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 CO 2 , 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 neuroinflammatio...

Effects of moderate and severe hypocapnia on intracerebral perfusion and brain tissue oxygenation in piglets

“…However, hypocapnia (2.7-3.3 kPa) and hypotension(27)(28)(29)(30)(31)(32)(33) mm Hg) goals were lower than those of the present trial. Also, a study investigating the effects of severe hypocapnia (3.3 kPa) combined with mild hypotension (50 mm Hg MAP) on PtO Mean and standard deviation values for brain tissue oxygen partial pressure (PtO 2 ), regional cerebral oxygen saturation (rSO 2 ), laser Doppler flow (normalized flow = divided by the baseline value), and heart rate (HR) of 29 piglets anesthetized with sevofluranemidazolam and divided into three groups: mHC (mild hypocapnia; PaCO 2 = 3.7-4 kPa, n = 10), sHC (severe hypocapnia; PaCO 2 = 3.1-3.3 kPa, n = 10) and HCT (combined hypocapnia and hypotension; PaCO 2 = 3.7-4 kPa and MAP: 35-38 mm Hg, n = 9).…”

“…Although a major limitation of the study is the use of a piglet instead of an infant model, it would be unethical to subject children to hypocapnia and hypotension for research purposes. Piglets are, however, proposed to be an ideal model to investigate anesthesia‐associated brain complications . Regarding brain growth, 4‐ to 6‐week‐old piglets correspond to 1‐ to 2‐year‐old children .…”

“…Piglets are, however, proposed to be an ideal model to investigate anesthesia-associated brain complications. 27 Regarding brain growth, 4-to 6-week-old piglets correspond to 1-to 2-year-old children. 28 No control group was included in the present experiment; this was decided in order to reduce the number of animals (3R principle), as control animals had shown stable PtO 2 , rSO 2, and HR over time in a previous study with a similar setup.…”

Adaptation of Microelectrode Array Technology for the Study of Anesthesia-induced Neurotoxicity in the Intact Piglet Brain

“…From the start of the experiment, the piglet's physiological homeostasis must be maintained as described in this lab's prior publication 21 . Minimal monitoring should include pulse oximetry, electrocardiography, capnography, non-invasive blood pressure, and temperature.…”

“…Perform the perfusion and brain tissue collection procedures as previously described 21 . For non-survival surgeries, animals are euthanized immediately after experimentation while still under general anesthesia.…”