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. Author manuscript; available in PMC: 2025 Aug 13.
Published in final edited form as: Neuropharmacology. 2025 Jul 23;278:110599. doi: 10.1016/j.neuropharm.2025.110599

Adenosine A2A receptor and glial glutamate transporter GLT-1 are synergistic targets to reduce brain hyperexcitability after traumatic brain injury in mice

Mariana Alves 1,2, Rogério Gerbatin 1, Rebecca Kalmeijer 1, Denise Fedele 1, Tobias Engel 2,3, Detlev Boison 1,4,5
PMCID: PMC12348367  NIHMSID: NIHMS2101118  PMID: 40712754

Abstract

Traumatic brain injury (TBI) is a leading cause of acquired epilepsy, with post-traumatic epilepsy (PTE) significantly contributing to morbidity and mortality. To date, there is no treatment capable to prevent the development of PTE, which remains an urgent unmet need. Previous studies suggest that adenosine A2A receptor (A2AR) activation and glutamate transporter 1 (GLT-1) dysregulation may contribute to epileptogenesis, however, it is unclear whether therapeutic targeting of the A2AR or GLT-1 can attenuate TBI-induced hyperexcitability, and whether there are synergistic interactions between the two. Here, we investigated the therapeutic potential of two FDA approved drugs istradefylline (A2AR inhibitor) and ceftriaxone (GLT-1 activator) in preventing long-lasting brain hyperexcitability in a clinically relevant rodent model of TBI. Adult male mice underwent controlled cortical impact (CCI)-induced TBI and were randomly assigned to istradefylline, ceftriaxone, istradefylline/ceftriaxone, or vehicle groups, receiving treatment during the first 24 hours post-injury. Susceptibility to chemoconvulsant-evoked seizures was quantified at 4–5 weeks after CCI. We show that CCI caused a reduction in GLT-1 and an increase in A2AR protein levels in the ipsilateral hippocampus. Transient acute treatment with istradefylline or ceftriaxone reduced brain hyperexcitability at 4–5 weeks post-TBI. Notably, mice treated with the combination of istradefylline and ceftriaxone exhibited increased GLT-1 levels, accompanied by further reductions in brain hyperexcitability, showing greater effects than either drug alone. Our findings identify a novel disease-modifying approach following TBI using a combination of two FDA-approved drugs which might be useful to mitigate the long-lasting brain hyperexcitability-induced by TBI.

Keywords: Traumatic brain injury, Seizures, A2AR, Glutamate transporter 1, Controlled cortical injury, Inflammation, Kainic acid, Pentylenetetrazole

Introduction

Epilepsy, a chronic neurological disorder affecting over 80 million people worldwide, remains a significant health concern. Approximately 30% of epilepsy cases are acquired, resulting from precipitating injuries such as traumatic brain injury (TBI) (Agrawal et al. 2006; Dewan et al. 2019). TBI is a leading cause of injury-related death and disability globally, with devastating consequences for patients and their families. Notably, 90% of hospital-presenting TBI cases are classified as mild, while 10% are moderate-to-severe (Maas et al., 2022). Moderate-to-severe injuries increase the risk to develop post-traumatic epilepsy (PTE), which significantly contributes to morbidity and mortality in people exposed to prior TBI. Despite the development of over 40 anti-seizure medications (ASMs), approximately 30% of patients experience uncontrolled seizures (Klein et al. 2018; Zack and Kobau 2017). Furthermore, ASMs often have undesirable side effects and only target symptoms without significantly impacting disease progression (Bialer and White 2010; Thijs et al. 2019). Critically, post-traumatic epilepsy (PTE) exhibits high pharmacoresistance rates (Pease et al., 2022) and lacks effective preventive treatments (Larkin et al. 2016; Pitkanen et al. 2015). Therefore, there is an urgent need for innovative therapeutic strategies to prevent epilepsy following a TBI and its progression.

Brain injuries, such as a TBI, trigger a large outflow of danger signal molecules, such as adenosine triphosphate (ATP) and adenosine (Lusardi 2009; Faroqi et al. 2021). Adenosine and its metabolites are substantially elevated in the cerebrospinal fluid of patients that suffered from a severe TBI, with adenosine levels showing a correlation with the severity of the injury (Strogulski et al. 2022; Robertson et al. 2001).These findings have been corroborated in experimental TBI animal models (Bell et al. 1998). Thus, an acute TBI-induced adenosine surge might be involved in the epileptogenic process. Following brain injury, A2AR expression increases in both neurons and glial cells, influencing the immune-inflammatory response (Gomes et al. 2011). Experimental TBI models demonstrate elevated A2AR protein levels from day 3 to several weeks post-injury (Zhao et al. 2017). Notably, blocking A2ARs has neuroprotective effects in various brain disorders such as Parkinson’s disease, Alzheimer’s disease, ischemic damage, and TBI (Moreira-de-Sa et al. 2021; Gomes et al. 2011). Importantly, because A2ARs physically interact with and regulate the expression of the glial glutamate transporter GLT-1 (Matos et al. 2013), increased activation of the A2AR can downregulate GLT-1, thereby compromising astroglial glutamate uptake which may result in increased excitotoxicity, neuronal death, and epilepsy (He et al. 2020; Matos et al. 2013).

