Transient P2X7 Receptor Antagonism Produces Lasting Reductions in Spontaneous Seizures and Gliosis in Experimental Temporal Lobe Epilepsy: A Review
Introduction
This research was conducted by a multiorganisational team into the role of P2X7 receptors in epilepsy, and the effect of targeting the receptor has on the epileptic brain(1). This paper was submitted to the Journal of Neuroscience in November 2015, accepted in March 2016 and published on 1 June 2016.
Significance of field of study
Epilepsy has been defined as a brain disease with a predisposition for enduring, unprovoked seizures(2). Latest available statistics suggest epilepsy affects over fifty million people worldwide(3) and , with approximately 8-9 in every 1,000 people in Ireland over the age of 5 suffering from the disease(4). Currently, treatment of the disease generally focusses on supressing seizures with no changes to the underlying pathophysiology that actually causes epilepsy; furthermore, up to 30% of patients do not respond to currently available antiepileptic drugs (AED)(5). At the time when this research was conducted, it was identified that a need existed to identify a novel method of treatment for epilepsy which not only targeted the underlying pathophysiology but which also had long term disease modifying characteristics(5).
Aim of Study
Interest has grown within the field of study of epilepsy in targeting of gliosis and neuroinflammation(5-7). While the role of P2X7 receptors in epilepsy was unclear at the time of research, it had emerged as a potentially important target for a variety of neurological disorders due to its role in neuroinflammation following the pathological release of ATP(8) following some form of insult to the brain. The hypothesis of the study was that not only microglia but neurons also expressed P2X7 receptors in epileptic brains and that targeting these receptors could reduce hyperexcitability and gliosis, therefore presenting a new method of treatment of patients suffering from epilepsy(1).
Methodology
The methodology chosen was in line with previous studies conducted into P2X7 receptor targeting(9). In order to identify cells that expressed P2X7 receptors, reporter mice which were engineered to express enhanced green florescent protein (EGFP) simultaneously with the P2X7 receptor gene were used. This method was chosen in order to try and mitigate limitations surrounding obtaining reliable staining patterns using by P2X7 receptor antibodies during immunohistochemistry(10, 11)
The mice were anesthetized and a number of procedures were performed so that researchers could conduct a number of different functions:
1. The installation of a guide cannula for intra-amygdala targeting and subsequent induction of Status Epilepticus.
2. The Affixation of cortical skull-mounted EEG electrodes to allow for continuous monitoring/recording.
Once the mice had recovered, baseline EEG readings were recorded and kainic acid was delivered to the basolateral amygdala through the guide cannula to induce Status Epilepticus; Lorazepam, an anticonvulsant, was then delivered 40 mins later to stop morbidity and mortality. This method of induction of Status Epilepticus damages the brain in specific regions; mainly the ipsilateral CA3 subfield with some limited damage to the CA1 and DG/hilar region(12). This method has advantages over other models due to its ability to generate unclustered spontaneous seizures displaying clinical features(13, 14).
Mice that developed spontaneous seizures were then randomly assigned into 2 groups, vehicle and treatment. From this point forward the groups were monitored throughout three distinct phases: Baseline (days 5-10), treatment (days 11-15), and washout (days 16-21). During these periods EEG was continuously recorded and up to 10 seizures per mouse (6 per mouse in the treatment group during the treatment and wash out phases), randomly selected, were scored according to their severity as being either partial or severe. The main parameters of interest other than severity that were measured were daily seizure frequency and average duration.
The treatment group received a newly available P2X7 receptor antagonist JNJ-47965567, which was chosen due to its potency and central availability after injection(15). Mice were killed at various time intervals of between 15 mins and 6 hours to not only confirm that the antagonist reached the brain but also how long it remained detectable in the brain. This was detected to be somewhere between 4 and 6 hours, making JNJ-47965567 suitable for systematic injections(1). The treatment group were injected twice daily with JNJ-47965567; the vehicle group were injected twice daily with a vehicle solution.
At different stages during the 21 day period mice were killed and their brains prepared for various different types of analysis such as immunochemistry, histopathology, western blot analysis, and electrophysiological recordings. During histopathology and immunochemistry antibodies and florescent indictors were used to determine what types of cells were expressing P2X7 receptors, this allowed for the hypothesis to be tested as cell types such as microglia and neurons could be differentiated. Control measures were also employed to confirm the specificity of these antibodies.
Electrophysiological recordings were used to detect and alteration in the electrical activity of the individual cell types.
Human tissue samples of the temporal lobe were received under consent from 12 patients undergoing surgery for intractable TLE at Beaumont Hospital Dublin. 12 control samples from brains with no known history of neurological disease were received from the University of Maryland Brain and Tissue Bank. The groups were balanced in terms of gender (6 males and 6 female); control samples were chosen so that the average age and over all age range of both groups were comparable. Human tissue samples were subjected to Western Blot analysis in an effort to try and confirm findings of the murine model in human epileptic brains.
Findings
There were a number of important findings from this study;
The role of expression of P2X7 Receptors in specific cells types is now somewhat clearer. The expression of the receptor in microglia was previously known, however its expression in astroglia and neurons was less understood(8); by determining cell-specific expression, P2X7 receptors’ role in modulating neuroinflammation and hyperexcitability could be better understood. Determination of specific cells was completed using a combination of specific antibodies and florescent indicators as previously described, 14 days after Status Epilepticus was induced. Indicators for neuronal expression were observed in all hippocampal layers and were the most observed cell type in the CA1 and DG sublayers; although in low numbers in the CA3 sublayer, it should be noted that this is also an area of extensive neuron loss. Microglia indicators were observed across each subfield. No astroglia indicators were observed, although in subsequent studies this finding has been challenged(16, 17). Furthermore to this P2X7 receptors were also found to be upregulated in the CA1 band. This proved to be significant as through a series of control measures and Western blot analysis involving both human epileptic and control brain tissue samples, it was confirmed that P2X7 receptor upregulation is a pathophysiological feature in Human Temporal Lobe Epilepsy.
