Raw data of hypothermia and seizure intensity scores

Preclinical and clinical studies show that mild to moderate hypothermia is neuroprotective in sudden cardiac arrest, ischemic stroke, perinatal hypoxia/ischemia, traumatic brain injury and seizures. Induction of hypothermia largely involves physical cooling therapies, which induce several clinical complications, while some molecules have shown to be efficient in pharmacologically induced hypothermia (PIH). Neurotensin (NT), a 13 amino-acid neuropeptide that regulates body temperature, interacts with various receptors to mediate its peripheral and central effects. NT induces PIH when administered intracerebrally. However, these effects are not observed if NT is administered peripherally, due to its rapid degradation and poor passage of the blood brain barrier (BBB). We conjugated NT to peptides that bind the low-density lipoprotein receptor (LDLR) to generate “vectorized” forms of NT with enhanced BBB permeability. We evaluated their effects in epileptic conditions following peripheral administration. One of these conjugates, VH-N412, displayed improved stability, binding potential to both the LDLR and NTSR-1, rodent/human cross-reactivity and improved brain distribution. In a mouse model of kainate (KA)-induced status epilepticus (SE), VH-N412 elicited rapid hypothermia associated with anticonvulsant effects, potent neuroprotection and reduced hippocampal inflammation. VH-N412 also reduced sprouting of the dentate gyrus mossy fibers and preserved learning and memory skills in the treated mice. In cultured hippocampal neurons, VH-N412 displayed temperature-independent neuroprotective properties. To the best of our knowledge, this is the first report describing the successful treatment of SE with PIH. In all, our results show that vectorized NT may elicit different neuroprotection mechanisms mediated by hypothermia and/or by intrinsic neuroprotective properties.

Methods
Forty-eight young adult male FVB/N mice (25-30 g; 9-10 weeks-old) were injected s.c. with a single dose of Kainate (KA, 45 mg/kg) to generate mice with spontaneous recurrent seizures as a hallmark of status epilepticus (SE). KA-injected mice were individually housed and received a 0,5 mL i.p. dose of glucose G5 and had free access to agarose-doliprane gel (2.4%) to avoid pain. Mice were observed for 9 hours for onset and extent of seizure activity. Seizure activity was scored by visual inspection according to a modified Racine scale: stage 0: exploration, “normal behavior”; stage 1: immobility, staring; stage 2: head nodding and/or extended tail; stage 3: forelimb clonus and/or circling behavior; stage 4: rearing with forelimb clonus and falling; stage 5: continuous rearing and falling; stage 6: severe tonic-clonic seizures. Only animals having displayed at least stage 5 were included in the study, and 5 animal groups were generated: 1) animals injected 30 min after the onset of the SE with one intravenous (tail vein) bolus injection of the VH-N412 compound at the dose of 4 mg/kg eq. NT (group “SE30 + VH-N412”, n=13); 2) animals injected 30 min after the onset of the SE with an intravenous bolus injection of NT at the dose of 4 mg/kg eq. NT (group “SE30 + NT(8-13)”, n=14); 3) animals injected 30 min after the onset of the SE with Diazepam (DZP) i.p at the dose of 15 mg/kg (group “SE30 + DZP”, n=5); 4) animals injected 30 min after the onset of the SE with saline 0.9% (vehicle control) (group “SE”, n=11); 5) a negative control group of age-matched mice was administered saline 0.9% alone (group “SHAM”, n=5). All animals were weighed and monitored daily. Body temperature was assessed using a digital thermometer rectal probe before KA injection and every 30 min during 3 H thereafter. Seizure intensity score was assessed following KA injection, and every 30 min during 3 H. To study mice in the chronic stage of epilepsy with spontaneous seizures, they were observed daily (at least 3 hours per day) for general behavior and occurrence of spontaneous recurrent seizures (SRS). These are highly reproducible in the mouse KA model, allowing for visual monitoring and scoring of epileptic activity. After 3 weeks, most animals exhibited SRS with 2 to 3 seizures per day, similar to previous observations. The detection of at least one spontaneous seizure per day was used as criterion indicating the animals had reached the chronic phase that was ultimately confirmed by mossy fiber sprouting.