Ifenprodil

NR2B receptor- and calpain-mediated KCC2 cleavage resulted in cognitive deficiency exposure to isoflurane

Xiaole Tang MD, Xue Zhang MD, Shiyong Li MD, PhD, Xiaohui Chi MD, PhD, PhD, Ailin Luo MD, PhD, Yilin Zhao MD, PhD

Highligts

• A significant increase in NR2B, excessive activation of calpain-2 and increased cleavage of plasmalemmal KCC2, are involved in isoflurane-induced neurotoxicity and long-term cognitive deficiency.
• Blocking NR2B and calpain-2 activity significantly attenuated these responses.
• The KCC2 cleavage mediated by NR2B and calpain-2 is a major determinant of isoflurane-induced long-term cognitive deficiency.

Abstract

Background During brain development, volatile anesthetic can rapidly interfere with physiologic patterns of dendritic development and synaptogenesis and impair the formation of precise neuronal circuits. KCC2 plays vital roles in spine development and synaptogenesis through its Cl- transport function and structural interactions with the spine cytoskeleton protein 4.1N. The aim of this study was to dissect the mechanism of volatile anesthetics, which impair dendritic development and synaptogenesis via mediation of KCC2 cleavage.
Methods Westernblotting was employed to assess the expression change of NR2B, NR2A, calpain-1, calpain-2, KCC2, and 4.1N protein of rat(PND 5). Co-immunoprecipitation was applied to demonstrate the interaction between KCC2 and 4.1N protein. Long-term cognitive deficiency was assessed by MWM. Lentivirus-calpain-2 was administered by hippocampus stereotaxic injection.
Results There was a significant increase in the level of NR2B instead of NR2A exposure to isoflurane. Calpain-2 was excessively activated via NR2B after 6h of isoflurane exposure. The expression of plasmalemmal KCC2 and 4.1N protein was significantly decreased treated with isoflurane. The isoflurane group showed longer traveled distance, prolonged escape latency, less time spent in the target quadrant, and decreased platform crossings. Pretreatment with ifenprodil and downregulated calpain-2 expression significantly alleviated these neurotoxicity responses and cognitive deficiency after isoflurane exposure.
Conclusions A significant increase in NR2B, excessive activation of calpain-2 and increased cleavage of plasmalemmal KCC2, are involved in isoflurane-induced neurotoxicity and long-term cognitive deficiency. Blocking NR2B and calpain-2 activity significantly attenuated these responses. The KCC2 cleavage mediated by NR2B and calpain-2 is a major determinant of isoflurane-induced long-term cognitive deficiency.

Introduction

During brain development, dendritic development and synaptogenesis are vulnerable to anesthetic exposure, which could result in impairment of neuronal circuits and long-term cognitive function(Briner et al. , 2010, Briner et al. , 2011, Milanovic et al. , 2017). Recently, the Mayo Anesthesia Safety in Kids (MASK) study has proven that multiple, but not single, exposures are associated with a pattern of changes in behavioral and learning difficulties(Warner et al. , 2018). Our preclinical studies have shown long-time or repeated isoflurane exposure in early life could result in calcium overload, activation of caspase-3, mitochondria dysfunction, and ultimately lead to neurotoxicity and long-term cognitive deficiency(Zhao et al. , 2011a, Zhao et al. , 2011b). However, the mechanism of how volatile anesthetics impaired the dendritic development and synaptogenesis remained fragmentary.
N-methyl-D-aspartate receptors (NMDARs), a major class of glutamate receptors, are mainly heteromers composed of NR1, NR2A, and NR2B subunits in the central nervous system (CNS)(Cull-Candy and Leszkiewicz, 2004, Furukawa et al. , 2005). However, neurons in the developing brain exhibit some characteristics different from those of adult neurons. One of the most prominent features of the immature brain is a developmental switch in the subunit composition of NMDA receptors from NR2B to NR2A(Gambrill and Barria, 2011, Sanz-Clemente et al. , 2010).
During early postnatal development, an increase in the NR2A/NR2B ratio plays vital roles in dendritic development, synaptogenesis, and synaptic plasticity(Liu et al. , 2004, Paoletti et al. , 2013). Our previous study proved that isoflurane enhances the current of NMDA receptor in developing rat hippocampal neurons(Zhao, Xiang, 2011b). However, it remains unclear whether the developmental switch from NR2B to NR2A NMDA receptor subunits is involved in isoflurane-increased activity of

NMDARs during the first weeks of postnatal brain development.