In line with the mechanisms discussed above, several groups have reported a reduction of GLT-1 expression following TBI (Romariz et al. 2023; Goodrich et al. 2013). Importantly, the FDA approved beta-lactam antibiotic ceftriaxone can be repurposed to increase GLT-1 (Rothstein et al. 2005) and accordingly, ceftriaxone reduced PTZ-induced seizures following CCI in rats (Romariz et al. 2023; Goodrich et al. 2013), and CCI-induced inflammation (Lim et al. 2021).

The long-term effects of maintaining a balance between GLT-1 and A2AR levels during the early stages of TBI remain unknown. Therefore, the combination of an A2AR antagonist and ceftriaxone offers a synergistic approach to neuroprotection in TBI. Antagonizing the A2AR is expected to mitigate excitotoxicity and inflammation, whereas ceftriaxone enhances glutamate uptake, reducing excessive glutamate release and subsequent excitotoxic neuronal damage. Together, these mechanisms complement each other, providing a two-pronged strategy to target the complex pathophysiology of TBI and potentially improve outcomes.

Here we propose that inhibiting A2AR activity with the FDA-approved inhibitor istradefylline and acutely increasing GLT-1 levels with ceftriaxone after controlled cortical impact (CCI)-induced TBI may effectively attenuate subsequent brain hyperexcitability and PTE development. To test this hypothesis, we investigated the synergistic therapeutic potential of two FDA-approved drugs: ceftriaxone (GLT-1 activator) and istradefylline (A2AR inhibitor) in a preclinical mouse model of TBI-induced brain hyperexcitability.

Material and Methods

Animals

All experiments were performed in accordance with protocols approved by Rutgers Institutional Animal Care and Use Committee (IACUC). All animals were treated according to American standards and regulations for animal experiments and all efforts were made to minimize animal suffering and reduce the numbers of animals used. A total of 149 mice were used in this study. All mice used in our experiments were CD1 wild type (WT) (Charles River) 8–10 week’s old males, with a weight range between 30 g – 35 g. All animals were housed in a controlled biomedical facility using Techniplast conventional cages and Lignocel, premium hygienic animal bedding with 2–5 mice per cage on a 12-hour light/dark cycle at 22 ± 1°C and humidity of 40–60% with food and water provided ad libitum. For each cage, enrichment was provided in the form of nesting material PVC tubes and red polycarbonate mouse houses. All in vivo studies were carried out during the light phase of the cycle.

Controlled Cortical Impact (CCI) model of TBI

A total of male 123 CD1 mice received a single right lateral cortical impact with an electromagnetic CCI device (Leica Biosystems) allowing precisely controlled cortical injuries as described previously (Guo et al. 2013). Mice were anesthetized with isoflurane (5% induction, 1 – 2% maintenance) and core body temperature was maintained at 37°C using a heating pad. Mice were placed in a stereotaxic frame (David Kopf Instruments) and the right stereotaxic arm an impact device was mounted at an angle of 15° from vertical. A 10 mm midline incision was made over the skull, and the skin and fascia were reflected to perform a craniotomy over the right fronto-parietal cortex using an electrical drill avoiding damage to the underlying dura. A 2 mm impact tip was positioned over the center of the craniotomy site, 3.0 mm anterior to lambda and 2.7 mm to the right of midline. The impounder tip of the injury device was extended to its full stroke distance, positioned to the intact surface of the exposed dura mater, and reset to impact the cortical surface. CCI was triggered using a computer controller. An impact injury was delivered to compress the cortex to a depth of 2.0 mm at a velocity of 5 m/s and 100 ms duration. After injury, the skin incision was closed with interrupted 5–0 silk sutures and anesthesia was terminated. The animal was placed into a heated incubator to maintain normal core temperature for 45 min post-injury before being returned to their home cage. 26 sham-injured mice underwent the same procedure as CCI mice (anesthesia and craniotomy) except for the impact.

Drug administration

The GLT-1 agonist ceftriaxone (200 mg/kg) (Romariz et al. 2023), the A2AR inhibitor istradefylline (5.3 mg/kg) (Purnell et al. 2023) or vehicle (20% DMSO in 0.9% Saline) were delivered via intraperitoneal (i.p). injection (10 ml/kg) at 6 h, 12 h and 24 h post-CCI.

Analysis of seizures

Susceptibility to chemoconvulsant-induced seizures following a TBI is frequently used to assess lasting disease modifying treatments in epilepsy research. However, disease modifying treatments may affect different mechanisms involved in the epileptogenic processes that turn a healthy into an epileptic brain capable of generating spontaneous recurrent seizures. To investigate disease-modifying effects of our interventions, we used two different chemoconvulsant agents with different mechanisms of action: kainic acid (KA) to model excitotoxicity and its effects on electroencephalogram (EEG) power, and pentylenetetrazol (PTZ) to assess generalized behavioral seizure thresholds. Therefore, the use of two chemoconvulsants provides complementary information, offering a more comprehensive understanding of post-traumatic brain changes and increasing model validity. Employing multiple seizure induction methods strengthens the validity of findings, as results can be compared and contrasted across different models. By using both KA and PTZ, we hope to gain a more nuanced understanding of the complex changes in brain susceptibility after controlled cortical impact.

