NKCC1 up-regulation contributes to early post-traumatic seizures and increased post-traumatic seizure susceptibility

Male C57bc/6j mice were obtained from Jackson Laboratories. Mice at 6–8 weeks old were subjected to a single, closed-head, unilateral cortical injury, as previously described (Petraglia et al. 2014a, b). Briefly, awake un-anesthetized mice were restrained in DecapiCones (Braintree Scientific, Inc.) for 10–20 s during the injury to achieve head restriction relative to the body (6 mm in diameter and 2 mm in thickness) covering the target area of injury (left parietal, center of the disc 5–7 mm from sagittal suture) by securing it to the mouse head to prevent skull fracture. The injury device includes a pneumatic-controlled impactor (Pittsburgh Precision Instruments, Inc.) with a rubber tip attached to a stainless steel piston used to create the impact, and a foam platform, on which the animals were positioned. Before producing the injury, the impactor tip was aligned with the metal disc to ensure the consistency of the injury. Brain injury was delivered by the injury device mounted at 15° from the perpendicular to create a head displacement of 1 cm at a velocity of 7.14 m/s during 100 ms period. Sham mice received identical treatment, but did not receive the TBI.

Slc12a2^−/− mice were generated by Delpire et al. (1999), and breeding and genotyping were carried out as per their descriptions (Delpire et al. 1999). As above, male mice, at 6–8 weeks old, were used. These mice (N = 26), and the wild-type littermates (N = 28) were bred in our facility and used in experiments comparing wild type with Slc12a2^−/− mice. All animal experiments were approved by the Animal Care and Use Committee of the University of Rochester.

Mice were video-taped for 4 h after the injury to characterize their early post-traumatic seizure behavior. Two to three random 5 min periods per hour of recordings were blindly analyzed (a total ten periods were analyzed per mouse). A scoring system adapted from Racine scale was used to quantify the seizure-related behaviors in the immediate phase: 1, immobility; 2, head nodding, stiff tail; 3, isolated myoclonic jerks; 4, clonic seizure; 5, tonic–clonic seizure; 6, convulsion with jumping; and 7, convulsion culminating in death.

Mouse preparations were modifications of the previous protocols (Dombeck et al. 2007; Thrane et al. 2012). EEG recordings were carried out with a tungsten wire implanted under the skull and touching the dura 1 day before the TBI, as previously reported (Petraglia et al. 2014a, b). These recordings were performed for half an hour sessions, randomly selected at 0, 3, or 24 h after TBI. EEG signals were externally filtered at 60 Hz (Filter Butterworth Model by Encore, Axopatch 200B by Axon Instruments), bandpass filtered at 1–100 Hz, and digitized (Digidata 1440A by Axon Instruments). Recordings were analyzed offline using pClamp 10.2. The total power at different frequencies was added together to get normalized power. Myoclonic seizures were defined on the EEG/ECoG as single or multiple 3–9 Hz polyspike and wave discharges of 0.2–2 s duration associated with myoclonic jerks determined by video recording or direct observation.

Unless otherwise noted, 6-week-old C57BL/6 (Jackson Laboratories) were used for the preparation of cortical brain slices as previously described (Wang et al. 2012a). The mice were anesthetized with isoflurane in a closed chamber (1.5 %) and decapitated. The brains were rapidly removed and immersed in ice-cold cutting solution that contained 230 mM sucrose, 2.5 mM KCl, 0.5 mM CaCl₂, 10 mM MgCl₂, 26 mM NaHCO₃, 1.25 mM NaH₂PO₄, and 10 mM glucose (pH 7.2–7.4). Coronal slices (400 mm) were cut with a vibratome (Vibratome Company) and transferred to oxygenated aCSF that contained 126 mM NaCl, 4 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 26 mM NaHCO₃, 1.25 mM NaH₂PO₄, and 10 mM glucose (pH 7.2–7.4, osmolarity = 310 mosM). Slices were incubated in aCSF for 1–5 h at room temperature before recording. Experiments were performed at room temperature (21–23 °C). During the recordings, the slices were placed in a perfusion chamber and superfused with aCSF gassed with 5 % CO₂ and 95 % O₂ at room temperature. Cells were visualized with a 40 × water-immersion objective and differential inference contrast (DIC) optics (BX51 upright microscope, Olympus Optical). Patch electrodes were fabricated from filament thin-wall glass (World Precision Instruments) on a vertical puller; the resistance of the pipette was about 6–9 MΩ with intracellular pipette solution added. GABA currents were recorded under voltage clamp with an Axopatch MultiClamp 700B amplifier (Axon Instruments). The pipette solution contained 140 mM K-gluconate, 5 mM Na-phosphocreatine, 2 mM MgCl₂, 10 mM Hepes, 4 mM Mg-ATP, and 0.3 mM Na-GTP (pH adjusted to 7.2 with KOH). In addition, gramicidin was diluted in methanol to make a stock solution at 10 mg/ml first and then made to the final 50–100 µg/ml in the pipette solution. The junction potential between the patch pipette and the bath solution was zeroed before forming a gigaseal. Patches with seal resistances of less than 1 GΩ were rejected. Data were low-pass filtered at 2 kHz and digitized at 10 kHz with a Digidata 1440 interface controlled by the pClamp Software (Molecular Devices).

