Isoflurane-induced apoptosis of oligodendrocytes in the neonatal primate brain

Abstract

Objective: Previously we reported that exposure of 6-day-old (P6) rhesus macaques to isoflurane for 5 hours triggers a robust neuroapoptosis response in developing brain. We have also observed (unpublished data) that isoflurane causes apoptosis of cellular profiles in the white matter that resemble glia. We analyzed the cellular identity of the apoptotic white matter profiles and determined the magnitude of this cell death response to isoflurane.

Methods: Neonatal (P6) rhesus macaques were exposed for 5 hours to isoflurane anesthesia according to current clinical standards in pediatric anesthesia. Brains were collected 3 hours later and examined immunohistochemically to analyze apoptotic neuronal and glial death.

Results: Brains exposed to isoflurane displayed significant apoptosis in both the white and gray matter throughout the central nervous system. Approximately 52% of the dying cells were glia, and 48% were neurons. Oligodendrocytes (OLs) engaged in myelinogenesis were selectively vulnerable, in contrast to OL progenitors, astrocytes, microglia, and interstitial neurons. When adjusted for control rates of OL apoptosis, the percentage of OLs that degenerated in the forebrain white matter of the isoflurane-treated group was 6.3% of the total population of myelinating OLs.

Interpretation: Exposure of the infant rhesus macaque brain to isoflurane for 5 hours is sufficient to cause widespread apoptosis of neurons and OLs throughout the developing brain. Deletion of OLs at a stage when they are just beginning to myelinate axons could potentially have adverse long-term neurobehavioral consequences that might be additive to the potential consequences of isoflurane-induced neuroapoptosis.

FIGURE 1

Bright field images of the corpus callosum of an isoflurane-exposed brain stained with AC3 antibodies (A,B,C), or by the DeOlmos silver stain (D). Some of the profiles stained by AC3 (panel A) display multiple small condensed fragments (arrows), a sign of advanced apoptotic degeneration. Cells in an earlier stage (boxed) are shown at higher magnification in B & C. All of the cells stained by silver show signs of disintegration and are surrounded by a halo of particulate debris, signifying that the silver stain detects late, but not early, stages of degeneration. Scale bar = 25 μM for A; 10 μM for B and C.

FIGURE 2

Isoflurane-resistant cell types in neonatal monkey cerebral white matter. A. Interstitial white matter neurons, identified with NeuN (green nuclei; arrows) were a distinct population from AC3-labeled cells (red; arrowheads). B. GFAP-labeled astrocyte somata and processes had normal-appearing morphology (green) and did not co-localize with cells labeled with AC3 (red). C, D. Morphological features of Iba1-labeled activated phagocytic microglia (green). Most degenerating nuclei in the white matter were co-localized to microglia with reactive-appearing features. In C, a fragmented nucleus (Hoechst-labeled; blue; arrowhead) is contained within a bleb of membrane. Note the intact-appearing nuclei (arrows) of this and an adjacent cell. In D, multiple fragments of condensed chromatin (arrowheads; Hoechst, red pseudo-color for improved contrast) were distributed within two blebs of membrane of this cell with an intact nucleus (arrow). Note the thickened processes of this activated-appearing cell. E, F. Numerous PDGFRα-labeled OL progenitors (green; thin arrows) were visualized throughout the corona radiata and did not stain for AC3 (red; arrowhead). Note that the nuclei of the OL progenitors appeared intact in contrast to adjacent nuclei visualized with Hoechst (blue, thick arrows in E, F) that displayed features of apoptotic degeneration with condensed and fragmented chromatin. Bars= 50 μm (A); 25 μm (B, C); 10 μm (D, E, F).