Calpain, as a Ca2+-activated neutral cellular cysteine protease, could be activated by NMDARs through Ca2+ influx(Baudry and Bi, 2016). Calpain has been found to be highly expressed in both the cytosol and the synaptic terminal of neurons(Mellgren et al. , 1989, Melloni and Pontremoli, 1989, Tomimatsu et al. , 2002). Calpain-1 and calpain-2 are the major calpain isoforms in the central nervous system (CNS)(Baudry and Bi, 2016). The activation of calpain-1 is required for micromolar calcium concentrations, induces long-term potentiation (LTP), and is generally neuroprotective, while calpain-2 activation requires millimolar calcium concentrations, limits the extent of potentiation and is neurodegenerative(Baudry and Bi, 2016, Stracher, 1999, Vosler et al. , 2008). During postnatal brain development, calpain is not only evident in synaptic transmission and plasticity but also in the neuronal apoptosis involved in neurodegenerative diseases induced by volatile anesthetic(Chiu et al. , 2005). Previous studies have proved that the interaction between NMDAR and calpain participates in dendritic development, synaptogenesis, and synaptic plasticity(Chiu, Lam, 2005, Dong et al. , 2004, Wu et al. , 2005). However, the specific signaling mechanisms involved in neurodegenerative diseases induced by volatile anesthetics remain unclear. K+-Cl- cotransporter (KCC2) has been found to be a key molecule in the regulation of the equilibrium between excitation and inhibition in the CNS(Blaesse and Schmidt, 2015). KCC2 extrudes Cl- from neurons to maintain intracellular Cl- homeostasis(Blaesse and Schmidt, 2015). However, KCC2 also acts as an extremely important structural protein via orchestrating the organization of the cytoskeleton. In the immature brain, KCC2 plays a crucial role in dendritic spine formation. In KCC2-deficient neurons, the morphology differs, and the number of normal dendritic spines decreases compared to wild-type neurons. Strikingly, the structural deficits in KCC2-deficient neurons can be rescued by the upregulation of a KCC2 N-terminal deletion(Blaesse and Schmidt, 2015, Li et al. , 2007). The interaction of KCC2 with the cytoskeleton-associated protein 4.1N has been reported to play pivotal roles in spine morphogenesis and structural plasticity during the development of synapses(Blaesse and Schmidt, 2015, Li, Khirug, 2007). However, the specific cellular signaling mechanisms involved in volatile anesthetic-induced synaptogenesis and synaptic plasticity remain unclear.
The current study aimed to investigate the effect of NMDAR, calpain, and KCC2 signaling pathway at the cellular by westblotting, co-immunoprecipitation and lentivirus-calpain2 microinjected and system levels by MWM on the mediation of isoflurane-induced synaptic plasticity and long-term cognitive deficiency. We proposed the hypothesis that isoflurane exposure increases NMDARs and induces calcium influx, then activates calpain activation and subsequently mediates cleavage of KCC2, which in turn results in synaptic formation and cognitive deficiency in neonatal rats. The study first revealed the role of the NRDAR/calpain-2/KCC2 signaling pathway in isoflurane-induced neurotoxicity, which regulates isoflurane-related synaptic plasticity and behavior in the immature brain.