Cells from epileptic tissue expressing P2X7 receptors were found to have an altered response to P2X7 receptor agonists, displaying a much larger agonist-evoked inward current when compared to non-expressing cells. This combined with the observation of a greater localization P2X7 receptors at presynaptic sites suggests that there may be enhanced or abnormal P2X7 receptor function in epilepsy; this offers an explanation as to why targeting P2X7 receptors has an effect on seizures.
The chosen P2X7 receptor antagonist JNJ-47965567 was observed to have a lasting effect on seizure suppression. As previously described mice from both groups were continuously monitored during all three phases using video EEG for daily frequency and average duration of seizures. There was no difference in seizure duration during any of the three phases. There was no observed difference in seizure frequency during the baseline phase; however the probability of spontaneous seizures was significantly reduced in the treatment group when compared to the vehicle group during both the treatment and washout phases by 64% and 69% respectively. It was hypothesised by the research team that the increased seizure suppression during washout may be linked to the time required for effect of receptor inhibition on the underlying pathophysiology to take effect. This serendipitous observation of seizure suppression post treatment is significant due to implications that P2X7 receptor antagonism may have disease-modifying effects.
Following further scoring of seizure severity, it was observed that the probability of having a severe seizure did not differ between the two groups during either the treatment or washout phases; indicating that JNJ-47965567 supresses seizures rather than reducing severity. Seizure suppression was similar to conventional AEDs(13).
Tissue samples from both the vehicle and treatment groups were examined on cessation of recording using immunostaining for both astrogliosis and microgliosis. All sublevels of the hippocampus examined in the vehicle group displayed widespread microgliosis and astrogliosis. This was in contrast to the treatment group in which the number and immunoreactivity of both microglia and astroglia in all examined hippocampal subfields was greatly reduced. This reduction may account for the observed disease modifying effects or P2X7 receptor targeting. Although the role of microglia in seizures is unknown(18), it has been observed that astrogliosis alone is sufficient to induce epilepsy in mice models(19, 20) through several different mechanisms.
Limitations
A number of limitations with the study have been highlighted in the study(1) as well in an interview with one of the co-authors(21):
1. Even though selection of mice for the vehicle and treatment groups was conducted randomly, mice selected for the treatment group did tend to display shorter, less frequent seizures during the baseline phase. This may have had an impact on the success of P2X7 receptor and skewing the results to a more positive outcome.
2. Measurements of brain levels of JNJ-47965567 was conducted using brain tissue from healthy mice. Use of epileptic mice brain tissue would provide a better insight into its relationship with seizure suppression.
3. At the time of writing, researchers lacked the ability to gather information in relation to ATP release in vivo during seizures. Such technology has since become available for use in future research(21).
4. This study used only one type of induced epilepsy model, in this case kainic acid was used. In such studies 2 different models are often used to validate findings. A model of epilepsy induced by a traumatic brain injury more closely mimics human TLE(21).
5. Further experimentation is needed to determine the exact causes of glial changes and whether or not they are as a direct or indirect result of P2X7 receptor inhibition.
Further applications of research
There are many further applications and ongoing studies relating to these findings which were highlighted in an interview with the author(21):
1. A proposed study of P2X7 receptors using mice with a traumatic brain injury induced model of epilepsy. As previously stated such a model would more closely mimic human TLE.
2. Further identification of exact cells expressing P2X7 receptors by using knockout mice.
3. Developing a test to identify persons most suitable for this type of treatment for epilepsy by testing purine levels in the blood.
As P2X7 receptors have an important role in neuroinflammation, this study has applications in studies into other neurological disorders, such as:
1. The reduction of diabetic retinopathy, which is one of the leading causes of blindness worldwide(22).
2. Localized inhibition of P2X7 receptors in the spinal cord of rats has been observed to improve neurogenic bladder dysfunction(23).
3. A potential anti-metastatic agent for use in the treatment certain types of cancers(24).
4. The treatment of many forms of inflammatory diseases affecting a variety of organs and systems such as the cardiovascular, kidney, airways, liver, and bladder(24).
Conclusion
The findings of this research may have lasting implications on both the research and treatment of epilepsy in the future. Findings with relation to P2X7 receptor expression, upregulation and activity have helped further understand its role in epilepsy, which was previously poorly understood. This will have a positive impact on our understanding of epilepsy as a whole.
As kainic acid produces a model of epilepsy which is resistant to conventional AED(13), the observed seizure suppression of targeting P2X7 receptors may provide an avenue of treatment for the approximately 30% of epilepsy patients not responding to currently available AED(5).
Arguably more importantly for the treatment of epilepsy is the potential for P2X7 receptor inhibition to produce lasting disease-modifying effects, which has been seen by many as being a paramount requirement for the treatment of epilepsy(5). This finding can be viewed as the most important outcome of this research.
Finally information with regards to P2X7 receptors inhibition has widespread applications in the study or other neurological and inflammatory disorders(24); meaning that this study is significant in fields far wider than just the study of epilepsy.