Anti-High Mobility Group Box-1 Monoclonal Antibody Attenuates Seizure-Induced Cognitive Decline by Suppressing Neuroinflammation in an Adult Zebrafish Model

Abstract

Epilepsy is a chronic brain disease afflicting around 70 million global population and is characterized by persisting predisposition to generate epileptic seizures. The precise understanding of the etiopathology of seizure generation is still elusive, however, brain inflammation is considered as a major contributor to epileptogenesis. HMGB1 protein being an initiator and crucial contributor of inflammation is known to contribute significantly to seizure generation via activating its principal receptors namely RAGE and TLR4 reflecting a potential therapeutic target. Herein, we evaluated an anti-seizure and memory ameliorating potential of an anti-HMGB1 monoclonal antibody (mAb) (1, 2.5 and 5 mg/kg, I.P.) in a second hit Pentylenetetrazol (PTZ) (80 mg/kg, I.P.) induced seizure model earlier stimulated with Pilocarpine (400 mg/kg, I.P.) in adult zebrafish. Pre-treatment with anti-HMGB1 mAb dose-dependently lowered the second hit PTZ-induced seizure but does not alter the disease progression. Moreover, anti-HMGB1 mAb also attenuated the second hit Pentylenetetrazol induced memory impairment in adult zebrafish as evidenced by an increased inflection ration at 3 and 24 h trail in T-maze test. Besides, decreased level of GABA and an upregulated Glutamate level was observed in the second hit PTZ induced group, which was modulated by pre-treatment with anti-HMGB1 mAb. Inflammatory responses occurred during the progression of seizures as evidenced by upregulated mRNA expression of HMGB1, TLR4, NF-κB, and TNF-α, in a second hit PTZ group, which was in-turn downregulated upon pre-treatment with anti-HMGB1 mAb reflecting its anti-inflammatory potential. Anti-HMGB1 mAb modulates second hit PTZ induced changes in mRNA expression of CREB-1 and NPY. Our findings indicates anti-HMGB1 mAb attenuates second hit PTZ-induced seizures, ameliorates related memory impairment, and downregulates the seizure induced upregulation of inflammatory markers to possibly protect the zebrafish from the incidence of further seizures through via modulation of neuroinflammatory pathway.

Naturally Occurring HMGB1 Inhibitor, Glycyrrhizin, Modulates Chronic Seizures-Induced Memory Dysfunction in Zebrafish Model

“…Standard husbandry settings were maintained in the animal facilities of Monash University Malaysia for conducting the entire experimental work where the water system of zebrafish tank is temperature (between 26 and 30 °C) and pH (between pH 6.8 and pH 7.1) maintained and further adjusted with a light and darkness cycle as stated in our earlier reported study. 80 TetraMin Tropical Flakes and live brine shrimps (Artemia) bought from Bio-Marine (Aquafauna, Inc. United States) were used to feed zebrafish (thrice a day). The standard zebrafish tanks (length 36 cm; a width 26 cm; and a height of 22 cm) were used to keep zebrafish, and the water circulation system is connected to offer continuous aeration.…”

“…76−78 Reduced level of NPY in our second hit PTZ group depicts the occurrence of seizure and impairment in learning and memory, which corroborates the reduced mRNA expression of NPY in PTZ and second hit PTZ induced seizure in adult zebrafish. 79,80 However, pretreatment with GL 25, GL 50, and GL 100 does not exert any significant effect in modulating the mRNA expression of NPY when compared to the second hit PTZ group.…”

“…Second hit PTZ induced chronic seizure model in adult zebrafish was developed as per the experimental protocol from our earlier documented finding. 80 Rather than dissolving in the water, pilocarpine (on day 1) and PTZ (on day 10) were injected ip as per the earlier reported study. 87 This is due to the fact that a small volume of water is used to limit the amount of proconvulsants required and having zebrafish in such a small volume of water may lead to negative impact on learning and memory.…”

“…The RT-PCR was performed as per our earlier documented study. 31,80 The detail on the primer sets used for the RT-PCR is provided in the Supporting Information.…”

Post-Translational Modification of HMGB1 Disulfide Bonds in Stimulating and Inhibiting Inflammation

“…Recent evidence indicates that stimulated sensory neurons actively secrete HMGB1 in an antidromic fashion by molecular mechanisms that remain elusive [ 35 ] ( Figure 2 D). The neuronally released HMGB1 is most likely disulfide HMGB1 since it acts via TLR4 to mediate inflammation and pain [ 13 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 ] and disulfide HMGB1 is the single redox form of HMGB1 capable of binding to the MD-2/TLR4 receptor complex [ 48 ]. Functional consequences regarding the neuronal HMGB1-regulation of inflammation will be discussed later in this review.…”