Materials and Methods Animals

Enceinte Sprague-Dawley (SD) rats (n=25) with litters containing pups (n=232, the numbers of animals per category in supplemental data table 1) were purchased from the Laboratory Animal Center of Tongji Medical College (Wuhan, China). The rats were housed and bred in the animal facilities under a 12-hour light and 12-hour dark cycle and temperature-controlled (22° ± 2°C) conditions(Zhang et al. , 2018). Ad libitum access to food and water was provided. Five-day-old pups used in this study were randomly divided into control group (CON, the percent of animals surviving: 94%), isoflurane group (ISO, the percent of animals surviving: 87.1%), ifenprodil group (IFEN, the percent of animals surviving: 88.9%), ifenprodil+ isoflurane group (IFEN+ISO, the percent of animals surviving: 82.8%), downregulation lentivirus+ isoflurane group (LV+ISO the percent of animals surviving: 82.8%), control lentivirus+ isoflurane group (CV+ISO, the percent of animals surviving: 85.7%). All animal protocols were approved by the experimental animal committee of Tongji Medical College. The maintenance and handling of rats was performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals.

Isoflurane exposure

Five days after birth, pups were placed in a temperature-controlled chamber before isoflurane or control gas exposure as we described previously(Fang et al. , 2017, Wang et al. , 2016). Pups in the control group received 60% oxygen (balanced with air) in a chamber for 6 h, while pups subjected to isoflurane exposures were placed in a similar anesthesia chamber and exposed to 1.8% isoflurane (1.8%, approximate 1 MAC for P5 rats)(Fang, Li, 2017, Orliaguet et al. , 2001) flushed with 60% oxygen (balanced with air) for 6 h. The chambers whose sizes were 20 x 20 x10 cm were kept in a homoeothermic incubator to maintain the experimental temperature at 37°C. After isoflurane treatment (n=4 per group), a single blood sample (100 ul) was collected by cardiac puncture from each to determine arterial blood gas with a blood gas analyzer (Kent Scientific Corp., Torrington, CT, USA). The rats subjected to Morris water maze (MWM) test were weaned at 22 days of age, and then kept under standard lab housing with a 12 h light/dark cycle.

Experimental protocol

Three experiments were conducted as shown in supplemental data figure1. In experiment one, P5 rats were allocated to exposure to isoflurane or air for 6 h randomly. At 6, 12, and 24 h after the termination of anesthesia, rats were killed by decapitation, and some of their hippocampi were used to collect total protein to measure the expression of NR2A, NR2B, calpain-1, calpain-2, and 4.1N protein; hippocampi of the other rats were used for subcellular fractionation to detect the expression of plasmalemmal KCC2 by Western blotting.
In experiment two, 1 mg/kg of ifenprodil (MCE, Wuhan, China), a potent NR2B inhibitor(Zhao et al. , 2012), or vehicle (saline solution), in 100 μl was administered by intraperitoneal injection 30 min before the exposure to isoflurane(Ebert et al. , 1997, Maroso et al. , 2010). The regimen of ifenprodil was selected based on our previous studies(Tan et al. , 2017). Some rats were sacrificed, and their hippocampi were used for Western blot studies 6, 12, and 24 h after anesthesia, as described in experiment one. The other rats underwent the MWM test at P31, and their hippocampi were collected after completing the behavior test.
In experiment three, rats received hippocampus stereotaxic injection of the calpain-2 downregulation lentivirus or control lentivirus at P3. The methods of injection were described in the section on “hippocampus stereotaxic injection”. Samples were collected in the same way as in experiment two.

Brain tissue and neuron harvest and protein level quantification

Six, 12, and 24 h post-isoflurane or -control gas exposure, the brain hippocampus was harvested. For the Western blot analysis, the harvest was homogenized at 4 ℃ for 30 min in radioimmunoprecipitation assay (RIPA) lysis buffer containing 150 mM of sodium chloride, Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM of Tris, pH 8.0, and 1 mM of phenylmethylsulfonyl fluoride (PMSF). The lysates were collected, centrifuged at 13,400 x g for 10 min, and quantified for total proteins with BCA assay kit (Boster, Wuhan, China).

