5.3.2.1 Total HMGB1 abundance in the hippocampus by western blotting
Twenty-eight days following the onset of SE, there was no significant difference in hippocampal HMGB1 abundance, as measured by western blotting, between the healthy controls (mean HMGB1/Actin ratio = 0.422 ± 0.09 arbitrary units, n=4) and those with spontaneous seizures resulting from pilocarpine-SE (0.5251 ± 0.057, n=18, figure 5.7).
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Figure 5.7 Hippocampal high mobility group box-1 (HMGB1) expression in the brain of mice treated with pilocarpine-status epilepticus (SE) compared to vehicle-only control. Results are expressed as the mean (± standard error of the mean) ratio of the optical densities of HMGB1 and actin bands, n=4 control and n=18 pilocarpine-SE.
5.3.2.2 Serum expression of HMGB1
In pilocarpine-treated epileptic mice, serum total HMGB1 was significantly higher than that of representative control NMRI mice (control 5.32±0.92ng/ml vs 16.59±2.072ng/ml, p=0.0011, figure 5.8).
Figure 5.8 Quantification of high mobility group box-1 (HMGB1) by ELISA in mouse serum from control (n=5) and mice exposed to pilocarpine-status epilepticus and experiencing spontaneous epileptic seizures (n=18). Results are expressed as the mean (± standard error of the mean, **p<0.01 by Mann Whitney test)
168 5.3.2.3 Relationship between serum HMGB1 and seizure frequency
Serum total HMGB1 was not influenced by (or indeed, did not influence) the frequency of convulsive seizures (Racine stage 3-5) occurring in the 14 days prior to sacrifice (figure 5.9).
5.3.2.4 Relationship between serum HMGB1 and time-since-last seizure
No relationship was identified between serum total HMGB1 concentration and the time since last seizure activity (Racine stage 3-5, Mann Whitney U, p=0.3564). Mice were subcategorized into those that had experienced a convulsive seizure within the preceding 72 hours prior to sacrifice and those that had been seizure-free in that period (figure 5.10). A 72-hour cut-off was chosen solely to ensure adequate numbers of animals in each of the comparator groups.
Figure 5.9 Relationship between total serum concentration of high mobility group box-1 (HMGB1) and total number of convulsive seizures (Racine stage 3-5) experienced across a 14 day period prior to sacrifice. Spearman’s rank correlation is illustrated by the solid line, with the corresponding correlation co-efficient reported. The dotted line represents the mean concentration of serum HMGB1 in healthy control mice (n=5, 5.32ng/ml)
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Figure 5.10 Box and whisker plots depicting serum high mobility group box-1 (HMGB1) concentrations in mice exposed to pilocarpine-status epilepticus. Each box represents the 25th and 75th percentiles. Lines outside the boxes represent the minimum and maximum limits. Lines inside the box represent the median. Time since last seizure comparison was performed by Mann Whitney test, ns: not-significant, p=0.3564.
5.4 Discussion
Both clinical and experimental evidence suggest that HMGB1 is involved in the pathogenesis of seizure disorders (Maroso et al., 2010; Zurolo et al., 2011). What remained unclear was whether up-regulation occurred as a consequence of brain insult, seizures, epileptogenesis or the chronic epileptic state. Together with work undertaken in the KA-model (chapter 4), the present study aimed to identify and clarify whether HMGB1 expression in brain and blood is involved in provoked seizures in normal brain (MES), provoked seizures following brain insult (KA), and/or spontaneous epileptic seizures (pilocarpine-epilepsy) and to provide evidence that HGMB1 expression in blood is not simply a marker of recent seizures.
Following an isolated MES-induced seizure, the non-acetylated form of HMGB1 in brain peaked at 24 hours, consistent with a purely necrotic release process. This is in contrast to the KA-SE model (chapter 4), wherein the non-acetylated form of HMGB1 in brain peaked much earlier, at 3-6 hours following KA-induced seizures. This was
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then followed by a delayed but significant rise in the acetylated form in brain at 24 hours in the KA model only. This difference in timing likely reflects the extreme difference in models of isolated seizures versus prolonged SE. The MES test is proposed to be a predictive model of generalized tonic-clonic seizures limited to hind-limb extension followed by clonus (Krall et al., 1978). The entirety of the seizure activity lasts less than a minute. In contrast, the excitotoxic glutamate analogue kainate causes widespread neuronal damage to pyramidal cells in the CA3 area of the injected hippocampus (Balosso et al., 2008; Ravizza et al., 2006e).
