Presented at ASA, October 27, 2015
Emmett E. Whitaker, M.D., Bruno Bissonnette, M.D., Joseph Tobias, M.D., Christopher Pierson, M.D.,Ph.D., Tanner Koppert, B.S., Fievos L Christofi, Ph.D.
Nationwide Children’s Hospital, Columbus, Ohio , United States
Piglets are recognized alternative to large animal research for neuroscience. Their gyrencephalic brains have striking similarities with human newborn brain structure and function. Although studies have examined apoptosis after anesthesia, none reported effects of anesthetics on neuroinflammation, an arguably more important process when considering lasting neurodegeneration that leads to severe neurocognitive deficits. CNS inflammatory response in children undergoing anesthesia has become our primary focus in an attempt to elucidate the true consequence on the brain in development.
35 neonatal piglets (10-14 days old, 2-3 kg) are studied. Animals are divided into three groups: 1) control, 2) LPS and 3) Isoflurane 2%. Sevoflurane 8% in 100% O2 via face cone is used to induce anesthesia followed by isoflurane 2% in 50% O2/50% air for 3 hours. An IV catheter is placed, the trachea intubated and lungs mechanically ventilated to ensure normocapnia and normoxia. A femoral artery catheter assures continuous blood pressure monitoring and allows for blood sampling. End-tidal carbon dioxide, rectal temperature, and arterial blood pressure are monitored continuously throughout the study. Arterial blood gases are measured hourly during anesthesia. At the end, vascular catheters are removed and the surgical incision is closed and infiltrated with bupivacaine. Piglets are awakened and the trachea extubated. Animals are returned to their temperature-controlled home cage for 72h. They are monitored every hour for the first 6 hours and every 4 hours thereafter. Piglets receive a nutritionally complete commercial piglet milk replacer. After 72 hours the piglets receive 8% sevoflurane in 100% O2. Peripheral blood is collected via venipuncture and cerebrospinal fluid (CSF) obtained via cisterna magna puncture. The CSF and blood are frozen and stored at -80oC within 30 min. A transaortic perfusion of heparinized phosphate buffered saline (PBS) and 4% paraformaldehyde (PFA) is given. Brains are harvested. Untreated animals serve as controls. The positive control group receives an E. coli endotoxin, lipopolysaccharide (LPS) intraperitoneally at a dose known to induce an acute phase response. Tissue in these animals is harvested as described above.
Brains from 29 animals were analyzed. One animal in the LPS group died due to an inadvertent intravascular injection. All animals were successfully anesthetized and survived until sacrifice. 4 micron sections were mounted on slides and stained with hematoxylin and eosin (H&E) (Figure 1). Microscopic brain examination showed: 1) significant morphological and structural changes in the entorhinal cortex and CA1 hippocampus in the isoflurane and LPS treated animals when compared to controls (Figure 1), and 2) an acute ischemic infarct in one of three isoflurane-treated animals not present in the either groups. Control brains showed clusters of small, round cells in the neuronal layers most likely immature microglial cells, astrocytes, or oligodendrocytes in development. They were virtually obliterated in every isoflurane- and LPS-treated animal.
We hypothesize that clusters of cells may be dying as a result of neuroinflammation, ischemia, or both when animals are treated with isoflurane or LPS. Identifying their implications and the mechanisms involved in their loss will be essential to determine the significance of this observation.