A novel, clinically relevant use of a piglet model to study the effects of anesthetics on the developing brain

Abstract

Background: Anesthesia-induced neurotoxicity research in the developing brain must rely upon an unimpeachable animal model and a standardized treatment approach. In this manner, identification of mechanisms of action may be undertaken. The goal of this study was to develop a novel, clinically relevant, translational way to use a piglet model to investigate anesthesia effects on the developing brain.

Methods: 29 newborn piglets were assigned to either: (1) control (no intervention, n = 10); (2) lipopolysaccharide (LPS; positive inflammatory control, n = 9); or (3) isoflurane anesthesia (n = 10). Positive inflammatory control animals were given 100 mcg/kg LPS from Escherichia coli intraperitoneally (IP) on the same day as those receiving isoflurane. Isoflurane was administered for 3 h while care was taken to ensure human perioperative conditions. To establish a clinical scenario, each animal was intubated and monitored with pulse oximetry, invasive and non-invasive blood pressure, electrocardiogram, temperature, end-tidal CO2, anesthetic concentration, and iSTAT blood analysis. All animals were sacrificed after 48 h using transcardiac perfusion of ice-cold, heparinized phosphate buffered saline (PBS) followed by 4 % paraformaldehyde (PFA). Brains were collected and histopathological analysis focused on the entorhinal cortex looking for degenerative changes due to its critical role in learning and memory. Reliable identification of entorhinal cortex was achieved by using colored ink on the surface of the brains, which was then cross-referenced with microscopic anatomy. Hematoxylin & eosin-stained high-power fields was used to quantify cells. ImageJ™ (National Institutes of Health, Bethesda, MD, USA) was used to count absolute number of progenitor glial cells (PGC) and number of PGCs per cluster. Immunohistochemistry was also utilized to ensure positive identification of cellular structures.

Results: Histopathological sections of 28 brains were analyzed. One animal in the LPS group died shortly after administration, presumably from inadvertent intravascular injection. There was an acute basal ganglia ischemic infarct in one isoflurane-treated animal. A large number of small, round nucleated cells were seen throughout layer II of the entorhinal cortex in all animals. These cells were identified as PGCs using immunohistochemistry and light microscopy. Although there was no difference in the absolute number of PGCs between the groups, animals given isoflurane or LPS demonstrated a significant increase in cells forming 'clusters' in the entorhinal cortex. An apparent change in the pattern of doublecortin labeling also suggests changes in neuronal precursors and undifferentiated neurons.

Conclusions: This study represents the first novel use of a clinically relevant neonatal piglet model to study anesthesia effects on the developing brain. LPS induces neuroinflammation, and this is a potential mechanism for LPS and perhaps isoflurane in causing a change in progenitor cell distribution. We postulate that the isoflurane-induced change in glial progenitor cell distribution could have important implications for cell differentiation, maturation and neural circuit behavior in the rapidly developing brain.

Keywords: Anesthesia; Hippocampus; Isoflurane; Neurocognitive outcome; Neurodevelopment; Neuroinflammation; Neurotoxicity; Piglets.

Fig. 1

One piglet being administered an inhalational induction of anesthesia with 8 % sevoflurane in 100 % oxygen via face cone mask

Fig. 2

The gyrencephalic brain of the neonatal piglet. Panel a shows the left hemisphere without surface dye. Panel b illustrates surface dyes used to identify anatomical structures and guide microscopic evaluation. Structures are labeled based on conclusions drawn from cross-referencing dyed surface anatomy with known microscopic features of the entorhinal cortex

Fig. 3

Microscopic anatomical brain tissue identification. Using dyed brain surface landmarks, the rhinal sulcus can be located at the junction of the blue and black ink (panels a and b). This information is used to assist in the identification of the entorhinal cortex, our primary area of interest

Fig. 4

Entorhinal Cortex. Microscopic demonstration of brain tissue in a control animal at a magnification of ×100. The red arrows indicate the normal distribution of progenitor glial cells

Fig. 5

Doublecortin staining of immature neurons in entorhinal cortex. A significant increase in doublecortin positive cells is seen in isoflurane-treated animals when compared with control

Fig. 6

Quantification of PGCs in entorhinal cortex. Box- and -whisker plot displaying total PGCs in entorhinal cortex for all three groups. There was no difference in number of total PGCs when the isoflurane and LPS groups were compared to control

Fig. 7

Clustering of PGCs. Microscopic view of layer II of the entorhinal cortex at ×100 magnification. The clusters of PGCs can be observed (red arrows)

Fig. 8

Quantification of PGCs in Clusters within the entorhinal cortex. There was a significant increase in the number of PGCs seen in clusters in both the isoflurane and LPS groups when compared to controls

Fig. 9

Basal ganglia infarct in an animal administered isoflurane. This composite of four panels shows a control animal (panels a and b) and an ischemic insult in an animal who was administered isoflurane (panels c and d). Panel c is characterized by the “shaggy” appearance of the brain tissue damage and hypereosiniphilic neurons (black arrows; panel d). Note the pericellular edema (white halos). This piglet suffered no periods of hypoxia or hypotension and electrolytes were normal at all times. It is critical to identify if this is an anesthetic effect