Oral Glutamine Supplementation Increases Seizure Severity in a Rodent Model of Mesial Temporal Lobe Epilepsy

Abstract

Background:

Glutamine synthetase (GS) is the only enzyme known to synthesize significant amounts of glutamine in mammals, and loss of GS in the hippocampus has been implicated in the pathophysiology of medication refractory mesial temporal lobe epilepsy (MTLE). Moreover, loss-of-function mutations of the GS gene causes severe epileptic encephalopathy, and supplementation with glutamine has been shown to normalize EEG and possibly improve the outcome in these patients. Here we examined whether oral glutamine supplementation is an effective treatment for MTLE by assessing the frequency and severity of seizures after supplementation in a translationally relevant model of the disease.

Methods:

Male Sprague Dawley rats (380 - 400 g) were allowed to drink unlimited amounts of glutamine in water (3.6% w/v; n = 8) or pure water (n = 8) for several weeks. Ten days after the start of glutamine supplementation, GS was chronically inhibited in the hippocampus to induce MTLE. Continuous video-intracranial EEG was collected for 21 days to determine the frequency and severity of seizures.

Results:

While there was no change in seizure frequency between the groups, the proportion of convulsive seizures was significantly higher in glutamine treated animals during the first three days of GS inhibition.

Conclusion:

The results suggest that oral glutamine supplementation transiently increases seizure severity in the initial stages of an epilepsy model, indicating a potential role of the amino acid in seizure propagation and epileptogenesis.

1. Introduction

Glutamine synthetase (GS, also known as glutamate-ammonia-ligase, GLUL) catalyzes the formation of glutamate and ammonia to glutamine and is the only enzyme known to synthesize significant amounts of glutamine in mammals (1). Glutamine is critical for several biological processes such as synthesis of the excitatory and inhibitor neurotransmitters glutamate and gamma-aminobutyric acid (GABA) (2) and ammonia detoxification (3). Not surprisingly, loss-of-function mutations of the GS gene are associated with considerable mortality and morbidity, and the small number of humans reported with such mutations have suffered from multi-organ failure, encephalopathy, and epilepsy (4). However, in one case study in which glutamine was enterically supplemented to an infant with GS deficiency, glutamine normalized the EEG, suggesting that the amino acid might prevent GS-associated seizures (5).

The activity of GS is also reduced in patients with mesial temporal lobe epilepsy (MTLE) (6), neocortical epilepsies (7), and glioblastoma-associated epilepsy (8). However, the reduction is limited to discrete brain regions, such as the hippocampus in the case of MTLE. We have demonstrated that chemical inhibition or genetic deletion of GS in the hippocampus and neocortex of rodents causes epileptic seizures and neuropathological changes similar to human MTLE (9, 10). This suggests that the focal loss of brain GS in human epilepsy is implicated in the causation of the disease. Thus, we now ask whether supplementation with glutamine can be used to treat MTLE. To this end, we used long-term video-intracranial EEG monitoring to quantify the effects of oral glutamine supplementation on the frequency and behavioral severity of epileptic seizures using a well-established rat model of GS-deficient MTLE (9, 11). Our working hypothesis is that glutamine supplementation will reduce the frequency and severity of recurrent seizures.

2. Materials and Methods

2.1. Chemicals and animals:

All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise noted. Male Sprague Dawley rats (380 - 400 g) were obtained from Harlan (Indianapolis, IN). Rats were housed in a temperature-controlled colony room (21 – 23°C) on a 12-hour light-dark cycle. Animals were allowed food and water ad libitum and underwent 1 week of acclimation prior to treatment. All procedures were approved by the Institutional Animal Care and Use Committee at Yale University.

2.2. Treatment:

One group of animals (n = 8) was given a 3.6% (w/v) solution of glutamine in water beginning 10 days prior to surgery and continuing until the end of the experiment. Another group of animals (n = 8) was given water during the experiment’s entirety. Blood was sampled at the time of surgery to determine blood glutamine concentrations.

