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

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.

Female rats are more vulnerable to lasting cognitive impairment after isoflurane exposure on postnatal day 4 than 7.

BACKGROUND: The factors determining peak susceptibility of the developing brain to anaesthetics are unclear. It is unknown why postnatal day 7 (P7) male rats are more vulnerable to anaesthesia-induced memory deficits than littermate females. Given the precocious development of certain regions in the female brain during the neonatal critical period, we hypothesised that females are susceptible to anaesthetic brain injury at an earlier time point than previously tested. METHODS: Female rats were exposed to isoflurane (Iso) 1 minimum alveolar concentration or sham anaesthesia at P4 or P7. Starting at P35, rats underwent a series of behavioural tasks to test their spatial and recognition memory. Cell death immediately after anaesthesia was quantified by Fluoro-Jade C staining in select brain regions, and developmental expression of the chloride transporters KCC2 and NKCC1 was analysed by immunoblotting in male and female rats at P4 and P7. RESULTS: Female rats exposed to Iso at P4 displayed impaired spatial, object-place, -context, and social recognition memory, and increased cell death in the hippocampus and laterodorsal thalamus. Female rats exposed at P7 exhibited only decreased performance in object-context compared with control. The ratio of NKCC1/KCC2 expression in cerebral cortex was higher in P4 females than in P7 females, and similar to that in P7 males. CONCLUSIONS: Female rats exposed to Iso at P4 are sensitive to anaesthetic injury historically observed in P7 males. This is consistent with a comparably immature developmental state in P4 females and P7 males. The window of anaesthetic vulnerability correlates with sex-specific cortical expression of chloride transporters NKCC1 and KCC2. These findings suggest that both sex and developmental age play important roles in determining the outcome after early anaesthesia exposure.