Dimethyl sulfoxide (DMSO) produces widespread apoptosis in the developing central nervous system

Abstract

Dimethyl sulfoxide (DMSO) is a solvent that is routinely used as a cryopreservative in allogous bone marrow and organ transplantation. We exposed C57Bl/6 mice of varying postnatal ages (P0-P30) to DMSO in order to study whether DMSO could produce apoptotic degeneration in the developing CNS. DMSO produced widespread apoptosis in the developing mouse brain at all ages tested. Damage was greatest at P7. Significant elevations above the background rate of apoptosis occurred at the lowest dose tested, 0.3 ml/kg. In an in vitro rat hippocampal culture preparation, DMSO produced neuronal loss at concentrations of 0.5% and 1.0%. The ability of DMSO to damage neurons in dissociated cultures indicates that the toxicity likely results from a direct cellular effect. Because children, who undergo bone marrow transplantation, are routinely exposed to DMSO at doses higher than 0.3 ml/kg, there is concern that DMSO might be producing similar damage in human children.

Figure 1. DMSO-Induced Caspase-3 Immunoreactivity at Different Post-Survival Intervals

Activated caspase-3 immunohistochemistry from P7 mouse pups treated with either saline (A–D) or DMSO (E–X) and sacrificed at various time points (2, 4, 8, 12, & 24 hours) after exposure to DMSO. Left column contains low-powered photomicrographs, all of which are taken from approximately the same rostro-caudal level. The three columns on the right show contain high-powered photomicrographs from caudate/putamen (CPu), lateral dorsal thalamus (LD Thal) and retrosplenial cortex (RSC). The approximate location from which each of the high-powered photomicrographs is taken is indicated by an asterisk is panel A. Subtle increases in activated caspase-3 activity could be seen by 2 hours. This increase is difficult to detect on high-powered images (black arrows = activated caspase-3 positive cells) but it is evident when comparing panels E and A. Peak damage occurred at 8 hours in most areas of the brain. However, some areas (e.g. CPu) showed peak staining at 4 hours. While the overall amount of activated caspase-3 appeared to be greater at 12 and 24 hours compared to 8 hours when comparing low-powered photomicrographs (Q and U vs. M), high-powered photomicrographs (R–X) showed that while some of the staining at 12 and 24 hours was localized to intact cell bodies (white asterisks), a larger amount of the staining appeared to come from debris (white arrows) and a general increase in background staining (white arrowheads). Prominent staining of white matter tracks was noted at 24 hours (U; black arrowheads). The Levels function of Photoshop^® was used to lighten the amount of dark staining in images T and X in order to allow the cell bodies to be more easily seen.

Figure 2. Activated Caspase-3 Immunoreactivity Varies by Region

P7 animals were treated with either DMSO 10 ml/kg (n=9) or saline (n=10) and sacrificed 8 hours later. Relative degeneration severity scores were determined for several different regions as indicated in Methods. Zero indicates similar degeneration relative to the same region in control animals. Regions have been ranked ordered by decreasing severity scores. The severity of activated caspase-3 immunoreactivity varied between different regions (F[11,88]=10.62, p<0.0001). The difference was mainly due to damage in the cortex (Ctx) and caudate/putamen (CPu) being greater than the other sampled regions of the brain (Newman-Keuls). Error bars = SEM.

Figure 3. DMSO-Induced Apoptotic Degeneration

Nissl (A) and De Olmos silver (B) stained sections of the CPu taken from a P7 animal exposed to DMSO (10 ml/kg) 24 hours previously. A) Numerous darkly labeled spheroid balls (black arrowheads), or apoptotic bodies, are present in the Nissl section. B) At 24 hours silver stain demonstrates that the degeneration is in an advanced stage. Cell bodies (white arrowheads) are amorphous indicating that cell membranes are no longer intact. Axons and dendrites are no longer discernible. Electron microscopic micrographs of the ultrastructure of degenerating cells after exposure to DMSO (C–F), demonstrate that the cells have the classical hallmarks of apoptotic degeneration. C) A neuron early in the apoptotic degenerative process. At this stage, the mitochondria (m) are slightly swollen. The nuclear membrane (black arrows) is still intact and separates the cytoplasm, which is slightly condensed, from the nucleoplasm. In the nucleus, chromatin has begun to clump into a chromatin ball (cb). D) Photomicrograph of a neuron in the later stages of apoptotic degeneration. The nuclear membrane is no longer intact, allowing the nuclear and cytoplasmic compartments to mix. The neuron has become more condensed and the chromatin ball is spherical and is in the center of the cell. The cell membrane (black arrows) remains relatively intact and the cell appears to be surrounded by processes from a phagocytic cell. E) Image of a neuron in a very late stage of apoptotic degeneration. The chromatin body has become marginalized and the material inside the cell is becoming degraded. While the dense degraded cell can still be distinguished from the surrounding neuropil, the cellular membrane is beginning to be compromised by processes from the surrounding phagocytic cell, which have begun to encroach into the degenerating cell (white arrow).

Figure 4. Dose-Dependency of DMSO-Induced Apoptosis

Animals were treated with one of several doses of DMSO and sacrificed 8 hours later. Total number of caspase-3 positive neurons was estimated with stereology. The number of degenerating cells increased with increasing doses of DMSO (p<0.0001) with R² of 0.82. The lowest dose tested produced a significantly greater amount of apoptosis than was present in the control animals (p=0.0017). Numbers in parentheses indicate number of animals studied at each dose. Error bars (SEM) for most points are too small to be seen.

Figure 5. DMSO-Induced Neuronal Loss in Hippocampal Culture

DMSO at either 0.5% or 1.0% produces a substantial decrease in the number of cultured hippocampal neurons (p<0.05). The loss of neurons is prevented by the addition of KCl. 0 (solid line) represents control counts (p<0.05). −1.0 (dotted line) represents 100% loss. +1.0 would represent doubling of cell numbers over control. Note that KCl increases neuronal survival over baseline (Moulder et al., 2002; Shute et al., 2005) and largely prevents the additional cell loss induced by DMSO. Error bars = SEM.

Figure 6. Comparison of PCP-Induced and DMSO-Induced Apoptosis

Activated caspases-3 immunohistochemistry of sections from three different rostrocaudal levels taken from animals treated with either DMSO (10 ml/kg; left hemisections) or PCP (50 mg/kg; right hemisections) and sacrificed 8 hours later. DMSO appeared to produce more apoptosis than PCP even though the doses for both agents were approaching toxic levels. The pattern of damage was similar in most regions of the brain, consistent with the agents producing apoptosis via a similar mechanism. There were some exceptions to this rule, however. A large number of neurons were degenerating in the Islands of Calleja (ICj; circled region) with DMSO. In contrast in the PCP-treated animals this region was not affected. Similarly, several regions of the hypothalamus (anterior [AntHypoTh] and medial [MedHypoTh] areas are illustrated here) were severely affected. In contrast, no damage above control levels was detected in the PCP-treated animals.