2.3. Surgery:

Following 10 days of glutamine supplementation in the treatment group and water consumption in the control group, rats were anesthetized with 1-2% isoflurane (Baxter, Deerfield, IL) in O₂ and placed in a stereotaxic frame (David Kopf Instruments, Tujunga, CA). A 30-gauge stainless steel infusion cannula (6.5 mm) attached to a plastic pedestal (Plastics One, Roanoke, VA) was stereotaxically targeted to the entorhinal cortex using the following coordinates, with bregma marking zero for the mediolateral (ML) and anteroposterior (AP) directions, and the top of the skull marking zero for the dorsoventral (DV) direction: AP 7.8 mm, ML 5.2 mm, DV −6.5 mm. The infusion cannula, lowered into the brain, was connected via a plastic tubing to a subcutaneously implanted Alzet osmotic pump (Model 2004, Durect Corp., Cupertino, CA), delivering a continuous flow of 0.25 μL/h for ~28 days. Pump reservoirs were filled with MSO [2.5 mg/mL, dissolved in Dulbecco’s phosphate buffered saline (PBS) to achieve a delivery of 0.625 μg of MSO per hour]. Following placement of the guide cannula, four epidural stainless-steel screw electrodes (Plastics One, Roanoke, VA) were implanted to record cortical EEG activity. The female socket contacts of each electrode were inserted into a plastic pedestal (Plastics One) and secured by acrylated urethane adhesive (Loctite 3106 Light Cure Adhesive, Henkel Corp., Rocky Hill, CT) to form a head cap.

2.4. Video-intracranial EEG monitoring:

Following surgery, rats were housed individually in custom plexiglass cages. EEG was measured continuously for 21 days with a digital recording unit (CEEGraph Vision LTM, Natus/Bio-Logic Systems Corp., Middleton, WI). The EEG was correlated with simultaneous video recordings and used to identify seizures by visual inspection. Seizure stage was classified according to a modified Racine scale(12), and was performed as described previously by Dhaher, Damisah (13).

2.5. Blood sampling for glutamine analysis:

Venous blood was collected in lithium-heparinized tubes at the time of surgery, 10 days after the start of glutamine consumption. The blood was centrifuged, and the plasma was processed for glutamine quantification using the AccQ-Tag Ultra Derivatization Kit (Waters, Milford, MA). The samples were processed according to the kit’s instructions and run on an Acquity-Xevo TQS mass spectrometer (Waters) as previously described(10).

2.6. Statistics:

Two-way repeated measures ANOVA was used to compare the seizure frequency (defined as the total number of seizures per day) between groups [glutamine vs. control] and between time-periods [early (Days 1 - 3) vs. late (Days 4 - 21)]. The justification for this separation of time periods is based on previous findings demonstrating that the seizure frequency during the first three days post-MSO treatment differs from post-treatment days 4 - 21(13, 14). Seizure frequency was calculated for the early time-period as the total number of seizures over days 1 - 3 divided by 3; and for the late time-period, as the total number of seizures over days 4 - 21 divided by 18. ANOVA of group over time-period was carried out separately to study the frequency of (1) all seizures [Figure 2a], (2) non-convulsive seizures [stage 1-2; Figure 2b], (3) and convulsive seizures [stage 3 through 5; Figure 2c]. ANOVAs were followed by a post hoc Fisher least significant difference (LSD) test. A two-sample t-test was used to compare glutamine levels between the glutamine-treated rats and the non-treated control group. Significance was defined as p < 0.05.

