Newly Published
Perioperative Medicine  |   June 2020
Androgenic Modulation of the Chloride Transporter NKCC1 Contributes to Age-dependent Isoflurane Neurotoxicity in Male Rats
Author Notes
  • From the Department of Anesthesia and Perioperative Care, University of California, San Francisco, California.
  • Submitted for publication February 1, 2020. Accepted for publication May 27, 2020.
    Submitted for publication February 1, 2020. Accepted for publication May 27, 2020.×
  • Supplemental Digital Content is available for this article. Direct URL citations appear in the printed text and are available in both the HTML and PDF versions of this article. Links to the digital files are provided in the HTML text of this article on the Journal’s Web site (www.anesthesiology.org).
    Supplemental Digital Content is available for this article. Direct URL citations appear in the printed text and are available in both the HTML and PDF versions of this article. Links to the digital files are provided in the HTML text of this article on the Journal’s Web site (www.anesthesiology.org).×
  • Part of the work presented in this article has been presented at the Society for Neuroscience in Anesthesiology and Critical Care symposium in Montreal, Canada, May 19, 2019, and at the University of California, San Francisco Anesthesia Research Day in San Francisco, California, November 29, 2018. An abstract with data from this article was accepted to the 2020 International Anesthesia Research Society meeting, but was not presented because the meeting was canceled due to the COVID-19 pandemic.
    Part of the work presented in this article has been presented at the Society for Neuroscience in Anesthesiology and Critical Care symposium in Montreal, Canada, May 19, 2019, and at the University of California, San Francisco Anesthesia Research Day in San Francisco, California, November 29, 2018. An abstract with data from this article was accepted to the 2020 International Anesthesia Research Society meeting, but was not presented because the meeting was canceled due to the COVID-19 pandemic.×
  • Correspondence: Address correspondence to Dr. Chinn: University of California, San Francisco, 1001 Potrero Avenue, 3C39, San Francisco, California 94110. Gregory.Chinn@ucsf.edu. Anesthesiology’s articles are made freely accessible to all readers on www.anesthesiology.org, for personal use only, 6 months from the cover date of the issue.
Article Information
Perioperative Medicine / Pharmacology
Perioperative Medicine   |   June 2020
Androgenic Modulation of the Chloride Transporter NKCC1 Contributes to Age-dependent Isoflurane Neurotoxicity in Male Rats
Anesthesiology Newly Published on June 25, 2020. doi:https://doi.org/10.1097/ALN.0000000000003437
Anesthesiology Newly Published on June 25, 2020. doi:https://doi.org/10.1097/ALN.0000000000003437
Abstract

Background: Cognitive deficits after perinatal anesthetic exposure are well established outcomes in animal models. This vulnerability is sex-dependent and associated with expression levels of the chloride transporters NKCC1 and KCC2. The hypothesis was that androgen signaling, NKCC1 function, and the age of isoflurane exposure are critical for the manifestation of anesthetic neurotoxicity in male rats.

Methods: Flutamide, an androgen receptor antagonist, was administered to male rats on postnatal days 2, 4, and 6 before 6 h of isoflurane on postnatal day 7 (ntotal = 26). Spatial and recognition memory were subsequently tested in adulthood. NKCC1 and KCC2 protein levels were measured from cortical lysates by Western blot on postnatal day 7 (ntotal = 20). Bumetanide, an NKCC1 antagonist, was injected immediately before isoflurane exposure (postnatal day 7) to study the effect of NKCC1 inhibition (ntotal = 48). To determine whether male rats remain vulnerable to anesthetic neurotoxicity as juveniles, postnatal day 14 animals were exposed to isoflurane and assessed as adults (ntotal = 30).

Results: Flutamide-treated male rats exposed to isoflurane successfully navigated the spatial (Barnes maze probe trial F[1, 151] = 78; P < 0.001; mean goal exploration ± SD, 6.4 ± 3.9 s) and recognition memory tasks (mean discrimination index ± SD, 0.09 ± 0.14; P = 0.003), unlike isoflurane-exposed controls. Flutamide changed expression patterns of NKCC1 (mean density ± SD: control, 1.49 ± 0.69; flutamide, 0.47 ± 0.11; P < 0.001) and KCC2 (median density [25th percentile, 75th percentile]: control, 0.23 [0.13, 0.49]; flutamide, 1.47 [1.18,1.62]; P < 0.001). Inhibiting NKCC1 with bumetanide was protective for spatial memory (probe trial F[1, 162] = 6.6; P = 0.011; mean goal time, 4.6 [7.4] s). Delaying isoflurane exposure until postnatal day 14 in males preserved spatial memory (probe trial F[1, 140] = 28; P < 0.001; mean goal time, 6.1 [7.0] s).

Conclusions: Vulnerability to isoflurane neurotoxicity is abolished by blocking the androgen receptor, disrupting the function of NKCC1, or delaying the time of exposure to at least 2 weeks of age in male rats. These results support a dynamic role for androgens and chloride transporter proteins in perinatal anesthetic neurotoxicity.

Editor’s Perspective:

What We Already Know about This Topic:

  • Experimental data in laboratory animals suggest sex-dependent differences in neurocognitive and behavioral vulnerability to early life anesthesia exposure

  • Steroid sex hormones play an important role in guiding sex-specific brain development

  • The relationship between steroid sex hormones and developmental anesthesia neurotoxicity is incompletely understood

What This Article Tells Us That Is New:

  • Blockade of androgen receptors in 7-day-old male rats protects against isoflurane anesthesia-induced behavioral deficits

  • Androgen receptor blockade results in a premature transition in the developmental expression profiles of chloride transporters NKCC1 and KCC2

  • These observations suggest that regulation of specific chloride transporters, determining functional modalities of γ-aminobutyric acid–mediated neurotransmission, by androgens is a critical component for developmental anesthetic neurotoxicity