Figure 2.
Rats exposed to repeated hypoxia from P1 to P3 had a significant loss of O4-positive pre-oligodendrocytes at P4, reduced cerebral myelin at P14 and P545 (i.e., 18 months of age), and a similar increase in myelin between P14 and P545 when compared with rats exposed to repeated normoxia from P1 to P3. A, Immunohistochemically identified O4-positive pre-oligodendrocyte (arrow) in the callosal white matter at P4. Because the O4-positive cells are located in a 50-μm-thick section, other cells are in focus at a different depth to the arrowed cell. Note also that this image was taken at 1000×, whereas in this study each O4-positive cell was identified at 3400× and counted as it clearly came into focus (see Materials and Methods). B, Total (i.e., absolute) number of O4-positive pre-oligodendrocytes in the callosal white matter at P4. Two groups of rats were used: (1) repeated normoxic (n = 4) and (2) repeated hypoxic (n = 4) (paired two-tailed Student's t test, *p = 0.032). C, D, E, I, Sagittal sections stained immunohistochemically with myelin basic protein for a P14 repeated normoxic rat (C), P14 repeated hypoxic rat (D, E), and P545 repeated normoxic rat (I). The area indicated with an * in D is illustrated at higher magnification in E. Note the staining in the internal capsule (ic) and optic tract (opt) is paler in the repeated hypoxic animal (compare D with C). The sections in C and D were immunostained at the same time, photographed at the same lighting intensity, and have the same section thickness. This qualitative difference reflects the quantitatively lower absolute lower surface area of cerebral myelin in the repeated hypoxic group versus the repeated normoxic group at P14 (F). Cycloid arcs intersecting myelinated axons to measure the Sv using an unbiased sampling frame are illustrated in E at P14 (395×) and I at P545 (330×). Note that the actual quantification of the Sv of myelin was completed at a magnification of 6800×. The stronger staining in I reflects the amount of myelin in a P545 brain. In I, a myelin basic protein-positive axon is indicated in the subcortical white matter (arrow) between the cerebral cortex (CCx) and caudate–putamen (CPu; striatum). F, Absolute (i.e., total) surface area of cerebral myelin at P14. Two groups of rats were used: (1) repeated normoxic (n = 8) and (2) repeated hypoxic (n = 8) (two-tailed Student's t test, **p = 0.021). G, Absolute (i.e., total) surface area of cerebral myelin at P545 (i.e., 18 months of age). Two groups of rats were used: (1) repeated normoxic (n = 9) and (2) repeated hypoxic (n = 10) (one-tailed Student's t test, *p = 0.045). H, Absolute (i.e., total) surface area of myelin at P14 and P545 (i.e., 18 months of age). Same rats as F and G (two-tailed Student's t test, *p < 0.0001, repeated hypoxic rats at P14 compared with P545; #p < 0.0001, repeated normoxic rats at P14 compared with P545). Reliable estimates were obtained for all stereological data in this figure (see also Results). Error bars show SEM.