KA-induced seizures: KA is an excitatory amino acid analogue that activates glutamate receptors, inducing excitotoxicity and seizures. Administering KA after CCI helps to evaluate changes in seizure susceptibility, neuronal excitability, and potential epileptogenic effects. Because of the excitotoxic nature of this model, our primary outcome measure was the elucidation of changes in EEG activity recorded from implanted cortical electrodes. 28 days following CCI, mice were anesthetized using isoflurane (5% induction, 1–2% maintenance) and maintained normothermic by means of a feedback-controlled heat blanket. The depth of the anaesthesia was frequently tested by checking the plantar nociception or corneal reflex. Once fully anesthetized, mice were placed in a stereotaxic frame and a midline scalp incision was performed to expose the skull. Then, two cortical electrodes, one on top of the undamaged, contralateral hippocampus and the reference electrode on top of the frontal cortex, were fixed in place with dental cement. EEG was recorded for 10 min before and for 90 min following an administration of KA (10 mg/kg, i.p.). To analyze seizure severity, data were uploaded onto Labchart7 software (AD Instruments) as before (Alves et al. 2019). EEG total power (μV2) is a function of EEG amplitude over time and was analyzed by integrating frequency bands from 0 – 50 Hz. Power spectral density heat maps were generated within LabChart7 (spectral view), with the frequency domain filtered from 0 – 40 Hz and the amplitude domain filtered from 0 – 50 mV. Seizure onset was defined as first seizure burst detectable on the EEG with consisting of high amplitude (> twice baseline) high frequency polyspiking of a minimum of 5 s in duration.

PTZ-induced seizures: PTZ is a GABA receptor antagonist that induces seizures by blocking inhibitory neurotransmission. Because the PTZ model triggers primary generalized tonic-clonic seizures our primary outcome parameter was the assessment of the latency to the development of generalized tonic clonic seizures. At day 32 post-CCI, mice were injected with a single subconvulsant dose of PTZ (35 mg/kg; i.p.), as described by (Gerbatin et al. 2019). Animals were monitored for the appearance of clonic and generalized tonic–clonic seizures for 30 min following PTZ injection and the latency to seizure onset was determined. Generalized convulsive seizures were considered as generalized whole-body clonus involving all four limbs and tail, rearing, wild running and jumping, followed by sudden loss of upright posture and autonomic signs. The behavioral analysis was carried out by two independent researchers, registering the time to the first myoclonic seizure, to the first generalized tonic-clonic seizure and the duration of generalized tonic-clonic seizure.

Western blotting

A total of 30 male CD1 mice (part of the cohort 1 in figure 1A) were subjected to CCI or sham injury, and the hippocampus was collected at different time points after CCI for protein level analysis by Western blot. Mice were randomly allocated to the 5 different groups as shown in Figure 1A (sham, 1, 3, 10, and 28 days post-CCI), with n = 6 per group. Western blot analysis was performed as described previously (Alves et al. 2019). RIPA lysis buffer containing a cocktail of phosphatase and protease inhibitors (Sigma-Aldrich, Massachusetts, USA) was used to homogenize ipsilateral hippocampal brain tissues and to extract proteins, which were quantified using a Tecan plate reader at 560 nm. 30 μg of protein samples were loaded onto an acrylamide gel and separated by 10–12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Following electrophoresis, proteins were transferred to a nitrocellulose membrane (Bio-Rad, California, USA) and immunoblotted with the following primary antibodies: A2AR (1:400, anti-rabbit IgG; Alomone AAR-002, Jerusalem, Israel), GLT-1 (1:400, anti-rabbit; Alomone AGC-022, Jerusalem, Israel) all prepared in 5% milk-tris buffered saline-tween (TBST). Membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit (1:1000, prepared in 5% milk-TBST; rabbit-IgG; HRP; Millipore-Sigma, Massachusetts, USA; Cat #: AP132P). Protein bands were visualized using iBright 1500 system (Invitrogen) with chemiluminescence (Clarity Western ECL substract, cat # 170–5060, Bio-Rad, California, USA) followed by analysis using Alpha-EaseFC4.0 software. Protein quantity was normalized to the loading control β-GAPDH (1:1000 prepared in 5% milk-TBST; anti-rabbit; Sigma G9545, Massachusetts, USA).

Figure 1. GLT-1 and A2AR expression changes following TBI.

Figure 1.

