Propofol-induced apoptosis of neurones and oligodendrocytes in fetal and neonatal rhesus macaque brain

Abstract

Background: Exposure of the fetal or neonatal non-human primate (NHP) brain to isoflurane or ketamine for 5 h causes widespread apoptotic degeneration of neurones, and exposure to isoflurane also causes apoptotic degeneration of oligodendrocytes (OLs). The present study explored the apoptogenic potential of propofol in the fetal and neonatal NHP brain.

Method: Fetal rhesus macaques at gestational age 120 days were exposed in utero, or postnatal day 6 rhesus neonates were exposed directly for 5 h to propofol anaesthesia (n=4 fetuses; and n=4 neonates) or to no anaesthesia (n=4 fetuses; n=5 neonates), and the brains were systematically evaluated 3 h later for evidence of apoptotic degeneration of neurones or glia.

Results: Exposure of fetal or neonatal NHP brain to propofol caused a significant increase in apoptosis of neurones, and of OLs at a stage when OLs were just beginning to myelinate axons. Apoptotic degeneration affected similar brain regions but to a lesser extent than we previously described after isoflurane. The number of OLs affected by propofol was approximately equal to the number of neurones affected at both developmental ages. In the fetus, neuroapoptosis affected particularly subcortical and caudal regions, while in the neonate injury involved neocortical regions in a distinct laminar pattern and caudal brain regions were less affected.

Conclusions: Propofol anaesthesia for 5 h caused death of neurones and OLs in both the fetal and neonatal NHP brain. OLs become vulnerable to the apoptogenic action of propofol when they are beginning to achieve myelination competence.

Keywords: anaesthetics i.v., propofol; developing brain; neurones; non-human primates; oligodendroglia; toxicity.

Fig 1

The total number of profiles (neurones+glia) undergoing apoptosis, as determined by AC3 staining, in fetal or neonatal NHP brain after exposure to propofol or to no drug (control). The difference between the mean apoptosis counts for propofol-exposed or control brain at each age was statistically significant (fetuses P=0.01; neonates P=0.007).

Fig 2

Computer plots showing the pattern of apoptotic cell loss in representative sections from a mid-rostrocaudal level of control or propofol-exposed fetal NPH brains. Apoptotic neurones (red dots) and apoptotic OLs (white dots) are more abundant in the propofol-exposed than control brain. Apoptotic neurones are most densely concentrated in several layers of the frontal cortices and temporal cortices, the caudate (Ca) nucleus, putamen (Pu), amygdala (Am), and in caudal regions not shown here (cerebellum and inferior colliculus). Apoptotic OLs are diffusely distributed throughout WM regions, including the centrum semi-ovale (CSO), corpus callosum (CC), and internal capsule (IC).

Fig 3

Histological appearance of degenerating neurones, as revealed by AC3 staining, in the frontal cortex of the NHP fetus after 5 h of propofol anaesthesia. The affected neurones are pyramidal, have a prominent apical dendrite and are distributed evenly across Layers II, III, and IV. Scale bar=50 µm.

Fig 4

Morphological and staining characteristics of degenerating glia in the centrum semi-ovale WM underlying the frontal cortex of a propofol-exposed fetal NHP brain. AC3 staining (a) displays the degenerating OLs as spider-like profiles surrounded by a smudgy halo comprised of disintegrating processes and perhaps AC3 protein that has leaked out of the cell. Silver staining (b) displays the cell body and a starburst pattern of fragmented debris shed by the degenerating processes. Fractin antibodies (c) detect fractin, a breakdown product formed by proteolysis of actin, which is present throughout the cell body and processes of OLs. Each of these cell death markers detects degenerating OLs in approximately the same numbers and distribution throughout the WM. (d) A section immunofluorescently double stained with antibodies to AC3 (red) and GFAP (green). Three cells are GFAP-positive (green), signifying that they are astrocytes. A fourth cell is AC3-positive, signifying that it is dying by apoptosis, but GFAP does not co-label with AC3 in this cell, signifying that it is not an astrocyte. Bar in (a)=50 µm for (a –c) and 30 µm for (d).

