Isoflurane exposure leads to apoptosis of neurons and oligodendrocytes in 20- and 40-day old rhesus macaques

Highlights

• Following 5 h of isoflurane anesthesia, brains of P20 and P40 rhesus macaques demonstrated similar levels of both neuronal apoptosis and oligodendrocyte apoptosis.

• The P20 and P40 rhesus macaques exposed to isoflurane had 3.6 times as many apoptotic cells (neurons + oligodendrocytes) as the control animals that were not exposed to anesthesia.

• In the P20 and P40 rhesus macaques exposed to isoflurane, approximately 66% of the apoptotic cells were oligodendrocytes and 34% were neurons.

Abstract

Previously we reported that a 5-hour exposure of 6-day-old (P6) rhesus macaques to isoflurane triggers robust neuron and oligodendrocyte apoptosis. In an attempt to further describe the window of vulnerability to anesthetic neurotoxicity, we exposed P20 and P40 rhesus macaques to 5 h of isoflurane anesthesia or no exposure (control animals). Brains were collected 3 h later and examined immunohistochemically to analyze neuronal and glial apoptosis. Brains exposed to isoflurane displayed neuron and oligodendrocyte apoptosis distributed throughout cortex and white matter, respectively. When combining the two age groups (P20 + P40), the animals exposed to isoflurane had 3.6 times as many apoptotic cells as the control animals. In the isoflurane group, approximately 66% of the apoptotic cells were oligodendrocytes and 34% were neurons. In comparison, in our previous studies on P6 rhesus macaques, approximately 52% of the dying cells were glia and 48% were neurons. In conclusion, the present data suggest that the window of vulnerability for neurons is beginning to close in the P20 and P40 rhesus macaques, but continuing for oligodendrocytes.

Introduction

Worldwide, millions of young children undergo surgery and anesthesia each year. It is becoming increasingly common for pediatric patients of all ages to receive general anesthesia not only for surgical procedures, but also to facilitate long diagnostic procedures and minimally invasive interventions. There is conflicting clinical evidence regarding a potential association between exposure to anesthesia in early childhood and deleterious neurodevelopmental outcomes. While children initially exposed to anesthesia in the first few years of childhood have been shown to have an increased risk of neurodevelopmental deficits in language and cognition, (Ing et al., 2012, Backeljauw et al., 2015) a recent cohort study found that an initial anesthesia exposure after age 3 had no effects on language or cognitive function, but did have deleterious effects on motor function (Ing et al., 2014). Results from two recently published clinical studies suggest that single, short anesthetic episodes may be of less concern (Sun et al., 2016, Davidson et al., 2016). Today, parents and caregivers often decide to postpone elective pediatric surgery until the child is older with the hope of avoiding any potential neurotoxic anesthetic effects; however, uncertainty remains as to when this window of vulnerability ends.

Substantial animal research suggests that exposure to anesthetics is neurotoxic to the developing brain causing acute apoptosis of neurons/oligodendrocytes and long-term behavior and learning impairment (Jevtovic-Todorovic et al., 2003, Creeley et al., 2014, Brambrink et al., 2012a, Brambrink et al., 2010). The rodent window of vulnerability for neuroapoptosis coincides with the brain growth spurt, a period that encompasses the first two weeks of life in rodents but extends from midgestation to several years after birth in humans. One investigation found that rats exposed to a combination of midazolam, nitrous oxide, and isoflurane on postnatal day 7 (P7) exhibit increased neuroapoptosis, synaptic dysfunction, and long-term cognitive deficits (Jevtovic-Todorovic et al., 2003). In another study, propofol exposure of P5 and P10 rats significantly decreased pyramidal neuronal spine density, whereas similar exposures induced an increase in spine density when administered to P15, 20, or 30 rats (Briner et al., 2011). However, differences in brain maturation rates between rats and humans make translation of data to the clinical setting challenging. Compared to the rodent, the nonhuman primate (NHP) brain more closely parallels human anatomy and neurodevelopment. In addition, the NHP model allows one to more closely approximate clinical conditions such as intubation, mechanical ventilation, and constant monitoring of vital signs and thus the ability to maintain physiologic homeostasis. In NHPs, a single study has investigated whether the vulnerability to anesthetic neurotoxicity decreases as animals age (Slikker et al., 2007). As part of a larger study, these authors compared the effects of a 24-hour exposure to ketamine on NHPs at P5 or P35 and found that the frontal cortices of the P5 animals showed apoptosis while those of P35 animals did not (Slikker et al., 2007). Of note, brain development in P5 and P35 monkeys approximates that of 5 and 9 months of age, respectively, in human infants (Dobbing and Sands, 1979, Workman et al., 2013).

We have previously shown that 5 hour isoflurane exposure of the P6 neonatal rhesus macaque (Macaca mulatta) triggered apoptotic neurodegeneration, and led to apoptosis of oligodendrocytes (Brambrink et al., 2012a, Brambrink et al., 2010). These histopathology findings are similar to those found following P6 NHP exposure to ketamine or propofol (Brambrink et al., 2012b, Creeley et al., 2013). Not only do these anesthesia exposures cause histopathologic changes, but they have also been found to lead to long-lasting cognitive and behavioral deficits in NHPs (Raper et al., 2015, Paule et al., 2011, Coleman et al., 2016). However, the effects of anesthetics on the brains of older NHPs remain unclear. In order to help further describe the window of vulnerability to the neurotoxic effects of anesthetics, the present study was performed to investigate whether the developing brain of nonhuman primates remains vulnerable on postnatal days 20 (P20) or 40 (P40). Of note, the brain development of the P20 rhesus macaque is equivalent to that of a 7-month-old human infant, whereas the P40 approximates that of a 9.5-month-old infant (Workman et al., 2013). Using the same experimental protocols as in our prior studies in P6 animals, we exposed P20 and P40 NHPs to 5 h of isoflurane anesthesia (Brambrink et al., 2012a, Brambrink et al., 2010).