In the majority of animal models of SE, marked gliosis, axonal sprouting, neuronal cell loss and consequent neurogenesis is seen. However, neuronal damage has been shown to most likely result from the seizure activity itself, rather than as a consequence of the metabolic disturbances that occur alongside SE (Walker et al., 2002). This was revealed by experiments using the GABA antagonist bicuculline to induce SE in adolescent baboons. Bicuculline-induced SE caused hyperpyrexia, severe hypotension, and profound hypoglycaemia in the baboons (Meldrum and Brierley, 1973). However, when the same study was undertaken in paralyzed and mechanically ventilated baboons, to prevent the systemic disturbances, significant neuronal damage was still observed as a consequence of seizures, indicating that prolonged seizure activity is the key pathogenic feature in these models (Meldrum et al., 1973). In addition, the intensity and duration of SE is critical in determining whether an animal will develop spontaneous seizures. Rescue therapy with diazepam within 30 minutes of SE onset limits the degree of neuronal damage and fewer of the animals exhibit spontaneous seizures (White, 2002). Therefore, it is unsurprising that the degree of HMGB1 expression in the brain following a single, brief (<1 minute) seizure was minimal and delayed compared to prolonged, recurrent seizures in the KA model (30 minutes). Furthermore, expression of the inflammatory isoforms of HMGB1 was almost absent in the MES-exposed brain. HMGB1 in its acetylated form cannot re-enter the nucleus and thus builds up in the cytosol (Bonaldi et al., 2003; Lu et al., 2012; Lu et al., 2014), which is consistent with active inflammatory production. In the KA-SE study (chapter 4), mixed expression of reduced and disulphide HMGB1 peaked at 24 hours. By contrast in this study,
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following an isolated seizure the rise was in the reduced form alone. Fully-reduced HMGB1 acts as a chemoattractant (Venereau et al., 2012) whereas disulphide HMGB1 has been shown to be the only form capable of inducing cytokine production (Yang et al., 2012). The relative lack of inflammatory isoforms of HMGB1 in this study of MES seizures showed that HMGB1-mediated inflammatory repair was not initiated as a consequence of a single seizure. The study is limited by a duration of 24 hours and it is therefore possible that inflammatory isoform expression is delayed beyond this time point. However, this is unlikely, given its appearance within 24 hours in the KA-SE model wherein seizure severity is much more severe. MES seizures are not known to induce lasting brain injury and indeed, MES-exposed rodents do not go on to develop spontaneous epileptic seizures (White, 2002). Therefore, the brain results of this study support the notion that HMGB1 inflammatory isoforms are relevant to the epileptogenic process and the subsequent development of epilepsy, and are not merely seizure-related phenomena.
In peripheral blood following single MES-seizure, a non-significant trend towards early release (4 hours) of HMGB1 was seen, and LCMS confirmed the isoform present to be non-acetylated and predominantly in the reduced form. This suggests that early release of HMGB1 following seizure activity is from damaged cells with the capacity to induce chemotaxis for the purpose of repair (Venereau et al., 2012). A later rise in the reduced and to a lesser extent, disulphide isoforms, was seen at 24 hours in the absence of a corresponding elevation in brain. This transient rise occurs likely as a result of monocyte and macrophage release consequent to seizure activity and is indicative of a recent seizure. Indeed, in the KA-SE model, 14 days following the initial SE, a significant rise in acetyl and disulphide HMGB1 was seen, possibly coinciding with the onset of spontaneous epileptic seizures. As discussed in detail in chapter 4, maturation of HMGB1 in peripheral blood, from the necrosis-released reduced form to the cytokine-activating disulphide form occurs following experimental stroke in mice (Liesz et al., 2015). A similar pathology appears to be happening here, with the reduced isoform released early following a seizure (examined in two distinct models, KA and ME) then replaced by the acetylated, disulphide pathological isoforms two weeks after the initial insult.
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Taken together, the findings of this study demonstrated that inflammatory isoforms of HMGB1 are not significantly released, in brain or blood, as a consequence of an isolated seizure on the background of normal brain. The study is limited in its time frame to 24 hours following seizures at which point a significant rise in total HMGB1 was seen. It is possible that HMGB1 release, and isoform expression, changes after the first 24 hours and therefore a longer study, up to 72 hours following a single seizure, would be required to exclude this possibility.
In the pilocarpine model of chronic epilepsy, total hippocampal HMGB1 did not differ significantly between mice experiencing recurrent seizures and the sham- operated controls, suggesting that by 28 days following the initial SE, changes in localised HMGB1 expression in brain have resolved. In the KA model, total hippocampal HMGB1 expression remained significantly elevated from 1 to 14 days following the initial SE; therefore resolution of the protective inflammatory reaction driven by HMGB1 likely occurs between 14 and 28 days after brain insult. Taken together, this suggests that HMGB1 is continuously expressed following both initial brain insult and during the critical epileptogenic period, wherein changes are occurring in the hippocampus leading to an excitable focus for future seizures (Wang et al., 2005). Further investigation is required to determine whether the isoforms released differ between healthy animals and those experiencing spontaneous seizures.