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A. Schematic showing the experimental design for tissue collection for the characterization of A2AR and GLT-1expression following CCI. B. Representative Western blot and corresponding graphs for A2AR and GLT-1 protein levels in the ipsilateral side of the hippocampus. C. Representative images of Nissl staining showing the lesion in the hippocampus induced by CCI at different time-points. Scale = 500 μm. D. Schematic showing the experimental design for KA evoked seizures. E. Representative EEG traces of Sham-exposed and CCI-exposed mice following KA-induced seizures. F. Graphs showing an increase in the EEG total power and amplitude in CCI-exposed mice when compared to sham injured mice following KA injection 28 days post-CCI. (B. n = 6 per group; One-way ANOVA parametric statistics with post hoc Fisher’s: A2AR: F(4,25) = 3.652; sham vs 3d, p = 0.0320, sham vs 10d, p = 0.0483, sham vs 28d, p = 0.0047; GLT-1: F(4,25) = 2.321; sham vs 1d, p = 0.0218, sham vs 3d, p = 0.0499, sham vs 10d, p = 0.0117, sham vs 28d, p = 0.0451. F. n = 6 sham and 5 CCI animals; unpaired t-test: EEG total power: t(9) = 3.411, p = 0.0077; Amplitude: t(9) = 3.762, p = 0.0045). Data presented as means ± SEM. *p<0.05, **p<0.01).

Nissl staining

A total of 12 male CD1 mice (part of the cohort 1 in figure 1A) were subjected to CCI or sham injury, and the brain was collected at different time points after CCI for Nissl staining. Brain sections were affixed to slides and left to air-dry overnight. Subsequently, the sections underwent rinsing in xylene followed by hydration through a series of alcohol dilutions (97%, 95%, 90%, 85%, 75%). Following a 7-min incubation in a 0.1% cresyl violet solution (Abcam, cat# ab246817, Massachusetts, USA), the sections were differentiated in alcohol, cleared in xylene, and finally mounted using mounting medium (Fisher Scientific, cat# SP15–100 UN1294, Waltham, Massachusetts, USA).

Immunofluorescence

Mice from three independent cohorts (cohort 2 in figure 1C, cohort 3 in figure 3A and cohort 4 in figure 4A) were used to perform immunofluorescence. Mice were transcardially perfused with 4% paraformaldehyde (PFA) and post-fixed for an additional 24 h. Brains were then transferred to 3% sucrose in PBS for 24 h and frozen before sectioning. 30 μm sagittal sections were cut using the cryostat (Leica Biosystems) and sections were stored at −20°C in PBS. For immunofluorescence staining, tissue sections were incubated with blocking with donkey blocking solution (2% normal donkey serum, 0.2% triton, 0.02% sodium azide made in 1x TBS) for 1 h. Sections were then incubated with the primary antibody GFAP (1:400; anti-mouse; 1:1000; Sigma-Aldrich G3893, Massachusetts, USA) and Iba-1 (1:400: anti-goat; Invitrogen cat # PA5–18039, Massachusetts, USA) overnight. After washing in PBS, tissue was incubated with fluorescent secondary antibodies, AlexaFluor488 or AlexaFluor568 (1:400; ThermoFisher, Massachusetts, USA). FluorSave™ with DAPI was used to cover the tissue and images were taken with a Leica DM4B confocal microscope using a 20x objective. Cell counts were taken as the average of two images (20x) from each brain regions and presented as an average for whole hippocampus.

Figure 3. Blockade of A2AR and activation of GLT-1 attenuates CCI-induced brain excitability and response to KA-evoked seizures.

Figure 3.

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A. Schematic showing the experimental design for KA-evoked seizures following CCI. B. Representative EEG traces of CCI-exposed mice treated with vehicle, CCI-exposed mice treated with istradefylline, CCI-exposed mice treated with ceftriaxone and CCI-exposed mice treated istradefylline+ceftriaxone following KA-induced seizures. C. Graph showing a decrease in the latency to seizure onset in CCI-exposed mice treated with the combination of istradefylline+ceftriaxone when compared to CCI-exposed mice treated with vehicle following KA injection 28 days post-CCI. D. Graph showing a decrease in the EEG total power in CCI-exposed mice treated either with istradefylline, ceftriaxone or combination of istradefylline+ceftriaxone when compared to CCI-exposed mice treated with vehicle following KA injection 28 days post-CCI. E. Graph showing a decrease in the EEG amplitude in CCI-exposed mice treated either with istradefylline, ceftriaxone or combination of istradefylline+ceftriaxone when compared to CCI-exposed mice treated with vehicle following KA injection 28 days post-CCI. Graph showing the survival percentage. F. Immunofluorescence images and corresponding graphs showing an increase in GLT-1 fluorescence intensity in contralateral side of the hippocampus in CCI-exposed mice treated with the combination of istradefylline+ceftriaxone when compared with CCI-exposed mice treated with vehicle. (C. n = 12 CCI-veh, 12 CCI-istra, 12 CCI-ceft and 9 CCI-istra+ceft; One-way ANOVA parametric statistics with post hoc Fisher’s: Total Power: F(3,41) = 3.088; CCI-Veh vs. CCI-istra+ceft, p < 0.0066). D. n = 12 CCI-veh, 12 CCI-istra, 12 CCI-ceft and 9 CCI-istra+ceft; One-way ANOVA parametric statistics with post hoc Fisher’s: Total Power: F(3,41) = 7.523; CCI-veh vs. CCI-istra, p = 0.0314; CCI-veh vs. CCI-ceft, p = 0.0009; CCI-veh vs. CCI-istra+ceft, p < 0.0001; E. n = 12 CCI-veh, 12 CCI-istra, 12 CCI-ceft and 9 CCI-istra+ceft; One-way ANOVA parametric statistics with post hoc Fisher’s: Amplitude: : F(3,41) = 2.462; CCI-veh vs. CCI-istra+ceft, p = 0.0102). F. n = 11 CCI-veh, 11 CCI-istra, 9 CCI-ceft and 8 CCI-istra+ceft; One-way ANOVA parametric statistics with post hoc Fisher’s: F(3,35) = 3.638; CCI-veh vs. CCI-istra+ceft, p = 0.0110; Scale bar = 100 μm). Data presented as means ± SEM. *p<0.05, **p<0.01, ***p<0.001).

