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Combined Effects of Macrophage Enrichment and Addition of Menadione on Myelinating Rat Spinal Cord Aggregate Cultures

Contact Person: Richard M. Devon (devonr@duke.usask.ca)

Discussion and Conclusion

Numerous pathologies exist in which CNS tissues may be damaged or destroyed. As difficulties exist in the in vivo study of these diseases, a number of in vitro CNS models have been established that include organotypic, dissociation and re-aggregate cultures of CNS tissue (reviewed in Raine, 1984b; Compston, 1997). In the experiments reported here, a model was required that contained both mature myelinating oligodendrocytes and abundant examples of myelinated axons. Day 15 (E15) rat spinal cord aggregate cultures were chosen since these cultures mimic normal morphological and biochemical development of rodent neocortex (Honegger and Richelson, 1976; Matthieu et al, 1978a & b; Trapp et al, 1979; Bourre et al, 1979; Guentert-Lauber et al, 1985) and spinal cord (Devon, 1987), and feature a population of centrally positioned mature neurons surrounded by a peripheral ring of macroglial cells (Figure 1). While myelinated axons could be identified as early as 14 DIV, substantial myelination occured throughout the aggregate by 28 DIV.

The purpose of menadione treatment was to determine the effect of O2.- on mature myelin and cell viability. Menadione generates intracellular O2.- via redox cycling, involving a NADPH-cytochrome P-450 reducatase catalyzed one electron reduction of menadione into a semiquinone radical (Comporti, 1989; Shi et al, 1996). While the O2.- has been shown to be weakly reactive in biological systems, it does possess the ability to cause cell damage (Halliwell and Gutteridge, 1990). Furthermore, two additional reactive oxygen species, H2O2 and the OH. radical, can be produced during the enzymatic scavenging of O2.- and have been shown to cause various forms of cell damage (Gutteridge, 1995; Halliwell and Gutteridge, 1990; Inoue and Kawanishi, 1995). Previous experiments have shown that menadione can induce cytotoxicity by reacting with and/or depleting cellular thiol and amine stores (in particular glutathione) leading to protein damage and decreased intracellular levels of Ca2+ (Comporti, 1989; Shi et al, 1996).

Exposing the aggregates to menadione resulted in neuropil disruption, cell necrosis and myelin alterations. These effects were localized predominately to the outer area of the aggregate (Figure 3A and 4A) and may reflect an inability of the menadione to diffuse or penetrate past the peripheral glial ring into the center of the aggregate. Alternatively, this finding may be interpreted as the successful scavenging of menadione by the cells in the peripheral glial ring. Morphologically, the most striking alteration was the presence of large cavities in the otherwise compact neuropil. These spaces contained the remnants of cellular debris and were scattered randomly throughout the aggregate. The observation of cavities within the neuropil suggests that menadione had a negative effect on cell viability. Menadione exposure also effected the myelinated axons of these cultures (see Table 1). EM analysis showed damage to both the neuritic components (see Figure 3C and Figure 4B & C), and the lamellae of the myelin sheaths of the aggregates. Other ultrastructural changes to the neuropil included the presence of intracellular lipid deposits and myelin whorls. While the appearance of these intracellular myelin whorls suggests that the myelin was being removed and perhaps recycled, a process that is known to exist in vivo (Peters et al, 1991), the exact nature of this mechanism remains unknown. These findings, along with the Nv data, reflect a general loss of cell viability and points to the oligodendrocytes and neurons as the susceptible populations. Earlier work by Husain and Juurlink (1995) has demonstrated that menadione exposure results in a loss of oligodendrocyte viability in mixed glial cultures and has been explained as a reduced ability by oligodendrocytes to scavenge free radicals (Thorburne and Juurlink, 1995; Juurlink, 1996).

Research into a role for macrophages in neurodegenerative and demyelinating diseases has been confusing. On the one hand these cells may be important for myelin removal (Bruck et al, 1994) and remodelling. In addition, it has been shown that free radicals can induce macrophages to selectively attack myelin sheaths resulting in demyelination of the axons (Bruck et al, 1995). Mediators released by macrophages include TNF-a, IL-1, O2.- , H2O2, NO. and ONOO- (Marcinkiewicz et al, 1995). It is also known that all phagocytic cells, including macrophages, produce H2O2, O2.- , and OH. by the partial reduction of O2 during the respiratory burst (Babior, 1978; Babior et al, 1987). Macrophages may play a contrasting beneficial role however since it has also been shown that the addition of macrophages to fetal brain aggregate cultures is associated with an increase in the levels of the myelin proteins, MBP and PLP (Loughlin et al, 1994; Loughlin et al, 1997). Additionally, the presence of macrophages have recently been associated with the promotion of nerve regeneration and remyelination ( Schwartz et al., 1997, Cuzner et al., 1994; Pavelko et al., 1998).

Since the role of macrophages in demyelinating lesions is unclear, these cells were added to spinal cord aggregate cultures to determine what effect they would have on menadione induced cell death and myelin damage. The architecture of the aggregates changed very little when macrophages were added in the absence of menadione (Figure 2). They retained their typical spherical form containing centrally scattered cell nuclei and myelinated profiles surrounded by a peripheral glial ring. The macrophages were located outside of and throughout this structure. Exposing the macrophage enriched cultures to even the lowest dose of menadione resulted in severe damage represented by widespread neuropil cavitation, shrinkage and cellular necrosis throughout the aggregate (see Figure 5). The damage was so extensive by 24 hours that little change was observed after 48 or 96 hours of exposure. Quantification of the Nv confirmed the widespread cell necrosis and revealed significant decreases in numerical density at all time points and with all concentrations of menadione (see Figure 6B).

A possible explanation for extensive damage of the neuropil is the initiation of a positive feedback loop of free radical generation (specifically O2.- and NO.) within the population of added macrophages (Marcinkiewicz et al, 1995). The continual redox cycling of oxygen within these stimulated macrophages would render them cytotoxic, ultimately leading to the destruction of the neighbouring cellular architecture. Additionally, cells damaged by the liberation of other cytotoxic molecules may further activate macrophages. Such continued activation of macrophages may ultimately result in the massive destruction of the aggregate’s components. Free radical induced neuropil, cell and myelin damage observed in our model may therefore be considered relevant to an understanding of inflammatory demyelinating diseases like MS. Other reports supporting this include the observations that free radicals localize to the demyelinating regions in MS brains (Bo et al, 1995), and that macrophages residing near the demyelinating lesions produce both nitrogen and oxygen based free radicals (Auger and Ross, 1992; Banati et al, 1993; Vladutiu, 1993; Brosnan et al, 1994). Recent reports indicate that the use of free radical scavengers such as catalase and superoxide dismutase in experimental allergic encephalomyelitis (an animal model of MS), markedly delay the onset and suppress the severity of demyelination (Ruuls et al, 1995; Malfroy et al, 1997). While further research is necessary to clearly define the mechanism of free radical damage, there is a growing body of scientific evidence that implicates free radicals in demyelinating diseases.

This research was supported by a grant from the Medical Research Council (MRC) of Canada (MT-13467) awarded to BHJ and RMD. The authors would like to thank the following for assistance with various aspects of this study: Drs. A. Richardson, and R. Doucette, Mr. V. Appl, Ms. T. McGowan.

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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