Extraction of membrane and cytosol protein

A Membrane and Cytosol Protein Extraction Kit (Promoter, Wuhan, China) was applied to extract plasmalemmal KCC2 protein following the instruction provided by the manufacturer(Zhou et al. , 2012). In brief, the harvested tissues were homogenized at 4°C for 30 min in lysis buffer including 1 mM of PMSF, 1 mM of Protease and Phosphatase Inhibitor Cocktail(P1010, Sigma), and 1 mM of dithiothreitol (DTT). The dounce homogenate was centrifuged at 18.200 x g for 30 min. The resulting supernatant was cytosol protein, while the pellet was resuspended with extraction buffer, incubated at 4°C for 15 min with gentle rotation, and then centrifuged at 9.300 x g for 5 min. The plasmalemmal protein was included in the supernatant.

Co-immunoprecipitation

The hippocampi were dissected from neonatal rats and then homogenized in RIPA buffer with protease inhibitors, and the protein concentration was adjusted to 2 mg/ml. Samples (500 μl) were precleared with nonspecific rabbit IgG (Sigma) followed by adsorption on protein 4.1N (Santa Cruz). Immunoprecipitation was performed overnight at 4°C using 5 μg of rabbit anti-KCC2 (Millipore) antibodies, followed by adsorption to protein 4.1N. In control experiments, the antibodies were replaced with nonspecific rabbit IgG (Sigma). For western blotting, immunoprecipitates were loaded in 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels and then transferred onto polyvinylidene difluoride (PVDF) membranes. The blots were incubated with the specific primary antibodies and then with peroxidase-conjugated species-matched secondary antibodies. The experiments were repeated in triplicate.

Western blot analysis

Equal amount of extracts (40 ug) were separated by 8% SDS–PAGE and transferred to PVDF membrane (Millipore, Bedford, USA) by electrophoresis. After blocked by 5% nonfat skim milk or BSA in TBST (0.1% Tween 20 in TBS) for 1.5 h at room temperature, the membranes were incubated overnight at 4℃with anti-β-actin antibody (1:500; Boster, China), anti-Na+-K+-ATPase antibody (1:2,000; Proteintech, China) anti-NR2A antibody (1:1,000; Abclonal, China), anti- NR2B (1:1,000; Abcam, USA), anti-calpain1 antibody (1:1,000; Abclonal, China), anti-calpain2 antibody (1:1,000; Abclonal, China), anti-4.1N antibody(1:1,000; Santa Cruz, USA), and anti-KCC2 antibody(1:1,000; Sigma, USA). Then, the membranes were further incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse or anti-rabbit IgG antibody (1:5,000; Boster, China) for 2 h at room temperature. Labeled proteins were detected by enhanced chemiluminescence (ECL) reagents (Thermo Scientific, USA) with the ChemiDocXRS chemiluminescence imaging system (Bio-Rad, Hercules, USA). Protein bands were quantified using lab imaging software. Experiments were repeated at least three times.

Hippocampus stereotaxic injection

The hippocampus injection was performed as we described before(Fang, Li, 2017). Briefly, at the third day after birth, pups received the injection regardless of gender. The injection was conducted under a hypothermic anesthesia by putting the rats on the ice(Cunningham and McKay, 1993). Pups were placed in the holder after anesthetized. A 0.5-cm incision was made in the center of the scalp, followed by the landmark of surface skull. Control lentivirus (TTCTCCGAACGTGTCACGT) or calpain-2 downregulation lentivirus (CTGGACGAAGATTCAGAA ATA) was injected into the hippocampal CA1 section with the aid of a 22-gauge needle. Stereotaxic coordinates were aimed toward the dorsolateral hippocampus (relative to Bregma: anterior-posterior, 3.0 mm; lateral, 2.7 mm; dorsal, 3.0 mm). To maintain hypothermic anesthesia, an ice bag was put on the back of rats during injection. After injection, the rats were placed in a warm blanket until full recovery from anesthesia, then returned to mother rats.