FIGURE 3

Immature oligodendrocytes in myelinating neonatal white matter tracts degenerate in response to isoflurane exposure. (A) Neonatal cerebral white matter tracts were heavily myelinated, as visualized with the O4 antibody and the O1 antibody (not shown). Myelination was not observed in the deep cerebral cortical gray matter (asterisk). (B) At the edge of myelinating white matter tracts, scattered immature OLs were visualized with both the O4 antibody (arrowheads) and the O1 antibody (not shown). (C) Within heavily myelinated tracts, O4-labeled cells were visualized (arrowheads). Hoechst 33342 visualized the nucleus within the cell bodies (not shown). (D-F) Confocal microscopy confirmed that O4-labeled immature OLs were AC3-positive. (D) Degenerating AC3-labeled cell with a halo of degenerating processes. (E) Distribution of O4 staining shows the abundant myelination corresponding to the region shown in D. Note the increased O4 staining around the plasma membrane of a cell (arrows). (F) Merge of D and E. Bars = 100 μm (A); 25 μm (B, C); 10 μm (D–F).

FIGURE 4

Mature oligodendrocytes in myelinating neonatal white matter tracts degenerate in response to isoflurane exposure. (A, C) As visualized by epi-fluorescence microscopy, AC3-labeled cells at various stages of degeneration (red) did not appear to co-localize with cells labeled with the OL-lineage specific markers, CC-1 (A) and Olig 2 (C). (B) By confocal microscopy, the mature OL marker CC-1 (green) was robustly visualized in the cytoplasm of intact appearing cells (double arrow). Two AC3-positive cells were visualized at different stages of degeneration. One cell at an earlier stage (arrowhead) had intact appearing processes (see AC3 black/white inset) and was less intensely stained for AC3 and stained more weakly for CC-1 than adjacent intact cells (double arrow). Another cell at a more advanced stage of degeneration (arrow) displayed degenerating processes, more robust AC3 staining than the cell at an earlier stage of degeneration (double arrows) and had no apparent CC-1 staining. (D) As shown by confocal microscopy, some intact appearing nuclei (blue) strongly co-localized with Olig2 (green) (arrows) and did not label for AC3 (red). By contrast (black and white insets), a degenerating OL (arrowhead) had a pyknotic nucleus with fragmented chromatin (Hoechst), more weakly labeled with Olig 2 and double-labeled with AC3. Bars = 100 μM (A, C); 15 μM (B) and 25 μM (D).

FIGURE 5

Total numbers of apoptotic neurons or glia, or neurons + glia (N+G) per brain (mean ± SEM) of P6 rhesus neonates exposed to isoflurane or to no drug (control). All of the values for the isoflurane-exposed brains were significantly higher than those for the control brains at the significance levels indicated. The mean number of apoptotic neurons (19.14 × 105) was slightly less that the mean number of apoptotic glia (20.89 × 10⁵), yielding a neuron to glia ratio of 48 to 52.

FIGURE 6

Computer plots showing the distribution of apoptotic neurons (red dots) and apoptotic glia (green dots) in sections cut at a mid rostro-caudal level (A and B), or at the level of the primary visual (occipital) cortex (C and D). The control brain has sparse numbers of apoptotic profiles and there is no pattern of concentration in any particular region, except that the glial profiles are more concentrated in white matter and the neuronal profiles are enriched in gray matter. In contrast, the apoptotic profiles in the isoflurane-exposed brain are much more abundant, with glia being heavily concentrated in white matter zones, and neurons showing dense packing in specific gray matter areas. Note the highly organized laminar pattern in some regions, especially the visual cortex, reflecting the known location of layer II and layer V neurons.

Figure 7

Isoflurane induced diffuse glial degeneration throughout the neuraxis as demonstrated by brightfield images of apoptotic glia stained with antibodies to AC3. All images were acquired at the same magnification and demonstrate the relative density of degenerating cells in the representative white matter tracts shown. The staining in the frontal corona radiata is representative of that in the prefrontal, parietal, temporal and occipital cortices. The cerebral peduncle is shown at the level of the midbrain basilar division. The cerebellar peduncle staining is from the middle division situated between the peduncle and the deep cerebellar nuclei. The optic tract is in the diencephalon and the medial lemniscus is in the medula oblongata. Scale bar in D = 25 μM for all panels.