![]() Oxidative Stress Poster Session |
Results Spinal cord aggregate cultures develop into round cellular spheres with an approximate diameter of 1 mm. At the LM level, control aggregates have a consistant appearance of a neuropil filled sphere comprised of a peripheral glial ring that surrounds a more centrally located neuronal cell region (Figure 1). Examples of myelination in the form of myelinated axons (profiles) can be found throughout the aggregate, but are localized mainly to the inner two thirds. At the EM level, the aggregate neuropil contains a mixture of astrocyte, oligodendrocyte and neuronal nuclei and processes as identified by Peters et al. (1991). Furthermore, typical cell to cell relationships such as synapses as well as myelinated profiles are readily identified (Figure 1C). The majority of myelinated profiles feature compact myelin lamellae surrounding healthy axons (Figure 1C).
Control macrophage enriched spinal cord aggregates (Figure 2A) develop similarly to standard spinal cord aggregate cultures in that macrophages readily assimilate into the aggregate neuropil. While these aggregate cultures are smaller (approximately half the size) than standard spinal cord aggregate cultures, they retain the consistant aggregate appearance (Figure 2B) and contain numerous myelinated axons (Figure 2C).
LM analysis of the standard spinal cord aggregates 24 hour after exposure to 5�M or 10 �M menadione, showed minor signs of neuropil disruption (Figure 3A) which included shrunken neuropil and cavities in which remnants of cells could be identified.
Most of this damage was restricted to the outer edges of the aggregates. Furthermore, the myelinated axons and more centrally located neuropil within the treated aggregates appeared unaffected. Damage to the neuropil appeared to increase in the aggregates 48 hours following menadione exposure (Figure 4A). There was also a clear concentration dependent effect. There was an increase in extracellular space, vacuoles and necrotic nuclei in all the aggregates exposed to menadione.
While the deleterious effects of menadione treatment appeared more obvious 48 hours following exposure, the 96 hour samples showed no further increase in the extent of damage at the LM level. At the EM level, 24 hour treatment with menadione led to the disruption of the neuropil in the outer edges of the aggregate (compare Figure 1A and 3A) as well as the appearance of various stages of cellular necrosis. Aggregates again showed a concentration dependent increase in the presence of myelin alterations (Table 1), characterised as bloated axons surrounded by delaminated myelin sheaths (Figure 3B and C). Time Dose 24 Hrs. 48 Hrs. 96 Hrs. - M� + M� - M� + M� - M� + M� Control 14 17 12 21 12 18 5 �M 12 100 12 100 14 100 10 �M 26 100 27 100 33 100 20 �M 26 100 26 100 41 100
Table 1: Table outlining the extent of myelin alterations to both the standard spinal cord aggregate cultures (- M� ) and the macrophage enriched spinal cord aggregate cultures (+ M� ) exposed to the low doses of menadione for 24, 48 and 96 hours. All the values were calculated as percentages of the total number of myelin profiles counted. Three sections of 3 different aggregates were counted from three different experiments (n=9). Intracellular myelin whorls were also present and many of these occurred associated with large lipid droplets (Figure 3C). Numerous extracellular whorls were located in this disrupted neuropil suggesting that these may be the remnants of myelinated axons or cell debris (Figure 3B and C). At 48 hours following exposure, the aggregates displayed further disruption to their outer architecture (Figure 4B and C). The inner areas of these aggregates however still contained healthy axons surrounded by compact myelin. At the highest dose the damage to myelin appeared more extensive (Table 1). There was no increase however in the extent of neuropil damage 96 hours following menadione exposure. LM analysis of the macrophage enriched aggregates treated with 5 �M menadione revealed severe neuropil destruction throughout the entire aggregate after 24 hours. Most of the cells within the aggregate contained shrunken nuclei and vacuolated cytoplasm (Figure 5A).
In addition, the neuropil featured large cavities that corresponded to and contained the remnants of destroyed cells. While the added macrophages were still recognizable, other cells (neurons and oligodendrocytes) could not be readily identified. Furthermore, myelinated axons could no longer be identified in the treated cultures. The aggregates that were exposed to menadione for 48 and 96 hour exposures exhibited similar amounts of destruction. EM analysis of these macrophage enriched aggregates treated with 5 �M menadione reinforced the finding of widespread non-specific destruction of the aggregates (Figure 5B), with completely shrunken neuropil, cellular necrosis and complete demyelination and axonal destruction (Figure 5B and C). Neuropil destruction appeared to increase with time following exposure so that by 96 hours, there was a complete loss of cellular detail in the aggregates. Occassionally, examples of delaminated myelin could be identified (Figure 5C), however, in all of the macrophage enriched aggregate cultures exposed to menadione, there were no examples of normal healthy myelin (Table 1). The Nv of the individual cell types and the total cell population in the control and menadione exposed aggregates was calculated to determine whether or not one cell type was preferentially affected. In the standard aggregate cultures, exposure to different concentrations of menadione caused a decrease in the Nv in all treatment groups as time increased (Figure 6A). Individual cell populations were also analysed for Nv. Changes to Nv for oligodendrocytes and astrocytes were not significant (data not shown). With respect to the macrophage enriched aggregates that were exposed to menadione, it was not possible to readily identify individual cell types. Accordingly, only changes to the Nv of the total cell population were calculated. A significant decrease to the Nv of the total cell populations in the macrophage enriched aggregates (Figure 6B), was observed in all treatment groups.
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Devon, R.M.; Bartnik, B.; Juurlink, B.H.J.; (1998). Combined Effects of Macrophage Enrichment and Addition of Menadione on Myelinating Rat Spinal Cord Aggregate Cultures. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Available at URL http://www.mcmaster.ca/inabis98/oxidative/devon0759/index.html | |||||||||||
© 1998 Author(s) Hold Copyright |