MWM test

The MWM test was performed as described in our previous studies(Fang, Li, 2017, Wang, Chen, 2016). A round and steel pool (diameter, 150 cm; height, 60 cm) was filled up with water until the water level reached 1.0 cm over the level of a platform (diametric distance, 10 cm). MWM was performed in a single room, with four graphic signals hung on the walls. The rats (n=10 per group) prepared for MWM were weaned at P22 and started training at P31. The training protocol for the task of the MWM test contained three trials (60 s maximum; interval 30 min) each day and lasted for 5 consecutive days. After 2 h at the end of the 5th day of training, the probe trial was performed. AVTAS v3.3 ( AniLab Software and Instruments Co., Ltd., Ningbo, China), an automated video-tracking system, was used to track the motion of rats in the pool. The time spent searching and mounting the platform (latency), the path length, and the duration of time spent in each quadrant and platform crossing were calculated and determined.

Statistical analysis

All values were represented as mean ± standard error of the mean (SEM), and analyzed by SPSS software version 20.0 (SPSS Inc., USA). Two-way analysis of variance (ANOVA) was used to analyze the expression of target proteins or receptors in different time points, the escape path length, and the escape latency in MWM. Other data were analyzed by a one-way ANOVA followed by the Tukey test. A P value < 0.05 was considered statistically significant. Results 3.1 Physiological parameters The value of pH, PaCO2, PaO2, glucose and SaO2 were all in normal range and there is no significant difference among these groups in this experiment. (Table 1) 3.2 A significant increase in the expression of NMDA subunit 2B (NR2B) after isoflurane exposure Followed by the exposure to 1.8% isoflurane for 6 h, neonatal rats (P5) were decapitated at 6, 12, and 24 h to detect the expression of NMDAR in the hippocampus via western blotting. As shown in Fig. 1A, after isoflurane exposure, there was a significant increase in the expression of NR2B at 6 (1.00±0.09 in CON group, 1.66±0.18 in ISO group, n=5 per group, df=8, p<0.05), 12 (1.63±0.116 in CON group, 2.33±0.18 in ISO group, n=5 per group, df=8, p<0.05), and 24 h (2.05±0.09 in CON group, 2.939±0.31 in ISO group, n=5 per group, df=8, p<0.01). However, compared with the Con group, the expression of NR2A was not significantly increased after isoflurane exposure (Fig. 1B). In addition, in the CON group (Fig.1A), as time went on the expression of NR2B increased, which expression increased at 12 and 24 h (2.05±0.09, p<0.001) (1.63±0.116, p<0.01) compared at 6 h (1.00±0.09). And compared at 12 h (1.63±0.116, p<0.01), NR2B expression increased at 24 h (2.05±0.09, p<0.05), suggesting the developmental increase in the level of NR2B receptor subunit10. 3.3 Isoflurane increased the expression of calpain-2 instead of calpain-1 Calpain can be stimulated by the calcium influx generated by NMDA receptor activation as reported in a previous article(Baudry and Bi, 2016). the expression of two major calpain isoforms, calpain-1 and calpain-2, in the hippocampus were assessed by western blotting. As shown in Fig. 1C, calpain-2 increased at 12 h in the ISO group (1.84 ± 0.27) compared with the CON group ( p < 0.05). Further increase of calpain-2 could be observed at 24 h following isoflurane exposure，increasing from 1.41 ± 0.20 in the CON group to 2.48 ± 0.23 in the ISO group (Fig. 1C, n=5 per group, df=8, p < 0.01). On the other hand, calpain-1 showed no significant change after isoflurane exposure (Fig. 1D). 