Surgery increases cell death and induces changes in gene expression compared with anesthesia alone in the developing piglet brain

Abstract

In a range of animal species, exposure of the brain to general anaesthesia without surgery during early infancy may adversely affect its neural and cognitive development. The mechanisms mediating this are complex but include an increase in brain cell death. In humans, attempts to link adverse cognitive development to infantile anaesthesia exposure have yielded ambiguous results. One caveat that may influence the interpretation of human studies is that infants are not exposed to general anaesthesia without surgery, raising the possibility that surgery itself, may contribute to adverse cognitive development. Using piglets, we investigated whether a minor surgical procedure increases cell death and disrupts neuro-developmental and cognitively salient gene transcription in the neonatal brain. We randomly assigned neonatal male piglets to a group who received 6h of 2% isoflurane anaesthesia or a group who received an identical anaesthesia plus 15 mins of surgery designed to replicate an inguinal hernia repair. Compared to anesthesia alone, surgery-induced significant increases in cell death in eight areas of the brain. Using RNAseq data derived from all 12 piglets per group we also identified significant changes in the expression of 181 gene transcripts induced by surgery in the cingulate cortex, pathway analysis of these changes suggests that surgery influences the thrombin, aldosterone, axonal guidance, B cell, ERK-5, eNOS and GABAA signalling pathways. This suggests a number of novel mechanisms by which surgery may influence neural and cognitive development independently or synergistically with the effects of anaesthesia.

Fig 1. Representative TUNEL sections.

At x20 magnification from the anesthesia group (A) and anesthesia + surgery group (B).

Fig 2. Quantification of TUNEL histology.

There was an overall increase in the estimated mean TUNEL-positive cells per mm² (pooled across regions and R0/ R1 levels) in the anesthesia + surgery group versus anesthesia alone. On regional assessment there was a significant increase in TUNEL positive cells in the anesthesia + surgery group versus anesthesia alone in the cingulate cortex (cing), motor cortex (mCtx), somatosensory cortex (sCtx), pyriform cortex (pyr), internal capsule (ic), caudate (caud) and thalamus (thal). No significant increases were observed in the insula cortex, periventricular white matter (pvwm) or putamen (put).

Fig 3. Pathway analysis illustrating the top ten canonical pathways affected by 15 mins of surgery.

A positive z-score suggests an increase in pathway activity, the darker the orange colour the higher the increase in pathway activity. Our data suggests that surgery increases signalling in the thrombin, aldosterone, B-cell, ERK-5, eNOS, iCOS-iCOSL signalling in T-helper cells and growth hormone signalling pathways. A grey colour indicates that axon guidance signalling and breast cancer signalling by stathmin-1 is perturbed by surgery but its direction is not-apparent. Values are derived by the change in relative expression compared with the control group (fold change). This data suggests that surgery affects endothelial function, neural plasticity and inflammatory processes.