showing that CNS axons will grow into PNS environments indicate that comparable growth into spinal cord lesions could be achieved if ischemic necrosis could be prevented and the lesion site repopulated by astrocytes and ependymal cells rather than by the macrophages, lymphocytes, and fibroblasts that generally accumulate at sites of CNS injury. To examine this possibility, we made a laminectomy at T5 in rats and crushed the spinal cord for 2 s with a smooth forceps (leaving the dura mater intact to prevent ingrowth of connective tissue). At 1 week, the lesion was filled with mononuclear cells, degenerating nerve fibers, and capillaries that were oriented parallel to the long axis of the spinal cord. By 2 weeks, longitudinally oriented cords of ependymal cells and astrocytes had migrated into the lesion from the adjacent spinal cord, and similarly oriented nerve fibers had begun to grow into the lesion along these capillaries and cellular cordons. The mononuclear cells had now assumed phagocytic activity and were engorged with myelin and other cellular debris. After 3 weeks, the astrocytes had elaborated thick cell processes. The nerve fibers in the lesion were still oriented longitudinally but had increased in number and were often arranged in small fascicles. These observations provide the first histological evidence of growth of nerve fibers into a lesion of the rat spinal cord. We conclude that the intrinsic regenerative capacity of the spinal cord can be expressed if ischemic necrosis and collagenous scarring are prevented and the spinal cord parenchyma is first reconstructed by its nonneuronal constituents.

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PubMed1985

Enhancement of axonal growth into a spinal lesion by topical application of triethanolamine and cytosine arabinoside.

We have demonstrated that a brief compression lesion of the rat spinal cord produces axotomy with minimal necrosis or scarring and that axons grow into such a lesion along longitudinally oriented capillaries and similarly oriented cordons of ependymal cells and astrocytes. Inasmuch as extensive, oriented growth of axons into a spinal lesion is never seen after transection, concussion, or other models of spinal cord injury, this new surgical procedure appeared to be applicable to the in vivo testing of pharmacological agents designed to promote neuritic outgrowth. The spinal cord of anesthetized rats was crushed extradurally for 1 s with a smooth jeweler's forceps. After 2 days when edema had subsided, the animals were reoperated. The dura mater was opened, and a polyethylene tube was implanted so that one end was fastened over the injury site and the other end was exteriorized at the back of the neck. The lesion site was superfused with 0.1 ml of control or test solutions four times daily for 2 weeks and then the animals were anesthetized and killed by vascular perfusion with fixative. After decalcification, the vertebral column and spinal cord were embedded in paraffin and stained by several histologic procedures including the protargol silver impregnation method for nerve fibers. Treatment with triethanolamine and cytosine arabinoside, substances which promote neuritogenesis in cultured spinal ganglia of chick embryos, markedly stimulated the growth of axons into the lesion of the rat spinal cord. We conclude (i) that it is possible to pharmacologically enhance the intrinsic growth capacity of CNS neurons and (ii) that brief compression provides a type of injury that is well suited to the evaluation of treatments aimed at promoting axonal regeneration.

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