Propylene glycol produces excessive apoptosis in the developing mouse brain, alone and in combination with phenobarbital

Abstract

Introduction: Propylene glycol (PG) is a common solvent used in medical preparations. It is generally recognized as safe at regulated concentrations; however, its apoptotic potential is unknown.

Results: PG triggered widespread apoptotic neurodegeneration with the greatest damage at postnatal day 7 (P7). Significant apoptosis was observed at doses as low as 2 ml/kg. These findings have implications for the safety of drug preparations used in pediatric medicine. The anticonvulsant phenobarbital (PB), which alone produces apoptosis in the immature central nervous system (CNS) is prepared in 68% PG and 10% ethanol (EtOH). We assessed whether PG contributes to the neurotoxic potential of PB. The agents (both at subtoxic doses) produce significantly more apoptosis when used in combination.

Discussion: In conclusion, finding an alternative non-apoptotic solvent that can be used as a substitute for PG may be beneficial to patients.

Methods: C57BL/6 mice (P4-30) were exposed to PG to examine whether PG could produce apoptosis in the developing CNS.

Figure 1

Sections from P7 mouse brain following 8h treatment with saline or PG stained immunohistochemically with antibodies to AC-3. Saline brains (A–D) showed a pattern of sparse AC-3 positive neurons (white arrows) that is typical of physiological cell death, whilst PG triggered widespread neuroapoptosis in many brain regions (E–H). In the CPu, cells appeared to be in a later stage of the apoptotic process (staining localized to intact condensed cell bodies; white arrow heads). Top images were viewed at 10× magnification. High-powered photomicrographs were viewed at 20×.

Figure 2

Histological sections from P7 mouse brain treated with saline (A–D) or PG (E–X) at various time points after exposure. Black arrows depict AC-3 positive-cells. Hemi-sections are taken from the same rostro-caudal level (10× magnification). High-powered photomicrographs (20× magnification) are taken from the CPu (*), LD Th (†), and retrosplenial cortex (RSC; §), as indicated by a symbol in panel A. Onset of damage appeared at 2h in the CPu (F). Peak levels of damage occurred at 8h in most regions (M–P). Overall AC-3 staining was decreased at 12h (Q–T) and 24h (U–X), where staining was specific to intact cell bodies (white arrow heads). In addition to localized staining, a non-specific diffuse staining was present at 24h.

Figure 3

De Olmos silver staining at 24h shows advanced-stage degeneration (A - image taken from LD Th of a P7 animal exposed to 10mL/kg PG (10× magnification). Cell bodies are amorphous, axons and dendrites are no longer visible, and cellular debris is scattered throughout the region. Electron Microscopy photomicrographs (10,000× magnification) (B–C) demonstrate cells with classical hallmarks of apoptosis (8h exposure to 10mL/kg PG). Image (B) shows the nuclear membrane is no longer intact (broken membrane remnant: black arrow), allowing nuclear and cytoplasmic compartments to mix. Mitochondria appear to be swollen (m), and a condensed spherical chromatin ball (cb) has formed. A very late stage apoptotic cell (C) shows migration of nuclear chromatin masses into the cytoplasm and disintegration of material inside the cell. The apoptotic cell can be distinguished from adjacent tissue cells; however the surrounding phagocyte has already started to encroach into the degenerating cell (white arrow).

Figure 4

P7 animals were exposed for 8h to various doses of PG. Total AC-3 positive neurons were estimated using stereology. Number of degenerating cells increased with increasing doses of PG (F[5,35]=42.55, P<0.0001; R² of 0.99; n=6 at each dose tested). Significantly greater damage was observed at 2mL/kg compared to saline (*P=0.0042). Error bars are too small to be seen.

Figure 5

The combined effect of PB and PG, both used at sub-toxic doses. 5mg/kg PB prepared in saline (n=8) produces similar levels of apoptosis to saline alone (n=8). PB prepared in 68% PG (n=10) produces significantly more apoptosis than PB prepared in saline (§P<0.05).

Figure 6

Histological sections from P4–24 mouse brain following 8h treatment with saline (left hemi-sections) or PG (right hemi-sections); viewed at 10×. Dentate gyrus (*) and LD Th (†) photomicrographs are taken from the same section as those indicated by a symbol in panel A. Caudate/putamen photomicrographs are taken from a more anterior section of the brain to demonstrate the extent of damage in this region. High-powered images were viewed at 20×. At P4 (A–E), severe damage was seen in the LD Th and CPu. These same regions were damaged at P7 (F–J), the age at which overall damage peaked. At P14 (K–O), overall damage was decreased and both LD Th and CPu showed similar levels of AC-3 positive cells as saline. The granule cell layer of the dentate gyrus was severely damaged at P14 (M), whereas this region was not damaged at P4 (C) or P7 (H). At P17 (P–T), AC-3 positive cells were still present in the dentate gyrus (R), however overall damage was further reduced. A new population of dying cells appeared in the CPu (white arrow heads). Closer examination of these cells showed morphology typical of oligodendrocytes (T; box at 40× magnification). At P24 (U–Y), damage was similar to saline. Staining was extremely sparse making it difficult to see at low magnification.