Figure 2:

Oral glutamine supplementation increases the severity of recurrent seizures in the MSO model of MTLE. (A) While the number of seizures were significantly higher during days 1-3 of MSO infusion compared to days 4-21 (p < 0.001 for glutamine-treated group [early vs late], p = 0.02 for water-control group [early vs. late]), there was no significant difference in seizure frequency between these two groups when the two time-periods were analyzed separately. (B) The water-control group showed a significantly higher number of non-convulsive seizures during the first three days of MSO treatment compared to days 4-21 (p = 0.04). The glutamine drinking treatment group showed a higher number of non-convulsive seizures during the first three days of MSO treatment compared to days 4-21 that approached significance (p = 0.06). There was no significant difference between the two groups in the frequency of non-convulsive seizures when time-periods were analyzed separately. (C) Glutamine-treated rats showed a significantly higher frequency of convulsive seizures during days 1-3 of MSO infusion compared to days 4-21 (p < 0.001), as well as compared to the first three days of MSO infusion in the water-control group (p = 0.04). The water-control group did not demonstrate a significant decrease in non-convulsive seizures over time. All values are provided as mean ± SEM.

3. Results:

After 10 days of glutamine consumption, the plasma concentration of glutamine was significantly higher in the rats consuming glutamine (630.1 ± 24.5 μM) compared to water controls (562.9 ± 16.1 μM; p = 0.03, Fig. 1). The effect of glutamine consumption on seizure frequency was evaluated, and repeated measures ANOVA indicated no significant effect of group, a significant effect of time-period [F (1,7) = 25.17, p < 0.001], and no significant group by time-period interaction (Fig. 2A). The post hoc Fisher LSD test indicated that the daily number of seizures for the water-treated rats was significantly higher during days 1 - 3 (5.08 ± 0.62) when compared to days 4 - 21 (1.43 ± 0.24; p = 0.02, Fig. 2A). Furthermore, the glutamine treated rats also showed a significantly higher frequency of seizures on days 1 - 3 (7.5 ± 2.15) than on days 4 - 21 (1.05 ± 0.23; p < 0.001). The total seizure frequency was not different between the two groups within each of the two time periods.

Figure 1:

Oral glutamine supplementation increases blood glutamine levels. Blood samples were collected at time of surgery, ten days into consumption of 3.6% glutamine solution (n = 8) or water (n = 8). Glutamine concentrations were higher in the rats that drank the glutamine solution versus the water control. All values are provided as mean ± SEM.

The effect of glutamine consumption on the frequency of non-convulsive seizures was next evaluated, and repeated measures ANOVA indicated no significant effect of group, a significant effect of time-period [F (1,7) = 9.50, p = 0.008], and no significant group by time-period interaction (Fig. 2B). The post hoc Fisher LSD test indicated that the water treated rats had a higher frequency of non-convulsive seizures during the early time period after surgery (2.58 ± 0.75) than during the late time-period after surgery (0.88 ± 0.16, p = 0.04, Fig. 2B). In the glutamine treated rats, the decrease in the frequency of non-convulsive seizures from days 1-3 (2.29 ± 0.90) to days 4-21 (0.79 ± 0.14) approached significance at p = 0.06 (Fig. 2B). The non-convulsive seizure frequency was not different between the two groups within each of the two time periods.

Finally, the effect of glutamine consumption on the frequency of convulsive seizures was evaluated, and repeated measures ANOVA indicated no significant effect of group, a significant effect of time-period [F (1,7) =15.24, p = 0.002], and no significant group by time-period interaction (Fig. 2C). The post hoc Fisher LSD test indicated that glutamine treated rats experienced more convulsive seizures during the first three days after surgery (5.21 ± 1.56) versus water treated rats during the same time-period (2.5 ± 0.92, p = 0.04), and when compared to days 4-21 after surgery (0.31 ± 0.11, p = 0.001, Fig. 2C). There was no decrease in the frequency of convulsive seizures over time in the water control group.

4. Discussion:

To our knowledge, this is the first study to illustrate the effects of oral glutamine supplementation on recurrent, mesial temporal lobe-onset seizures. The main findings of this study are that oral glutamine supplementation causes increased plasma glutamine concentrations and worsens the severity of seizures during the early phase of hippocampal GS inhibition. There was a 10 percent increase in plasma glutamine concentration when compared to the water treated rats. This is mainly due to the rats drinking a saturated solution of glutamine ad libitum, with no signs of aversion, for several days prior to the blood measurement. The glutamine increase was modest and likely limited by effective metabolism of the amino acid by intestinal cells, liver and muscle (15). Similarly, in a case glutamine supplementation study carried out in a Sudanese child with congenital GS deficiency, relatively high doses of enteral glutamine were required for several weeks in order to elevate blood glutamine just above the lower limit of normal (5).