Figure 4. Blockade of the A2AR and activation of GLT-1 attenuates CCI-induced brain excitability and seizures following PTZ-evoked seizures.

Figure 4.

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A. Schematic showing the experimental design for PTZ-evoked seizures 32 days following CCI. B. Graph showing the latency to myoclonic seizures in mice challenged with PTZ 32 days post-CCI. C. Graph showing the percentage of myoclonic seizures vs no-myoclonic seizures in mice challenged with PTZ 32 days post-CCI. D. Graph showing the latency to tonic-clonic seizures in mice challenged with PTZ 32 days post-CCI. E. Graph showing the percentage of tonic-clonic seizures vs non-tonic-clonic seizures in mice challenged with PTZ 32 days post-CCI. (B. n = 11 Sham-veh, 10 CCI-veh, 10 CCI-istra, 10 CCI-ceft and 10 CCI-istra+ceft; non-parametric Kruskal-Wallis test: F(5,53) = 7.523; Sham-veh vs CCI-veh, p = 0.0015; CCI-veh vs. CCI-istra+ceft, p = 0.0026). C. n = 11 Sham-veh, 10 CCI-veh, 10 CCI-istra, 10 CCI-ceft and 10 CCI-istra+ceft; Chi-square test, a pairwise comparison of 2 by 2 table: Sham-veh vs CCI-veh, p < 0.0001; CCI-veh vs. CCI-istra, p < 0.0001; CCI-veh vs. CCI-ceft, p = 0.0035; CCI-veh vs CCI-istra+ceft, p < 0.0001. D. n = 11 Sham-veh, 10 CCI-veh, 10 CCI-istra, 10 CCI-ceft and 10 CCI-istra+ceft; Kruskal-Wallis test: F(5,51) = 24.95; Sham-veh vs CCI-veh, p < 0.0001; CCI-veh vs. CCI-istra, p = 0.0026; CCI-veh vs. CCI-istra+ceft, p = 0.0153. E. n = 11 Sham-veh, 10 CCI-veh, 10 CCI-istra, 10 CCI-ceft and 10 CCI-istra+ceft; Chi-square test, a pairwise comparison of 2 by 2 table: Sham-veh vs CCI-veh, p < 0.0001; CCI-veh vs. CCI-istra, p < 0.0001; CCI-veh vs. CCI-ceft, p < 0.0001; CCI-veh vs CCI-istra+ceft, p < 0.0001). Data presented as means ± SEM.*p<0.05, **p<0.01, ***p<0.001).

Statistical analysis

Statistical analysis of data was carried out using GraphPad Prism 10 and STATVIEW software (SAS Institute, Cary, NC, U.S.A). Data are presented as means ± standard error of the mean (SEM). One-way ANOVA parametric statistics with post hoc Fisher’s protected least significant difference test or the Kruskal-Wallis non-parametric test were used to determine statistical differences between three or more groups. Unpaired Student’s t-test was used for two-group comparison. A pairwise comparison of 2 by 2 table, using the chi-square test was performed to analyze the percentage of myoclonic seizures and tonic-clonic seizures. Normality and lognormality test were used to verify the normal distribution between groups. Significance was accepted at *p < 0.05.

Results

GLT-1 and A2AR expression changes following TBI

We hypothesized that early activation of the A2AR after TBI enhances subsequent brain activity through interaction with glial glutamate transport. To understand the temporal sequence of dysregulated adenosine signalling and glutamate transport following TBI, we performed a time-course study to assess the expression levels of the A2AR and of GLT-1 in the hippocampus ipsilateral of the controlled cortical impact in mice (Figure 1A). The CCI model induces gradual cortical damage, which extends to the hippocampus by 10 days post-CCI (Figure 1B). Our results showed that GLT-1 expression was significantly reduced (40%) in CCI-exposed mice when compared to sham treated mice from day 3 to 28 post-CCI (Figure 1C). In contrast, the A2AR was found to be 40% up-regulated from day 3 until day 28 following CCI when compared with the sham control (Figure 1C). We conclude that dysregulation of adenosine signalling and glutamate transport is an early and lasting consequence of TBI.

