Impact of inflammation on brain subcellular energetics in anesthetized rats

Abstract

Background Emerging data suggests that volatile anesthetic agents may have organ protection properties in the setting of critical illness. The purpose of this study was to better understand the effect of inflammation on cerebral subcellular energetics in animals exposed to two different anesthetic agents—a GABA agonist (propofol) and a volatile agent (isoflurane). Results Forty-eight Sprague–Dawley rats were anesthetized with isoflurane or propofol. In each group, rats were randomized to celiotomy and closure (sham) or cecal ligation and puncture (inflammation [sepsis model]) for 8 h. Brain tissue oxygen saturation and the oxidation state of cytochrome aa 3 were measured. Brain tissue was extracted using the freeze-blow technique. All rats experienced progressive increases in tissue oxygenation and cytochrome aa 3 reduction over time. Inflammation had no impact on cytochrome aa 3 , but isoflurane caused significant cytochrome aa 3 reduction. During isoflurane (not propofol) anesthesia, inflammation led to an increase in lactate (+ 0.64 vs. − 0.80 mEq/L, p = 0.0061). There were no differences in ADP:ATP ratios between groups. In the isoflurane (not propofol) group, inflammation increased the expression of hypoxia-inducible factor-1α (62%, p = 0.0012), heme oxygenase-1 (67%, p = 0.0011), and inducible nitric oxide synthase (31%, p = 0.023) in the brain. Animals exposed to inflammation and isoflurane (but not propofol) exhibited increased expression of protein carbonyls (9.2 vs. 7.0 nM/mg protein, p = 0.0050) and S -nitrosylation (49%, p = 0.045) in the brain. RNA sequencing identified an increase in heat shock protein 90 and NF-κβ inhibitor mRNA in the inflammation/isoflurane group. Conclusions In the setting of inflammation, rats exposed to isoflurane show increased hypoxia-inducible factor-1α expression despite a lack of hypoxia, increased oxidative stress in the brain, and increased serum lactate, all of which suggest a relative increase in anaerobic metabolism compared to propofol. Differences in oxidative stress as well as heat shock protein 90 and NF-κβ inhibitor may account for the differential expression of cerebral hypoxia-inducible factor-1α during inflammation. Electronic supplementary material The online version of this article (10.1186/s12868-019-0514-8) contains supplementary material, which is available to authorized users.

Anesthesia-Sepsis-Associated Alterations in Liver Gene Expression Profiles and Mitochondrial Oxidative Phosphorylation Complexes

“…As we described previously (23),Arterial blood was placed in BD Vacutainer tubes (Becton Dickson, Franklin Lakes, NJ) after collection, hand-mixed, and stored at room temperature for 45 min, after which it was centrifuged (2,000 g) for 20 min at 4 • C. The serum was removed with a micropipetter and stored at −80 • C for later analysis. Interleukin (IL)-6 (RAB0311, Sigma, St. Louis, MO), Interleukin (IL)-1β (RAB0277, Sigma), and tumor necrosis factor (TNF)-α (ab100785, Abcam, Cambridge, UK) were measured in accordance with the manufacturer's instructions.…”

“…Rat liver tissue (50 mg), were used either for nuclear protein extraction or for whole cell lysate preparation, depending on the protein to be analyzed. As we described previously (23), Nuclear proteins were extracted using a commercially available nuclear extraction kit (Abcam ab113474, Cambridge, MA) in accordance with the manufacturer's protocol, with the exception of adding 100 uM of the prolyl hydroxylase stabilizer CoCl 2 to the extraction buffer (RIPA) to prevent hypoxia-inducible factor-1α (HIF-1α) degradation in the presence of atmospheric oxygen.…”

“…Protein carbonyls levels are widely used to measure protein oxidation. As previously described (23). In brief, 50 mg of Liver tissue powder was suspended in ice cold PBS + 0.5 mM EDTA buffer containing a protease and phosphatase inhibitor cocktail (Halt TM ), followed by sonication and centrifugation at 4 • C. To remove nucleic acids from the supernatant, 10% streptozocin was added and the supernatant was incubated at room temperature.…”

Data_Sheet_1.docx

“…General anesthesia was maintained for 8 h post-celiotomy, after which 2 mL of arterial blood was withdrawn. Bilateral burr holes were placed and the supratentorial brain was ejected onto a liquid N 2 -cooled copper plate by applying a high-pressure air hose to one of the burr holes (freeze blow technique, see brain data published previously) (23). Immediately after death of the animal, the abdominal closure was re-opened, a piece of liver (∼25% by volume) was removed and placed immediately into liquid nitrogen.…”

“…Tissue pooling was performed as previously described (23) and is reported in Supplementary Table 1. In brief, we used a commercial kit for total RNA isolation (RNeasy plus mini kit, Qiagen) and NanoDrop (ThermoFisher Scientific) to estimate the RNA concentrations.…”