To assess post-traumatic seizure susceptibility, we administered PTZ (10 mg/kg; i.p.) at 3 days after TBI. For the first series of experiments, we only used those mice that did not experience any early post- traumatic seizures (N = 5, slc12a2^+/+ and N = 5 slc12a2^−/−). The latency and duration of seizures were assessed with EEG recording. We also used PTZ challenge to determine if bumetanide (Sigma Aldrich; 30 mg/kg, i.p., dissolved in DMSO, then diluted with 0.9 % saline) would protect against post-traumatic seizure susceptibility. Mice were subjected to TBI, and 3 days after, bumetanide was injected i.p., 30 min before application of PTZ (10 mg/kg, i.p.) to allow bumetanide to pass through the blood–brain barrier (Tollner et al. 2014). Behavioral and electrophysiological analysis of seizures was performed as above.

A third set of PTZ seizure challenge experiments were performed to assess the effects of the TGF-β blocker LY-364947 on post-traumatic seizure susceptibility. For these studies, mice were subjected to TBI. LY-364947 (TOCRIS, 10 mg/kg, i.p.) was injected once daily for 3 days, with the first injection occurring immediately after the TBI. At 3 days after TBI, PTZ (10 mg/kg, i.p.) was injected and the analysis of seizures was performed as above.

Mice were anesthetized under ketamine (100 mg/ml), xylazine (20 mg/ml), and cocktail (0.1 ml per 10 g of body weight), and perfused transcardially with 4 % paraformaldehyde, and the brains were post-fixed overnight. Serial 20 µm coronal cryostat sections were cut after overnight cryoprotection in 15 % and then 30 % sucrose. Sections were incubated with single or combination of following primary antibodies overnight at 4 °C: rabbit anti-NKCC1 primary antibody (1:500, ABCam), mouse anti-KCC2 (1:500, ABCam), mouse anti-TGF beta 2 (1:1000, ABCam), mouse anti-NeuN (1:1000, Millipore), and rabbit anti-GFAP (1:1000 Sigma). The primary antibody detection was followed by incubation with secondary fluorescent antibodies. DAPI (Vector) was used for mounting.

Images were taken with a 20× lens in a BX53 Olympus system microscope attached to a DP72 Olympus digital camera, or with an Olympus laser-scanning confocal microscope (IX-82). The brightness and contrast of all images were adjusted using Adobe Photoshop (V. CS5). No other adjustments were made to any of the images. We captured 3–5 images per slice, in the areas between 1.2 and −1.8 from bregma, and assured that the regions of interest were sampled equally. Quantification of NKCC1- and KCC2-expressing neurons was performed using computer-assisted analysis (ImageJ software V. 2.0.0-beta-7.8). To randomly select regions of interest for analysis, a 2500 µm box (50 × 50 µm) was randomly placed bi-laterally, over the images-containing somatosensory and motor cortex. Pooled data from both hemispheres were used in the final analysis. For each time point, and for sham, we analyzed four mice per group. We placed three random boxes per slice and analyzed 3–5 images per slice, per mouse, per group.