CCI leads to increased brain excitability and inflammation

The CCI model is commonly used to study drug effects on TBI-induced epilepsy with mice subjected to CCI typically developing epilepsy after a seizure-free latent period of 2–3 months (Di Sapia et al. 2021). To determine whether CCI leads to a lower seizure threshold during the seizure-free latent period, 11 mice were administered KA (10 mg/kg, i.p.) at 4 weeks post-CCI (or a sham treatment) (Figure 1C) (Alves et al. 2025). EEGs were then recorded for 90 min and seizure severity analyzed (Figure 1C). Mice from the CCI group (n = 6) showed a significant increase in seizure severity including seizure total power (nearly 3-fold increase) and amplitude (almost 2-fold increase) when compared to sham injured mice (n = 5) during a 90 min recording period starting at the time of the KA injection (Figure 1D, E). Gliosis is a hallmark of brain hyperexcitability and epilepsy. To evaluate if in our model markers of inflammation, such as microglia and astrocytes are increased, we analysed the expression of GFAP and Iba-1 by immunofluorescence at the conclusion of the experiment. As expected, our results showed a significant increase in GFAP (p = 0.004) (Figure 2A) and Iba-1 (p = 0.0001) (Figure 2B) expression in the contralateral side of the hippocampus in animals from the CCI group when compared with sham controls. It is important to note that immunohistochemistry analysis was only performed in the contralateral side of the hippocampus, because the ipsilateral side shows progressive cell loss and tissue deterioration as a consequence of the CCI injury. We conclude that CCI leads to increased brain hyperexcitability and gliosis during the seizure-free latent period, which can increase the risk to develop epilepsy.

Figure 2. Gliosis is increased 28 days after TBI.

Figure 2.

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A. Immunofluorescence images and corresponding graphs showing an increase in GFAP positive cells in the ipsilateral side of the hippocampus in CCI-exposed mice. B. Immunofluorescence images and corresponding graph showing an increase of Iba-1 positive cells in ipsilateral side of the hippocampus in CCI-exposed mice. (n = 6 sham and 5 CCI animals; unpaired t-test: GFAP: t(9) = 3.837, p = 0.004; Iba-1: t(9) = 6.333, p = 0.0001). Scale bar = 100 μm. Data presented as means ± SEM. ** p<0.01, ***p<0.001).

Blockade of the A2AR and activation of GLT-1 attenuates CCI-induced brain hyperexcitability

To obtain functional evidence that GLT-1 and the A2AR play roles in TBI-induced brain hyperexcitability, 45 mice subjected to CCI were randomly treated with istradefylline (A2AR inhibitor, 3.5mg/kg) or ceftriaxone (GLT-1 agonist; 200mg/kg) or a combination with both istradefylline and ceftriaxone, or vehicle (20% DMSO in 0.9% saline) at 6 h, 12 h and 24 h after the injury (acute treatment). Mice were then challenged with KA (10 mg/kg, i.p.), a glutamatergic agonist, at day 28 post-CCI and EEGs were recorded for 90 min starting at the time of KA injection (Figure 3A).

Mice with controlled cortical impact (CCI) injuries treated with a combination of istradefylline and ceftriaxone showed a significant delay in seizure onset when challenged with KA four weeks later. Compared to CCI-vehicle treated mice, the combination therapy group had nearly twice the time to the first seizure (Figure 3B, C). In contrast, mice treated with istradefylline or ceftriaxone alone did not exhibit a significant delay in seizure onset (Figure 3B, C). Both istradefylline and ceftriaxone treatments reduced the severity of the electrographic seizure by 40% and 50%, respectively, during the 90-minute recording period (Figure 3B, D, E). However, the combination therapy group showed a more pronounced effect, with a 75% reduction in total EEG power, indicating a significant reduction in seizure severity (Figure 3B, D, E). Furthermore, mice treated with the combination of istradefylline and ceftriaxone had significantly increased levels of GLT-1 24 hours after KA-induced seizures, compared to CCI-vehicle treated mice (p = 0.0110) (Figure 3F).