Fig 5

Histological appearance of MBP-stained OLs. (a) The appearance of a typical premyelinating OL in the rostral WM of a control fetal NHP brain that stains with antibodies to MBP because it has accumulated MBP in its cytoplasm and processes in preparation for myelinating axons. This cell is in a state of readiness, but has not yet become engaged in myelination. In propofol-exposed brains there were numerous MBP-positive premyelinating OLs that appeared to be dying. The typical appearance of these MBP-positive premyelinating OLs in an advanced stage of degeneration is depicted in (b). (c) From a mid-rostrocaudal section where myelination is occurring, this micrograph illustrates the appearance of an MBP-positive OL that is engaged in early myelination. MBP antibodies stain the cell body and processes of the OL, and also stain multiple myelin sheaths with which its processes are in continuity. (d) When an OL succumbs to cell death while it is engaged in early myelination, MBP staining reveals that the degeneration products cluster in the central compartment (remains of the cell body) and line up in a linear pattern conforming to the configuration of the patch of axons it was myelinating. Scale bar in (d)=15 µm and pertains to all panels.

Fig 6

Immunofluorescent double staining of premyelinating OLs with antibodies to MBP and fractin. The micrograph depicts cell degeneration in the rostral CSO WM of a propofol-exposed NHP fetal brain. (a) MBP (green) stains, with varying degrees of intensity, about nine cellular profiles (one example marked by arrowhead). (b) Fractin antibodies also mark the same profiles with varying degrees of intensity. One of the fractin-positive profiles (arrow in a and b) is in an advanced stage of degeneration, as evidenced by the starburst halo of fractin-positive degeneration products surrounding the profile that represents dense accumulation of fraction in the centre of the disintegrating cell body. The remaining fractin-positive cells are in earlier stages and have less fractin-positive debris in their cytoplasm. The merged image in (c) reveals that MBP and fractin co-localize in all of the profiles that were MBP-positive in (a), but the cell that has the greatest display of fractin positivity (arrow in a and b) has relatively faint MBP positivity (arrow in a), suggesting that the degenerative process may tend to extinguish MBP positivity. Profiles in (b) that are only faintly stained with fractin (asterisk) are in an early stage of injury and therefore have generated only small amounts of fractin. Scale bar in (a)=25 µm for all panels.

Fig 7

Immunofluorescent double staining with antibodies to MBP and AC3. (a) A myelinating OL in a propofol-exposed fetal NHP brain stained with the MBP-antibody (green). The cell body is densely positive for MBP and its MBP-positive processes are in contact with axons that it is myelinating. (b) This same cell co-labels for AC3 including its processes, which is confirmed by the merged image in (c). MBP positivity of the cell body signifies that this is an OL; contact and confluence of its processes with MBP-positive myelin sheaths confirms that it is engaged in myelinating axons; AC3 positivity signifies that this OL is undergoing apoptotic degeneration. DAPI counterstaining (d, blue colour) shows at increased magnification that the nucleus of this cell has a pattern of nuclear chromatin clumping that proves apoptotic cell death. Scale bar in (c)=15 µm for (d –c); scale bar in (d)=5 µm.

Fig 8

Immunofluorescent double staining with antibodies to MBP and AC3 (a) or MBP and fractin (b) of degenerating OLs in a heavily myelinated caudal WM pathway (cerebellar peduncle) from a propofol-exposed fetal NHP brain. MBP staining in both of these panels reveals that myelinated axonal bundles are strongly immunoreactive for MBP antibodies, but there are no MBP-positive cell bodies. Both cell death markers (AC3 and fractin) vividly stain dying cells that are intermingling with the MBP-positive fibre bundles, but none of these dying cell bodies are immunoreactive for MBP. Scale bar in (a)=15 µm for both panels.

Fig 9

Pattern and appearance of dying neurones in the primary visual cortex of anaesthesia-exposed neonatal NHP brain. Computer plots in (a–c) show the location of apoptotic neurones in sections from the primary visual cortex of neonatal NHP brain after exposure to no anaesthesia (control), or to propofol (present study) or isoflurane (prior study). The control brain (a) has a sparse display of randomly scattered apoptotic neurones representing the magnitude and pattern of natural apoptosis in this brain region at this age. After isoflurane exposure (c) there is a highly organized laminar display of degenerating neurones densely concentrated in cortical Layers II and V. Propofol-exposed brain (b) shows a laminar pattern of neuronal degeneration that is not as great in magnitude but selectively impinges on the same cortical layers as are affected by isoflurane. (d and e) The appearance of AC3 stained neurones in Layers II and V of the neonatal NHP visual cortex after propofol exposure. Scale bar in (d)=50 µm for (d and e).