Western blot analysis for NKCC1 expression change in the posttraumatic cortex was done as previously described (Tong et al. 2014). Briefly, mice were anesthetized and perfused with PBS. Cortex was dissected from the rest of brain tissue in ice-cold PBS using a fine-straight and a fine-angled dissecting forceps. The brain tissue was then lysed on ice in RIPA lysis buffer (150 mM sodium chloride, 1.0 % Triton X-100, 0.5 % sodium deoxycholate, 0.1 % SDS, 50 mM Tris, and pH 8.0) with protease inhibitor (Roche). Protein concentration was determined by Pierce BCA Protein Assay Kit (Thermo scientific). Samples each containing 100 µg of total protein were resolved on SDS-PAGE (10 % gel) in Laemmli Sample Buffer (Bio rad) and transferred to PVDF membranes (Immobilon-FL, Millipore) in transfer buffer for 1 h at room temperature. Membranes were blocked with Odyssey Blocking Buffer at room temperature for 2 h, then incubate with anti-NKCC1 primary antibody (1:500, AbCam), anti-KCC2 primary antibody (1:1000, ABCam), and anti-β-actin at 4 °C overnight. Blots were then probed with florescent secondary antibodies and imaged using the LI-COR Odyssey system. Band intensity was quantified using the LI-COR Odyssey software. Relative protein expression levels were calculated by normalizing the band intensity of the target protein to its respective β-actin loading control.

Mice were anesthetized under ketamine (100 mg/ml), xylazine (20 mg/ml), and cocktail (0.1 ml per 10 g of body weight), and perfused transcardially with PBS. Cortex and hippocampus were carefully dissected from each hemisphere in ice-cold PBS and immediately transferred to dry ice. Total RNA were prepared using TRIzol^® reagent (life technologies) and treated with Turbo DNA-free™ (Life Technologies) to remove DNA contamination. RNA samples were reverse transcribed by TaqMan Reverse Transcription Reagents (life technologies). Relative transcription level of NKCC1 was detected by TaqMan probe (life technologies, Mm01265951_m1) on an ABI Prism 7000 apparatus (Applied Biosystems) in three independent experiments. GAPDH (life technologies, CAT No. Mm99999915_g1) was used as an internal control. The comparative threshold (ΔC _t) method was used and results were converted to fold of expression relative to sham group.

Images captured using the Olympus IX82 microscope were analyzed using Image J (1.48V), as previously described. Briefly, a standard cutoff is used, such that pixels above a pre-determined intensity cutoff are counted by the program, and those that fall below the threshold are omitted from counting. Prior to thresholding the image to detect NKCC-labeled structures, a background image was created using median filtering. This background image was subtracted from the densitometric analysis. We analyzed 4–6 sections per animal and 3–4 animals per group. Slides were coded and images were captured from corresponding regions of the hippocampus. The analysis was then performed on the coded images, after which the codes were broken and the data analysis performed.

All analysis was performed using the SPSS 19 software (IBM), and all tests were two tailed, where significance was achieved at α = 0.05 level. Where appropriate, either an unpaired t test (≤2 variables) or one-way ANOVA (>2 variables) was used for independent samples. Where N < 10 or the data were non-normally distributed, we employed non-parametric tests, including Mann–Whitney U (≤2 variables) or Kruskall–Wallis (>2 variables), that were used for independent samples, and Wilcoxon signed ranks test for paired samples. For all graphs, error bars represent ±standard error of the mean (SEM).

We used western blot, and immuno-fluorescent labeling to characterize the expression of NKCC1 and KCC2 after TBI and compared the results with that from sham controls. Quantitative analysis of NKCC1⁺ neurons in cortex revealed a significant increase by 1 day after TBI (Fig. 1a–e). The results from the analysis of western blots (Fig. 1c, e) show a significant increase in NKCC1 in neocortex (F (4,15) = 1.7164; P < 0.05 at 3 days post-TBI). Quantification of KCC2 expression in neocortex revealed a significant decrease (F (4,15) = 4.868, P < 0.01 at 1 day; and P < 0.001 at 3 days) post-TBI (Fig. 1f–j). Consistent with these findings, qPCR also revealed a significant increase in NKCC1 expression and a decrease in KCC2 expression in neocortex after TBI (Supplementary Fig. 1). These results were similar to those observed in the hippocampus (Supplementary Fig. 2). Therefore, using multiple methodologies, we demonstrate elevated NKCC1 and decreased KCC2 after TBI in neocortex and archicortex. We also demonstrate that NKCC1 peaks in neocortex at 3 days post-injury (Fig. 1).

