Androgenic Modulation of the Chloride Transporter NKCC1 Contributes to Age-dependent Isoflurane Neurotoxicity in Male Rats

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 What This Article Tells Us That Is New

Testosterone attenuates sevoflurane-induced tau phosphorylation and cognitive impairment in neonatal male mice

“…Both human 49 and animal 50 experiments suggest dose-dependent effects of testosterone on cognitive function. Although testosterone may have neuroprotective effects via inhibiting tau phosphorylation (present study), androgen receptor antagonism and gonadectomy may have neuroprotective effects via changes in chloride transporters 46 and being more developmentally similar to females, 47 respectively. Testosterone plus isoflurane could cause cognitive impairment in female neonatal rats via changes in chloride transporters.…”

“…Previous studies showed that an androgen receptor antagonist 46 or gonadectomy 47 attenuated isoflurane-induced behavioural deficiencies in neonatal male rats. Treatment with testosterone plus isoflurane caused cognitive impairment in neonatal female rats.…”

“…The reason for such difference remains unknown. In the aforementioned studies, neonatal rats received isoflurane for 6 h at P7; androgen receptor antagonist flutamide at P2, P4, and P6 46 ; or testosterone at P1eP6. In the present study, neonatal mice received sevoflurane 3% for 2 h with and without testosterone on P6, P7, and P8.…”

Amide Proton Transfer-Weighted Magnetic Resonance Imaging for Detecting Severity and Predicting Outcome after Traumatic Brain Injury in Rats

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“…The higher level of KCC2 and lower level of NKCC1 expression in the cerebral cortex of female rats compared with male rats are important evidence 76 . In another trial using androgen inhibitors, androgen regulation of KCC2 and NKCC1 expression levels was an important factor in the cognitive dysfunction caused by anaesthetic 77 …”