3.4 Isoflurane induced the repression of plasmalemmal KCC2 and total 4.1N protein in neonatal rats Previous studies have proven that the binding of KCC2 to the cytoskeleton-associated protein 4.1N plays an important role in spine development(Li, Khirug, 2007). Therefore, in this part of the experiment, the interaction between KCC2 and the 4.1N protein was confirmed through a co-immunoprecipitation experiment (Fig. 2A). After isoflurane exposure, the membrane protein of KCC2 and 4.1 N protein was detected by western blotting. At 24 h after isoflurane treatment, plasmalemmal KCC2 decreased from 2.02 ± 0.18 in the CON group to 1.38 ±0.142 in the ISO group (Fig. 2B and D, n=4 per group， df=6, p < 0.01), and total 4.1N protein reduced from 1.74 ± 0.28 in the CON group to 1.06 ± 0.15 in the ISO group (Fig. 2C and E, n=6 per group，df=10, p < 0.05), suggesting that KCC2 could interact with the 4.1N protein and be repressed after isoflurane treatment. 3.5 Inhibition of NR2B reversed the increase of calpain-2 as well as the reduction of KCC2 and 4.1N protein in neonatal rats To investigate whether increased NR2B induced by isoflurane contributes to the changes of calpain-2, KCC2 and 4.1N protein levels, NR2B inhibitor, ifenprodil, was applied 30 min before isoflurane exposure. As compared with the ISO group (1.71±0.13, n=6 per group, df=20, p<0.05, Fig. 3A and B), ifenprodil pretreatment significantly attenuated the level of calpain-2 (1.16±0.12). As shown in Fig. 3B-C, the reduction of KCC2 and 4.1N induced by isoflurane was reversed by ifenprodil administration. Compared with the ISO group (0.49±0.45), there was a significant increase in the expression of KCC2 in the IFEN+ISO group (0.87±0.39, n=6 per group, df=16, p<0.05, Fig. 3C and E). Compared with the ISO group (0.46±0.34), the expression of 4.1N protein was elevated in the IFEN+ISO group (0.88±0.89, n=6 per group, df=16, p<0.05, Fig. 3D and F). Additionally, there was no significant difference in the expression of calpain-2, KCC2, and 4.1N protein among the CON, IFEN, and IFEN+ISO groups (Fig. 3A-F). 3.6 Inhibition of NR2B alleviated the spatial memory deficits induced by long-time exposure to isoflurane in early stages To verify the effects of extended isoflurane (1.8%, 6 h) and ifenprodil (pic) exposure on long-term cognition, the MWM task was performed at 26 d after isoflurane exposure (P31). Rats exposed to isoflurane in their neonatal age displayed a cognitive impairment in MWM as compared with the CON group, which were indicated by longer traveled distance (Fig. 4A and B, df=27, p < 0.05, n = 10 per group, from the training trials), prolonged escape latency (Fig. 4C, df=27, p < 0.05 or p < 0.01 or p < 0.001, n = 10 per group, from the training trials), less time spent in the target quadrant (Fig. 4D, df=27, p < 0.001 or p < 0.05, n = 10 per group, from the probe trials), and decreased platform crossings (Fig. 4E, df=27, p < 0.001, n = 10 per group, from the probe trials). In contrast, pretreatment with ifenprodil (pic) alleviated spatial and learning memory impairments induced by isoflurane The evidence was shown by shorter traveled distance (Fig. 4B, df=27, p < 0.05, n = 10 per group, from the training trials), decreased escape latency (Fig. 4C, df=27, p < 0.001 or p < 0.01 or p < 0.05, n = 10 per group, from the training trials), more time spent in the target quadrant (Fig. 4D, df=27, p < 0.05, n=10 per group, from the probe trials), and increased platform crossings (Fig. 4D, df=27, p < 0.05, n = 10 per group, from the probe trials). Compared with the CON group (1.00±0.08), the expression of calpain-2 was significantly elevated in the ISO group (2.05±0.16), and this increase was reversed in the IFEN+ISO group (1.14±0.10, df=30, p < 0.001, n = 7 per group, Fig. 4F). Compared with the CON group (1.00±0.09), the expression of KCC2 was significantly reduced in the ISO group (0.56±0.44), and this decrease was reversed in the IFEN+ISO group (0.92±0.04, df=21, p < 0.001 or p <0.01, n = 7 per group, Fig. 4G). Compared with the CON group (1.00±0.11), the expression of 4.1N protein was significantly reduced in the ISO group (0.37±0.04), and this decrease was reversed in the IFEN+ISO group (0.85±0.10, df=18, p < 0.001 or p <0.01, n = 7 per group, Fig. 4H). 