Connexin 43 gap junction-mediated astrocytic network reconstruction attenuates isoflurane-induced cognitive dysfunction in mice

Abstract

Background Postoperative cognitive dysfunction (POCD) is a common complication following anesthesia and surgery. General anesthetic isoflurane has potential neurotoxicity and induces cognitive impairments, but the exact mechanism remains unclear. Astrocytes form interconnected networks in the adult brain through gap junctions (GJs), which primarily comprise connexin 43 (Cx43), and play important roles in brain homeostasis and functions such as memory. However, the role of the GJ-Cx43-mediated astrocytic network in isoflurane-induced cognitive dysfunction has not been defined. Methods 4-month-old male C57BL/6 mice were exposure to long-term isoflurane to induce cognitive impairment. To simulate an in vitro isoflurane-induced cognitive dysfunction‐like condition, primary mouse astrocytes were subjected to long-term isoflurane exposure. Cognitive function was assessed by Y-maze and fear conditioning tests. Western blot was used to determine the expression levels of different functional configurations of Cx43. The morphology of the GJs-Cx43 was evaluated by immunofluorescence staining. Levels of IL-1β and IL-6 were examined by ELISA. The ability of GJs-Cx43-mediated intercellular communication was examined by lucifer yellow dye transfer assay. Ethidium bromide uptake assays were used to measure the activity of Cx43 hemichannels. The ultrastructural morphology of astrocyte gap junctions and tripartite synapse were observed by transmission electron microscopy. Results After long-term isoflurane anesthesia, the GJs formed by Cx43 in the mouse hippocampus and primary mouse astrocytes were significantly reduced, GJs function was impaired, hemichannel activity was enhanced, the levels of IL-1β and IL-6 were increased, and mice showed significant cognitive impairment. After treatment with the novel GJ-Cx43 enhancer ZP1609, GJ-Cx43-mediated astrocytic network function was enhanced, neuroinflammation was alleviated, and ameliorated cognition dysfunction induced by long-term isoflurane exposure. However, ZP1609 enhances the astrocytic network by promoting Cx43 to form GJs without affecting hemichannel activity. Additionally, our data showed that long-term isoflurane exposure does not alter the structure of tripartite synapse. Conclusion Our results reveal a novel mechanism of the GJ-Cx43-mediated astrocytic network involved in isoflurane-induced neuroinflammation and cognitive impairments, which provides new mechanistic insight into the pathogenesis of POCD and identifies potential targets for its treatment.

Enhancement of astrocytic gap junctions Connexin43 coupling can improve long‐term isoflurane anesthesia–mediated brain network abnormalities and cognitive impairment

“…The mice were given three intraperitoneal Danegaptide injections (300 μg/kg) per day for three consecutive days according to the previous study. 11 There was no significant difference among the three…”

“…The mice were given three intraperitoneal Danegaptide injections (300 μg/kg) per day for three consecutive days according to the previous study 11 . There was no significant difference among the three groups in the freezing time during the training phase of the fear conditioning tests (Figure S2A, F [2,21] = 0.386, p = 0.682).…”

“…Hippocampal tissues and primary astrocytes were collected and the total protein was extracted using RIPA Lysis Buffer (P0013B, Beyotime, China) on ice. The extraction method of Cx43 forming GJ configuration is based on the ability of GJs‐Cx43 to be insoluble in 1% Triton X‐100 11,22 . In brief, hippocampal tissues and primary astrocytes were collected and homogenized on ice with a homogenizer in immunoprecipitation buffer containing 1% Triton X‐100, incubated on ice for 30 min, and centrifuged at 12,000 rpm (revolutions per minute) for 30 min at 4°C and the supernatant (soluble fractions of Cx43) was collected.…”

“…Subsequently, the homogenate was centrifuged and supernatants (insoluble fractions of Cx43) were collected. The increase of insoluble fraction indicates that Cx43 forming GJs is increased 11 …”

“…The process by which one GJ breaks and forms two Cx43 molecules is called GJs‐Cx43 decoupling. Our previous studies showed that long‐term isoflurane exposure causes GJs‐Cx43 decoupling, resulting in cognitive dysfunction 11 . However, the effects of GJs‐Cx43 decoupling on brain functional networks and structure remain unclear.…”