DOI: 10.5061/dryad.b5mkkwhq1
title: Raw data of hypothermia and seizure intensity scores

description: 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.
description: 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<em> </em>(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.
description: # Raw data of hypothermia and seizure intensity scores 
 [https://doi.org/10.5061/dryad.b5mkkwhq1](https://doi.org/10.5061/dryad.b5mkkwhq1) ## Description of the data and file structure We assessed our VH-N412 conjugate that induces hypothermia in a model of Kainate (KA)-induced seizures using adult male FVB/N mice. KA was administered subcutaneously (s.c.) in FVB/N mice at the dose of 45 mg/kg. Five groups of mice were generated: SHAM, SE, SE + VH-N412, SE + NT(8-13), SE + diazepam (DZP). Body temperature was monitored using a rectal probe before KA injection and every 30 min during 2.5 H thereafter. SE occurred around 2 H after KA injection (KA-2H) and was characterized by stage 5-6 seizures and often associated with some hyperthermia (non-significant). VH-N412 administered at the dose of 4 mg/kg eq. NT 30 min after SE onset (SE30), hence 2.5 H after s.c administration of KA (KA-2H), invariably led to transient hypothermia which persisted at least 2 H. This hypothermia was associated with a significant decrease of seizures in the SE + VH-N412 group compared with the SE group. A subset of animals was administered i.p. a high dose of DZP (15 mg/Kg), used as a positive control for its anticonvulsant effects in seizure models and its hypothermic effects. No significant variations of body temperature or seizure intensity score were observed when the NT8-13 alone was administered at SE30, as compared with SE animals at all time points. ### Files and variables #### File: Hypothermia\_seizures\_RAW\_DATA\_Final.xlsx **Description:** Body temperature of mice injected with the proconvulsant agent Kainate, with and without administration of VH-N412 molecule that induces hypothermia. ##### Variables * Body temperature was measured in degrees Celsius (°C) as a function of time measured in minutes. #### File: Seizure\_intensity\_scores\_RAW\_DATA\_Final.xlsx **Description:** Seizure intensity scores of mice injected with the proconvulsant agent Kainate, with and without administration of VH-N412 molecule that induces hypothermia. ##### Variables * Seizure activity was scored as a function of time measured in minutes, by visual inspection according to a modified Racine (1972) 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.