We used a closed-head mouse injury model (Fig. 2a) that is adapted from the controlled cortical impact (CCI) model, as previously described (Petraglia et al. 2014a, b). Within hours after injury, the majority of the animals (73.7 %, 75 mice from total 102) exhibited behavioral manifestation of seizures (Fig. 2b), including chewing and head bobs, tonic–clonic forepaw, and hind-paw activity, and brief episodes of muscle jerks as defined with the modified Racine Scale (Medina-Ceja et al. 2012; Bergstrom et al. 2013). These episodes were typically accompanied by seizure-like EEG activity (Fig. 2c–e) based on frequency, amplitude, intensity, and waveform abnormalities (Abidin et al. 2011; Beamer et al. 2012). Interictal, tonic, and clonic discharges can also be characterized, as can periodic ictal discharges and power of EEG activity (Dzhala et al. 2005; Ferrie 2005). Of the mice (26.3 %, N = 27) that did not display behavioral signs of seizures, EEG recordings still showed seizure-like activity (Fig. 2f) in 21 out of the 27 mice that did not exhibit behavioral seizures. In sham-operated mice (N = 12), neither behavioral manifestations nor EEG signs of seizures were noted.

Considering that NKCC1 is increased after TBI, we next wondered if knocking out NKCC1 would reduce the early post-traumatic seizures. To accomplish this, we used a transgenic mouse strain, in which the exon 9 of the gene-encoding NKCC1, Slc12a2 was disrupted. Immunohistochemistry for anti-NKCC1 confirmed the deletion of NKCC1 expression in the brains of the Slc12a2^−/− mice (Fig. 3a). Following TBI, none of the homozygous Slc12a2^−/− mice (N = 6) exhibited behavioral seizures, whereas >70 % of WT mice experienced the early post-traumatic seizures. Thus, the Slc12a2^−/− mice seem to be less susceptible to the early post-traumatic seizures.

To further assess the seizure susceptibility of the Slc12a2^−/− mice, we compared the Slc12a2^−/− mice with their wild-type litter mates on a PTZ (10 mg/kg, i.p.) challenge at 3 days post-injury. For these studies, we selected the six Slc12a2^−/− mice from above, as well as the six WT mice that neither experienced behavioral or EEG seizures. After PTZ injection, an expected pattern of behavioral seizures emerged in all of the animals that were accompanied by a 3–9 Hz polyspike EEG discharge typical of myoclonic seizures (Fig. 3b). Despite the fact that all of the animals exhibited seizure-associated behaviors, the results showed that PTZ-induced seizure severity was significantly decreased in Slc12a2^−/− mice compared with the wild-type mice. The average latency of PTZ-induced seizures in Slc12a2^−/− mice (11.2 ± 1.7 min) was significantly (F = 9.64 and P < 0.001) longer than in WT mice (3.4 ± 0.5 min). We also examined the seizure latency after a PTZ challenge in Slc12a2^−/− mice (12.7 ± 1.8 min) that were only exposed to a sham TBI (N = 6), and observed no significant differences compared with the Slc12a2^−/− mice that received a TBI and then PTZ challenge (Fig. 3b–d). The maximum amplitude was also significantly (F = 5.27 and P < 0.001) lower in the Slc12a2^−/− mice, from 2.4 ± 0.7 mV in wild type to 1.1 ± 0.2 mV in Slc12a2^−/− mice, and 1.2 ± 0.4 mV in Slc12a2^−/− mouse following TBI (Fig. 3b–d). There was no significant difference in maximum amplitude between Slc12a2^−/− TBI group and Slc12a2^−/− sham group.

We assessed Cl⁻ by measuring the reversal potential of GABA_A currents using perforated patch on cortical slices prepared from animals at day 3 after TBI and using voltage-clamp recording from layer II cortical neurons. The results showed that E _GABA was −69.4 ± 1.9 mV in wild-type sham mice (mean ± SEM, N = 6 slices, Fig. 3e, f). In contrast, the reversal potential of neurons from the same layer II of wild-type TBI mice was only 58.3 ± 1.5 mV (N = 7 slices, *P < 0.01, one-way ANOVA). No significant differences in reversal potential were observed comparing the Slc12a2^−/− with wild-type littermates (P = 0.272, N = 5, Fig. 3g, h).

