Ventral and dorsal horn acetylcholinesterase neurons are maintained in organotypic cultures of postnatal rat spinal cord explants

Delfs, John R.; Friend, Judith; Ishimoto, S; Saroff, Daniel

doi:10.1016/0006-8993(89)90690-2

Cited by 37 publications

(19 citation statements)

References 45 publications

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“…In our previous studies relating to the effect of platelet products on the neurons of spinal explants, we had quantified by morphometric techniques the number and the size distribution of neurons in the ventral horns. We found, as reported by Delfs et al, 10 that >90% of the neurons in the ventral horns, both in the treated and control groups, were <500 fim 2 , and the major portion of this was in 100-200 /im 2 range. Although the number of neurons per ventral horn in the explants varied depending on the age of the animal, spinal level, and duration in culture before the experiment, these were strictly controlled for within each experiment.…”

Section: Methodssupporting

confidence: 88%

“…Maintaining central nervous system neurons, as we have in our study, in organotypic culture, is viewed by most as being closer to the in vivo condition than is dissociated neuronal culture systems. 10 The use of spinal cord explants had the added advantages of having recognizable neuroanatomical landmarks with intact cross-sectional outlines that were crucial during analysis. The specimens were strictly matched for age, spinal level of explants, and duration of incubation before exposure to platelets or their secretory products.…”

Section: Discussionmentioning

confidence: 99%

“…Unlike cerebral tissue, these explants have relatively well-defined neuroanatomical landmarks that permit better morphometric evaluation. Explants were prepared using modifications of the method described by Delfs et al 10 Lumbosacral spinal cord of postnatal Sprague-Dawley rats were dissected out under aseptic conditions and the spinal cord cut serially into twelve 400 -^,m thick sections from the thoracolumbar junction downwards. Each section (explant) was transferred to 20 /il chicken plasma (Cocalico Biologicals, Reamstown, Pa.) placed on a coverslip.…”

Section: Methodsmentioning

confidence: 99%

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Study of platelet-mediated neurotoxicity in rat brain.

Joseph

Tsering

Welch

1992

Stroke

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Background and Purpose:The mechanism of ischemic neuronal injury is not fully resolved. The present view is that vascular occlusion per se does not fully account for the extent of neurological dysfunction. We previously hypothesized that platelet secretory products might contribute to neuronal injury in the central nervous system. Our preliminary studies using organotypic rat spinal cord cultures exposed to human platelet and its secretory products revealed that platelet products had neurotoxic properties.Methods: Further studies, using the same methods, were conducted, with the addition of several refinements such as use of gel-filtered platelets (as opposed to washed platelets) and adding additional relevant controls including platelet membranes, red blood cells, and washed rat platelets.Results

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Section: Methodssupporting

confidence: 88%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Study of platelet-mediated neurotoxicity in rat brain.

Joseph

Tsering

Welch

1992

Stroke

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“…Organotypic spinal cord cultures were prepared using lumbar spinal cord slices from 8-day-old rat pups. Neonatal rat pups were decapitated, and the spinal cords were rapidly harvested and cultured by a modification of described methods (10,11 (14). …”

Section: Methodsmentioning

confidence: 99%

Chronic inhibition of glutamate uptake produces a model of slow neurotoxicity.

Rothstein

Jin

Dykes-Hoberg

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

566

350

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ABSTRACTprior to the addition of drugs. To produce chronic toxicity, long-term inhibition of glutamate uptake was effected by incubating slices with culture medium containing various concentrations of THA or PDC (5, 6). At very high concentrations (>1 mM), THA can have weak actions at the N-methyl-D-aspartate (NMDA) receptor, a property not shared by PDC (5). Transport inhibitors were maintained in cultures by replenishing them at each change in culture medium. In all experiments, potentially neuroprotective drugs were added repeatedly, either in the presence or absence of a glutamate transport inhibitor, again beginning after 8 days in culture. Motor-neuron toxicity was monitored by two methods: (i) biochemical analysis of tissue choline acetyltransferase activity (ChAT) and (ii) microscopic morphology. ChAT activity is largely restricted to ventral motor neurons in rat lumbar spinal cord, and assays of ChAT activity have been used as a reliable marker for motor neurons (7-9). Motor neurons were also visualized in organotypic cultures by histological analysis of stained semithin plastic sections and by immunohistochemistry.Organotypic Spinal Cord Cultures. Organotypic spinal cord cultures were prepared using lumbar spinal cord slices from 8-day-old rat pups. Neonatal rat pups were decapitated, and the spinal cords were rapidly harvested and cultured by a modification of described methods (10, 11). Lumbar spinal cords were collected under sterile conditions and sectioned transversely at 350-,um intervals with a McIlwain tissue chopper (Mickle Laboratory Engineering, Gomshall, Surrey, U.K.). Sections were then transferred to sterile Gey's balanced salt solution (GIBCO) containing glucose (6.4 mg/ml) and gently separated at room temperature. Slices were carefully placed on the surface of 30-mm Millipore Millicell-CM porous (0.4 ,um) membranes (five slices per membrane). Such tissue grows optimally at an air/fluid interface, so it was important to remove any excess medium on the membrane surface around slices. The membranes were placed in 35-mm culture wells (Nunc) containing 1 ml of incubation medium [50% (vol/vol) minimal essential medium-25 mM Hepes/ 25% (vol/vol) heat-inactivated horse serum/25% (vol/vol) Hanks' balanced salt solution (GIBCO) supplemented with D-glucose (25.6 mg/ml) and glutamine (2 mM), at a final pH of 7.2]. Initial studies using pH 7.4 or 7.8 revealed similar results. Antibiotic and antifungal agents were not used.Cultures were incubated at 37°C in a 5% C02/95% air humidified environment (Forma Scientific, Marietta, OH). Culture medium, along with any added pharmacological agents, was changed twice weekly. By using this technique, >95% of the explants can be maintained in culture for >3 months with excellent organotypic cellular organization.

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“…After dissection of the lumbar spinal cord and removal of the meninges, 200 to 400µm-thick transversal sections are sectioned and transferred into membrane inserts fitting six-well or 12-well culture plates (Caldero, et al, 2010). These organotypic cultures can be used for more than 2 months (Delfs, et al, 1989). Various types of molecules can be added in the culture medium, such as kainate or lithium, to modulate neurotoxicity (Caldero, et al, 2010;Mazzone and Nistri, 2011).…”

Section: Organotypic Cultures Of Spinal Cord Slicesmentioning

confidence: 99%