3.7 Calpain-2 downregulation lentivirus reversed the reduction of KCC2 and 4.1N protein in neonatal rats induced by isoflurane exposure To further determine the upstream and downstream relationship of calpain-2 and KCC2, a lentivirus system injected into the hippocampi of P3 rats was used to downregulate calpain-2 expression. The lentivirus (2 μl/site) was microinjected into the CA1 region. The brain tissues were analyzed 24 h after isoflurane exposure in P5. As traced by enhanced green fluorescent protein, lentivirus was found to result in CA1-specific expression. Quantitative analysis showed that calpain-2 levels were remarkably reduced by the downregulation lentivirus (1.01 ± .11) in comparison with the ISO group (Fig5.A and B, n=7 per group, df=24, 2.02 ± 0.22). Also, as we expected, downregulating the expression of calpain-2 increased the level of KCC2 gray value from 0.45 ± 0.07 in the ISO group to 0.92 ± 0.11 in the LV+ISO group (Fig. 5C and E, n=6 per group, df=20, p < 0.05). In addition, the gray value of 4.1N protein band increased from 0.51 ± 0.62 in the ISO group to 0.98 ± 0.78 in the LV+ISO group (Fig. 5D and F, n=6 per group, df=20, p < 0.01). 3.8 Spatial memory deficits mediated by isoflurane exposure were mitigated by calpain-2 downregulation lentivirus injection Rats received calpain-2 lentivirus injection at P3 before isoflurane exposure indicated by improved spatial memory with shorter traveled distance (Fig. 6B, df=24, p < 0.05, n = 9 per group, from the training trials), decreased escape latency (Fig. 6C, df=24, p < 0.05, n = 9 per group, from the training trials), more time spent in the target quadrant (Fig. 6D, df=24, p < 0.05 or p<0.01, n = 9 per group, from the probe trials), and increased platform crossings (Fig. 6E, df=24, p<0.05, n = 9 per group, from the probe trials) as compared with the rats in the ISO group. The elevated expression of KCC2 and 4.1N protein persisted to nearly 30 days after isoflurane exposure by downregulating calpain-2 (Fig. 6F-G). Compared with the CON group (1.00±0.09), the expression of KCC2 was significantly reduced in the ISO group (0.56±0.05), and this decrease was reversed in the LV+ISO group (1.05±0.08, df=21, p < 0.01 or p <0.001, n = 7 per group, Fig. 6F). Compared with the CON group (1.00±0.11), the expression of 4.1N protein was significantly reduced in the ISO group (0.37±0.04), and this decrease was reversed in the LV+ISO group (0.85±0.10, df=18, p < 0.05, n = 7 per group, Fig. 6G). Discussion Our data suggest an increase in the level of NMDAR subunit NR2B in connection with isoflurane neurotoxicity. Blockade of NR2B significantly increased isoflurane-induced calpain activation, KCC2 cleavage and spatial memory deficiency. In the CNS, functional NMDARs mainly consist of two NR1 subunits and two NR2A or NR2B subunits(Furukawa, Singh, 2005, Gambrill and Barria, 2011, Sanz-Clemente, Matta, 2010). Subunit composition varies across brain regions during development and in disease states(Paoletti, Bellone, 2013). In immature brains, NR1/NR2B receptors are predominantly expressed. With development, NR1/NR2A receptors gradually increase(Paoletti, Bellone, 2013). Our present study implied that both the type of NR2 subunit and the location of NMDAR were related to isoflurane-induced neurotoxicity, as the expression of NR2B increased after isoflurane exposure. Despite the controversy and criticism, it has been proposed that NR2A-containing NMDARs are more likely to occupy the central portion of the synapse, while NR2B-containing NMDARs are preferentially targeted to sites(Yashiro and Philpot, 2008). Taken together, we conclude that the NR2B should be connected