To confirm that our results were not dependent on KA, we used a mechanistically different chemoconvulsant, pentylenetetrazol (PTZ), a GABA receptor antagonist, to induce seizures in the animals (Gerbatin et al. 2019; Romariz et al. 2023). As before, we used the A2AR inhibitor (istradefylline), GLT-1 agonist (ceftriaxone) and a combination of both drugs (istradefylline and ceftriaxone). A total of 51 mice were randomly allocated to the 5 different groups and treated 3 times at 6 h, 12 h and 24 h after CCI injury. A systemic injection of PTZ (35 mg/kg) was used to test brain susceptibility to tonic-clonic seizures at day 32 after CCI, and latency to myoclonic and to tonic-clonic seizures was analysed (Figure 4A). Following PTZ-induced seizures, CCI-vehicle treated mice showed a marked decrease in the latency to myoclonic and tonic-clonic seizures compared to sham-vehicle treated mice, indicating an increase in CCI-induced brain hyperexcitability, which mirrored the results observed in the KA-induced seizure model (Figure 4B–E). CCI-exposed mice treated with the combination of ceftriaxone and istradefylline exhibited a reduced seizure phenotype. This result is supported by a significantly longer time to develop myoclonic seizures (7-fold increase) and tonic-clonic seizures (4-fold increase) in CCI-exposed mice treated with the combination therapy compared to the CCI-exposed and vehicle-treated group (Figure 4B–E). Istradefylline- or ceftriaxone-treated CCI-exposed mice also showed reduced seizure severity, but only istradefylline-treated mice showed a significant delay (p = 0.0026) in the latency to develop tonic-clonic seizures, while ceftriaxone-treated mice displayed only a trend to delay the time to develop tonic-clonic seizures when compared to CCI-exposed vehicle-treated animals (Figure 4C, D). The levels of GLT-1 were significantly increased in mice treated with ceftriaxone alone (p = 0.0105) and in combination with Istradefylline (p = 0.0011), compared to the CCI-vehicle control group after PTZ-induced seizures (Figure 5A). Notably, GLT-1 levels in these treatment groups were restored to levels comparable to those in the sham-vehicle group (Figure 5A).

Figure 5. Acute combined istradefylline / ceftriaxone treatment restores GLT-1 levels 28 days following CCI.

Figure 5.

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A. Immunofluorescence images and corresponding graph showing GLT-1 fluorescence intensity in the contralateral side of the hippocampus of mice from the PTZ group 32 days post-CCI. (n = 11 Sham-veh, 10 CCI-veh, 10 CCI-istra, 10 CCI-ceft and 10 CCI-istra+ceft; One-way ANOVA parametric statistics with post hoc Fisher’s: F(4,47) = 3.534; CCI-veh vs. CCI-ceft, p = 0.0111; CCI-veh vs. CCI-istra+ceft, p = 0.0012; Scale bar = 100 μm). Data presented as means ± SEM. *p<0.05, **p<0.01.

We conclude that a combined treatment with an A2AR antagonist and a GLT-1 activator provides protection against CCI-induced brain hyperexcitability and restores GLT-1 levels using two different seizure induction approaches (KA vs PTZ) that provide complementary information and offer a more comprehensive understanding of post-traumatic brain changes.

Discussion

Here we provide evidence for the efficacy of a novel disease modifying therapeutic strategy to ameliorate the downstream sequelae of TBI. Using a CCI mouse model of TBI, we demonstrate that pharmacological blockade of the A2AR in combination with activation of GLT-1 within the first 24 h post-injury: i) significantly reduces brain hyperexcitability; and ii) restores GLT-1 levels following injury. Notably, these therapeutic benefits persist for at least 32 days post-CCI. Our findings identify a novel and translationally relevant treatment paradigm to reduce TBI-induced brain hyperexcitability, leveraging two FDA-approved drugs. This approach may provide a translatable therapeutic avenue to mitigate the long-term neurological consequences of TBI.

TBI encompasses a primary mechanical insult to the brain, followed by secondary pathogenic events driven by molecular cascades that propagate damage to adjacent tissue. The secondary injury contributes to the progression of TBI over weeks and months, influencing behavioral outcomes such as memory deficits and seizures. These alterations include inflammation, brain hyperexcitability, dysregulation of adenosine metabolism and excitotoxicity, due to the excessive release of glutamate into the extracellular space (Kumar, Boles, and Wagner 2015; Sharma et al. 2019; Mukherjee et al. 2020; Klein et al. 2018).

Our first finding supports a mechanism in which there is an early activation of the A2AR after TBI followed by downregulation of GLT-1 which lasts at least 28 days after CCI, suggesting that dysregulation of adenosine signalling and glutamate transport is an early and lasting consequence of TBI. These results are consistent with previous studies, which showed a downregulation of GLT-1 (Cui et al. 2014; Goodrich et al. 2013), and upregulation of the A2AR following injury in experimental TBI models (Zhao et al. 2017). Furthermore, studies have demonstrated that A2AR activation reduces GLT-1 expression and function, thereby decreasing glutamate uptake and increasing excitatory neurotransmission (He et al. 2020; Matos et al. 2013).

The interaction between A2ARs and GLT-1 seems to play a crucial role in regulating seizure susceptibility in epilepsy. Although it is known that blocking the A2AR reduces excitatory neurotransmitter release and activating GLT-1 enhances metabolic glutamate clearance, a combination therapy to exploit both mechanisms synergistically had not been explored previously. One of the major findings of our study is that a combination of A2AR blockade and GLT-1 activation significantly decreased brain hyperexcitability, a hallmark of epilepsy development. More importantly, our study shows for the first time that these effects extend well beyond the time of active dosing, suggesting a lasting disease-modifying effect.