The finding of increased seizure severity in the glutamine-treated rats is unexpected and suggests that orally delivered glutamine augments the propagation of the seizures from the seizure focus in the GS-inhibited hippocampus to other regions of the brain during the first days of GS inhibition. These results direct us to an important hypothesis that an increased seizure load during early stages of epileptogenesis may lead to a future escalation in a variety of downstream epileptogenic events currently seen in our model, such as altered neuronal connectivity (11), increased neuronal loss (9), and long-term changes in the frequency and severity of recurrent seizures (11, 13, 14).

The pro-convulsant effect of glutamine on the seizures can be explained by several mechanisms. Firstly, in the event that the excess glutamine enters the brain, as has been previously established (16), some of the amino acid may be converted to glutamate and ammonia via the phosphate-activated glutaminase reaction (16), and if glutamate and ammonia are allowed to accumulate, mitochondrial damage, seizures, and neuron loss could occur (17). Even though the net flux of glutamine normally is directed from the brain to the blood (18), because of the established role of glutamate in epilepsy (6), testing the brain glutamate/ammonia accumulation hypothesis is necessary. It is also possible that the elevated extracellular glutamine may be converted to neurotransmitter GABA by glutamate decarboxylase (GAD) containing, inhibitory neurons. However, considering the fact that the number of excitatory neurons are nine times higher than inhibitory neurons (19) and 90% of all axon terminals release glutamate(20), it is most likely that the net effect of the enhanced neurotransmitter synthesis is increased excitatory transmission.

Secondly, it is possible that sustained high levels of enteric glutamine might affect seizures via a novel gut-brain signaling mechanism involving the vagus nerve or the formation of seizure-modulating, microbial metabolites. In fact, supplementation with enteric glutamine can alter the gut microbiota composition (21) Moreover, Olson et al. (22) showed that the effects of the ketogenic diet on seizures is mediated by the gut microbiota. Thus, we speculate that seizure-modulating effect by glutamine supplementation may, at least in part, be mediated by the gut/microbiota-brain axis.

It is of interest to note that the MSO paradigm is not the only epilepsy model in which glutamine is associated with seizures. Using the intrahippocampal kainate injection model of MTLE, Kanamori et al demonstrated that during seizures, there was an increase in extracellular brain glutamate that corresponded with a decrease in extracellular brain glutamine (23). When they inhibited neuronal glutamine uptake by blocking the sodium-coupled neutral amino acid transporter, the frequency of electrographic seizures was reduced (24). These data suggest that neuronal uptake of glutamine is important for triggering of seizures, possibly through increased synthesis of neurotransmitter glutamate.

Collectively, our study shows that, contrary to our original hypothesis, oral glutamine supplementation does not decrease the frequency and severity of seizures caused by hippocampal inhibition of GS, but rather transiently increases the severity of recurrent seizures. Because hippocampal GS-inhibition induces a state of progressive epileptogenesis (11, 14, 25), the seizure promoting effect of glutamine during the initial stages of our model, suggests that glutamine could modulate the epileptogenic process, particularly how seizures propagate from the primary seizure focus (the GS-inhibited hippocampus) to other parts of the brain. However, the exact long-term consequences of such modulation remain to be established.

Lastly, there is an increasing interest in the dietary therapies of the epilepsy. Dietary regimes such as ketogenic diet (26), modified Atkins diet (26) and medium chain triglyceride diet (27) have shown promise in the fight against epilepsy. More studies, like the present, are required to successfully identify commonly consumed metabolites, which may have an effect on epilepsy.