with isoflurane neurotoxicity. Calpain plays an important role in both synaptic plasticity and neuronal degeneration. In the CNS, two major calpain isoforms, calpain-1 and calpain-2, play the respective roles and exhibit opposite functions in synaptic plasticity, learning, and memory(Baudry and Bi, 2016, Chiu, Lam, 2005, Vosler, Brennan, 2008). Activation of calpain-1 is required for the induction of LTP and plays a neuroprotective role in the CNS, while activation of calpain-2 limits the extent of potentiation and plays a neurodegenerative role(Baudry and Bi, 2016). Previous studies have proven that calpain could be activated by the calcium influx mediated by NMDAR(Zhou, Chen, 2012). However, different effects of calpain on neuronal survival were due to different isoform activation and mediated via different NMDAR signaling pathways(Baudry and Bi, 2016). Our data suggest that there is a significant increase in the expression of calpain-2 instead of calpain-1 after isoflurane anesthesia, accompanied with the increasing expression of NR2B (Fig.2). Comparing the expression in different time points, we found that although NR2B increased from 6 h after anesthesia, the calpain-2 level was elevated at the same time that NR2B increased. Pretreatment with NR2B blocker could reverse the change of calpain-2 induced by isoflurane. These data further confirmed the interaction between calpain-2 and NR2B. Our finding suggests that KCC2 is target of NMDAR and calpain-2. In immature brains, KCC2 has two independent biological functions: one is as an ion transporter to keep the balance of the extracellular and intracellular chloride concentration; the other is as a structural protein via the binding of the KCC2 C-terminal domain to the cytoskeleton-associated protein 4.1N(Blaesse and Schmidt, 2015, Li, Khirug, 2007). KCC2 has been reported to play pivotal roles in spine morphogenesis and structural plasticity during development synapses(Blaesse and Schmidt, 2015, Li, Khirug, 2007). In this study, we found that the level KCC2 and 4.1N were significantly decreased after isoflurane exposure (Fig. 2B and 4C), and these responses could be reversed by NR2B antagonist ifenprodil or calpain-2 downregulation lentivirus (Fig.5). Our data collectively suggest that KCC2 downregulation is due to calcium influx through NMDAR. Increased NMDAR activity results in calcium influx, and subsequent calpain can be activated by increases in intracellular calcium concentration. In parallel with these findings, pretreatment with NR2B antagonist ifenprodil or calpain-2 downregulation lentivirus was found to significantly mitigate the cognitive deficiency (Fig.4, Fig.6). Compared with the CON group, rats exposed to isoflurane in their neonatal age displayed longer traveled distance, prolonged escape latency, less time spent in the target quadrant, and decreased platform crossings in MWM. Application of NR2B antagonist ifenprodil or calpain-2 downregulation lentivirus could significantly reverse these responses in isoflurane-treated rats. Our data suggest that the NMDAR-calpain-KCC2 signal pathway plays pivotal roles in isoflurane-induced cognitive deficiency in immature brains.
In conclusion, our study put forward an undiscovered mechanism implying that a significant increase of NR2B, excessive activation of calpain-2, and increased cleavage of plasmalemmal KCC2, are involved in isoflurane-induced neurotoxicity and long-term cognitive deficiency. Blockade of NR2B and calpain-2 activity significantly attenuated these responses. The KCC2 cleavage mediated by NR2B and calpain-2 is a major determinant of isoflurane-induced long-term cognitive deficiency.
This finding may provide new precautionary and therapeutic measures of cognition deficiency mediated by anesthetic exposure.

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