Previous studies support the use of ceftriaxone for treating neurological conditions, but these studies only demonstrate symptomatic relief, not disease modification (Cui et al. 2014; Jagadapillai et al. 2014; Zumkehr et al. 2015; Krzyzanowska et al. 2017; Tikhonova et al. 2017; Romariz et al. 2023). For example, Romariz et al. (2023) showed that delayed ceftriaxone treatment (55d post-CCI) enhances glutamate transporter mRNA and reduces PTZ-induced seizures in CCI mice (Romariz et al. 2023). This supports the use of ceftriaxone to treat evoked seizures after a brain injury, but unlike our study, it did not demonstrate a long-lasting disease-modifying effect. By restoring GLT-1 expression in the injured cortical tissue, ceftriaxone has been shown to attenuate post-traumatic astrogliosis, and to partially prevent chronic post-traumatic seizures (Goodrich et al. 2013). As described above, our results document GLT-1 downregulation as an immediate consequence of the injury, which was maintained for at least 28 days. The loss of GLT-1 following TBI and the resultant deficiency in glutamate clearance may be sufficient to promote several mechanisms, which enhance brain excitability. Our results suggest that ceftriaxone can rescue GLT-1 levels in the acute post-traumatic period and thereby attenuate brain excitability.

On the other hand, the A2AR is known to modulate astrocyte activity, thereby influencing glutamate uptake. For example, A2AR activation reduces glutamate uptake by decreasing Na+/K+−ATPase activity in astrocytes (Matos et al., 2013). In addition, studies have shown that A2AR inhibition can restore normal endothelial GLT-1 function, potentially being an effective treatment for brain injury (Bai et al., 2018). Moreover, A2AR antagonists have shown protective effects against seizures (Canas et al. 2018; Nasrallah et al. 2024; Xu et al. 2022) and brain inflammation (Marti Navia et al. 2020; Pedata et al. 2014; Orr et al. 2009). In line with these findings our study demonstrated a protection against brain hyperexcitability when mice subjected to CCI were treated with an A2AR antagonist immediately following the injury, suggesting an important role for A2AR in TBI-induced brain alterations.

The novelty of our study lies in testing a combination therapy of istradefylline and ceftriaxone to make synergistic use of their potential benefits on TBI-induced brain hyperexcitability. Currently, there are no approved pharmacotherapies for disease modification after TBI. Therefore, there is an urgent need for novel therapies to mitigate brain damage from acute insults, preventing subsequent hyperexcitability and ongoing neurodegeneration. The A2AR inhibitor istradefylline) and the GLT-1 activator ceftriaxone (a are FDA-approved drugs for the adjunct treatment of Parkinson’s disease (Chen and Cunha 2020) and amyotrophic lateral sclerosis, respectively, offering an expedited development timeline based on established safety profiles. Thus, our findings hold clinical relevance, as transient early intervention with our combination therapy commencing 6 hours after a TBI has lasting disease modifying effects and the potential to mitigate long-term neurological consequences of TBI. Early intervention strategies have the potential to reduce subsequent seizure frequency, severity, and related complications, improving patient outcomes. Our study provides a proof-of-concept study for the therapeutic modulation of A2AR and GLT-1 signalling in TBI-induced brain hyperexcitability.

Outlook

In summary, our findings may offer a path for a novel therapeutic strategy to prevent TBI-induced brain hyperexcitability, leveraging two FDA-approved drugs. Early pharmacological intervention targeting the A2AR and GLT-1 presents a promising avenue to mitigate long-term neurological consequences of TBI, warranting further investigation, and further validation in PTE models.

Supplementary Material

1

Acknowledgements

We would like to thank Dr. Kao-tai Tsai for statistical analysis support.

Funding

This work was supported by funding from the National Institutes of Health (NIH) through grants R01NS127846, to DB; from the US Department of the Army through contract W81XWH2210638 to DB; and the EpiPurines grant 101032321 from the European Commission to MA. We would also like to apologize to those authors whose relevant work was not cited here for space reasons.

Abbreviations:

TBI

Traumatic brain injury

PTE

Post-traumatic epilepsy

A2AR

Adenosine A2A receptor

GLT-1

Glutamate transporter-1

KA

Kainic acid

PTZ

Pentylenetetrazol

CCI

Controlled cortical impact

ATP

Adenosine triphosphate

EAATs

Excitatory amino acid transporters

IACUC

Institutional animal care and use committee

EEG

Electroencephalogram

SDS-PAGE

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis

TBST

Tris buffered saline-tween

PFA

Paraformaldehyde

SEM

Standard error of the mean

NKAs

Na+/K+-ATPases

TLE

Temporal lobe epilepsy

SE

Status epilepticus

Footnotes

Ethics approval

All animal experiments were approved by the Experimental Animals Welfare and Ethical Inspection of the Institutional Animal Care and Use Committee of Rutgers, The State University of New Jersey and were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

CRediT authorship contribution statement

Conceptualization and methodology: M.A. and DB; validation, formal analysis, investigation and data curation: M.A., R.R.G., R.K., D.F, T.E. and D.B.; resources: M.A. and D.B.; writing - original draft preparation: M.A.; writing - review and editing: All; supervision: T.E. and D.B.; funding acquisition: M.A. and D.B.; All authors have read and agreed to the published version of the manuscript.

Declaration of Competing Interest:

DB is co-founder and CDO of PrevEp Inc.

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