In contrast, in the peripheral blood, the epileptic mice experiencing recurrent spontaneous seizures expressed significantly more HMGB1 than control, seizure-free animals. This suggests that ongoing release of HMGB1 may occur as a consequence of recurrent seizure activity, but that the source of the HMGB1 may not be spill over from the CNS and may in fact represent peripheral synthesis. Indeed, it is possible that peripheral immune production of HMGB1 is a driver for seizure activity, crossing the disrupted BBB and entering the brain to aggravate the hyperexcitable focus and induce seizures. This may then contribute to an ongoing cycle involving lowered seizure threshold and consequently, seizures. This is followed by local release of inflammatory mediators including IL-1β and HMGB1. Inflammatory mediators in turn induce intracellular Ca2+ influx and further seizure activity. Alternatively, it is also
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possible that peripheral release of HMGB1 occurs as a consequence of muscle injury resulting from recurrent seizure activity. However, no relationship was demonstrated between serum HMGB1 and either the total number of spontaneous convulsive seizures experienced or the time since last seizure. Furthermore, there is data on file at UCB from accelerometry measurements in these animals which would suggest that they were less active (and moved less far each day) following pilocarpine-treatment than they had done previously, despite experiencing recurrent seizures. Together, this argues against the possibility that HMGB1 is simply a marker of recent seizure activity, released as a consequence of muscle insult. This requires further exploration in humans experiencing both complex partial and generalised seizures, in order to identify the relative contribution of the seizure type. Furthermore, analysis of the muscle injury marker creatine kinase (CK) would help to elucidate whether HMGB1 is released as a consequence of muscular involvement. The white blood cell count (WBC) is known to increase following vigorous physical exercise (McCarthy and Dale, 1988; Tossige-Gomes et al., 2014) and could potentially be the source of peripheral HMGB1.
It is possible that there are other, non-seizure related factors that are involved in the expression of HMGB1 in peripheral blood in chronic epilepsy. Various brain insults are associated both with disruption of the BBB (Abbruscato and Davis, 1999; Betz et al., 1994; Brown and Davis, 2002; Banks, 1999) and a high risk of developing epilepsy (Annegers et al., 1988; Annegers et al., 1998; Jennett, 1975; Burn et al., 1997; Richardson and Dodge, 1954; Sander et al., 1990). Therefore, activation of central immunity in response to brain injury can foreseeably trigger activation of the peripheral immune response, through spill over of inflammatory mediators from the CNS. Indeed, within 24 hours of clinical stroke, stroke patients have been shown to have significantly higher HMGB1 serum concentrations than controls (Muhammad et al., 2008). Similarly, in experimental stroke, elevated HMGB1 serum levels occur 4 hours following occlusion of the middle cerebral artery, without a concomitant rise at the mRNA level (Muhammad et al., 2008). This suggests that, in the early stages, HMGB1 is probably attributable to a spill-over from necrotic neural cells. In contrast, delayed appearance of the disulphide form of HMGB1 in serum (but not brain) has
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been shown 24 hours following experimental stroke, suggesting a delayed maturation in the periphery which may well contribute to ongoing release in some individuals. Therefore recurrent seizure activity may be perpetuating release of inflammatory mediators, including HMGB1, which have the demonstrated capacity to provoke and exacerbate seizures (Maroso et al., 2010). Additionally, SE in humans has been associated with excessive muscular activity and relative leucocytosis (Simon, 1985). However, few studies have examined the relationship between single seizure and WBC. In a study involving various seizure types (38 simple partial seizures, 109 complex partial seizures and 91 generalized tonic–clonic seizures) one third of generalized seizures were associated with a significant increase in WBC count. Mean post-seizure sampling time was 21.62 ± 19.33 hours, indicating that in those in which WBC is elevated, the response is delayed. A clear correlation between the length of a seizure and increase in WBC count was seen. Further analysis is required to determine the isoforms present in both brain and blood in the pilocarpine epilepsy model, in particular the disulphide and acetylated isoforms that were shown to be significantly elevated 14 days following KA-SE (Chapter 4). This will help to determine whether release is driven by peripheral immune activation.
In conclusion, the results of this study show that inflammatory isoforms of HMGB1 are not significantly involved within the first 24 hours following isolated MES- induced seizures in mice. In addition, serum, but not brain, total HMGB1 is significantly elevated in chronic epileptic mice experiencing regular spontaneous seizures, the underlying driver for this release remains unclear at present.
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6
High-Mobility Group Box-1 as a
Mechanistic Biomarker in Drug
Resistant Epilepsy; Comparing
Drug-resistance with Drug-
176 6.1 Introduction