Most rapid synaptic inhibition in the vertebrate forebrain is mediated by GABA acting via GABA_A and GABA_B receptors (Wilcox and Dichter 1994). Here, we used the paired-pulse stimulation paradigm to test the in vivo excitability of the network in the TBI mice. Figure 3i shows a typical response for two inter-deflection intervals (200 ms). Note that, in general, there was an attenuated response to the second pulse relative to the response to the first pulse, even though the two deflections were identical in structure (Fig. 3i). However, after TBI, the paired pulse changed significantly, from 0.54 ± 0.07 to 0.88 ± 0.05 (*P < 0.01, N = 5, t test). Alternatively, in Slc12a2^−/− mice, the TBI did not induce any changes, from 0.46 ± 0.05 to 0.44 ± 0.03 (P = 0.373, N = 5, t test, Fig. 3j). These results suggest the intriguing possibility that TBI compromises cortical inhibition via an NKCC1-mediated mechanism.

NKCC1 up-regulation may contribute to increased seizure susceptibility which occurs after TBI. NKCC1 is selectively blocked by low micromolar concentrations of the loop diuretic bumetanide (Isenring et al. 1998). By reducing intracellular Cl⁻ accumulation, this diuretic shifts E _GABA to negative potentials, resulting in more effective inhibition (Dzhala et al. 2005). To determine if NKCC1 protects against the early post-traumatic seizures, bumetanide (30 mg/kg, dissolved in DMSO, then diluted with 0.9 % saline) was injected 1 h before TBI. Analysis of behavioral seizures and EEG recordings following bumetanide treatment revealed a significant decrease in seizures compared with controls (41.6 %, N = 12 in bumetanide-treated group, as compared with 73.7 % in control, N = 102).

To further determine if NKCC1 inhibition with bumetanide would alter post-traumatic seizure susceptibility, we again used the second-hit PTZ seizure challenge. After application of PTZ, the animals showed behavioral seizures and EEG recording spikes characteristic of myoclonic seizures (Fig. 4a). Statistical analysis of overall seizure activity revealed a significant difference (Fig. 4c). The latency to seizure onset was delayed in the TBI mice treated with bumetanide prior to PTZ (mean 6.98 ± 0.50 min), compared with TBI mice treated with vehicle prior to PTZ (mean 3.12 ± 0.23 min). The mean duration of the ictal episode in the bumetanide-treated group decreased significantly to 23.08 ± 1.95 and 24.8 ± 2.3 min, respectively (F = 13.5, P < 0.01, one-way ANOVA, Fig. 4d, e). The maximum amplitude of the spikes was also significantly decreased in the bumetanide group (F = 11.3, P < 0.01, one-way ANOVA, Fig. 4f). Thus, inhibiting NKCC1 with bumetanide reduced the early post-traumatic seizures and decreased susceptibility to second-hit PTZ-induced seizures.

TGF-β released from astrocytes or microglial cells has been shown to have neuroprotective effects, including improved function and decreased lesion size (Mannix and Whalen 2012; Logan et al. 2013). In the peripheral nervous system, the previous studies have shown that WNKs [with no lysine (K)], interact with TGF-β (Lee 2007) and modulate NKCC1 and KCC2 activity (Richardson and Alessi 2008). Another study concluded that the interaction between WNKs and NKCC1 might play an important role in spinal cord injury (Lee et al. 2013). Therefore, we sought to determine if TGF-β might be related to TBI-induced alterations in NKCC-1. We performed immunolabeling and western Blot, and found that TBI resulted in increased TGF-β in both cortex and hippocampus (Fig. 5a–h). To further explore the relationship of TGF-β with TBI-induced seizures and the expression of NKCC1, we used the TGF-β blocker LY-364947 (10 mg/kg, i.p., once daily for 3 days). It was found that after injection of LY364947, the animals showed less PTZ-induced seizures 3 days after TBI (Fig. 6a, b). More specifically, both the latency and duration of seizures were reduced (Fig. 6c–e). Decreased expression of NKCC1 in the brain was also observed following the LY364947 treatment (Fig. 6f–h).