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Figure 1. Characterization of primary cultures of neural cells from newborn rat cortex for expression of ASPA mRNA, enzyme activity and co-immunostaining of ASPA protein with neural cells markers. The in situ detection of ASPA mRNA in oligodendrocytes cells in cultures mixed glia probed with sense (A) and anti-sense (B) ASPA digoxigenin labeled probes. The oligodendrocyte cells show dark staining (arrows) on the bed layer of unstained astrocytes. Optimal ASPA enzyme activity (mU/mg protein) can be seen in culture of oligodendrocytes (C) and about 29% ASPA activity can also be seen in mixed glial cultures. The pure cultures of astrocytes and cortical neurons exhibit insignificant level of ASPA activity. Immunostaining of mixed glial cultures from normal rat brain shows a co-localization of ASPA (red) with oligodendrocyte cell specific markers A2B5 (D) and CNP (F), both green, or exhibiting a yellow staining for co-expression. The immunostaining of ASPA with GFAP (green), an astrocyte cell specific marker shows a discrete localization of the two markers (H). (Epifluorescence images D, F, H; Phase image E, G, I; see color insert that appears between pages 364 and 365)

Figure 2. Immunocytochemistry of mixed glia cultures from ASPA wt and KO mice cerebral cortex. The immunostaining of markers for progenitor and mature oligodendrocytes were carried out in 6 and 15d-old cultures. The immunostaining for marker NG2 (green) and GD3 (Red; A and B), A2B5> (red) and GPDH (green; C and D), and O4 (red; E and F) showed the status of oligodendrocytes, while GFAP (green; E and F), the marker for astrocyte shows no co-localization with oligodendrocyte marker O4. A delay in MBP (green) and PLP (red) expressing oligodendrocyte cells was detected in 15day old ASPA KO mice cortical cultures (G, I and K). The double immunostaining of MBP astrocyte, showed a discrete staining for ASPA (red) and GFAP (green) in mixed glial cultures (Fig. 1G and panel H as phase image; see color insert that appears between pages 364 and 365)

Figure 2. Immunocytochemistry of mixed glia cultures from ASPA wt and KO mice cerebral cortex. The immunostaining of markers for progenitor and mature oligodendrocytes were carried out in 6 and 15d-old cultures. The immunostaining for marker NG2 (green) and GD3 (Red; A and B), A2B5> (red) and GPDH (green; C and D), and O4 (red; E and F) showed the status of oligodendrocytes, while GFAP (green; E and F), the marker for astrocyte shows no co-localization with oligodendrocyte marker O4. A delay in MBP (green) and PLP (red) expressing oligodendrocyte cells was detected in 15day old ASPA KO mice cortical cultures (G, I and K). The double immunostaining of MBP astrocyte, showed a discrete staining for ASPA (red) and GFAP (green) in mixed glial cultures (Fig. 1G and panel H as phase image; see color insert that appears between pages 364 and 365)

(32) and marker for immature oligodendrocyte, O4 was also detected in both wt and KO cultures (Fig. 2 C-F). The immunostaining of these early markers was significantly higher in cultures of KO mouse as compared to wt, suggesting accumulation of oligodendrocyte progenitors in KO cultures, while progenitors in the wt cultures may have progressed to different level of development. In all these instances KO oligodendrocyte cell morphology appeared to be distinctly different. While wt oligodendrocyte cells showed fine processes with a round cell body, the KO oligodendrocytes appear to have more globular structure with rough processes. The cause and effect of these alterations are unknown at this time. In general, a fewer oligodendrocytes stayed attached on the bedlayer of astrocytes by day 15 in the KO cultures. Of those, only a few cells showed expression of late markers, MBP and PLP as compared to the wt (Fig. 2 G-K).

Our findings suggest an overall delay in maturation of ASPA KO oligodendrocytes. It is, however, evident that the surviving oligodendrocytes in the KO cultures can potentially express MBP and PLP. More comprehensive study is needed such as cell proliferation analysis and cell survival vs. cell death that may help delineate the outcome of oligodendrocyte cell developmental progression. Most importantly, in combination to the postnatal culture study, an age matched in vivo study will help elucidate the causal and temporal development of oligodendrocytes in ASPA KO mouse. These studies are currently in progress.

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