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.

Olfactory Tubing Maze (OTM)

The hippocampus-dependent learning and memory performance of control mice injected with saline solution (SHAM), SE and SE + VH-N412 mice were assessed 3 weeks after SE (12-week-old male mice). Behavioral assessment, based on the OTM, was carried out as we previously described (Roman et al., 2002; Girard et al., 2014) and we have shown that the FVB/N mouse strain used in the present study performed very well in this task (Girard et al., 2016). Briefly, mice were trained to learn odor-reward associations. The mice placed on water restriction were submitted to the presentation of odors in testing chambers (TC). Two synthetic odors (strawberry and jasmine), coming from 2 distinct arms and randomly assigned to these arms, were simultaneously presented to the mice. Each of these 2 odors was arbitrarily associated either with a drop of water (the reward) or with a non-aversive but unpleasant sound, which were delivered depending on the arm chosen by the mice. The OTM was composed of 4 identical TC joined to each other forming a circular structure in which mice could move freely only in a clockwise direction. The procedure was fully automated. The movement of the mice was detected by photoelectric cells. Odors, water, sound delivery, and the automatic doors separating the different TC were controlled by a microcomputer running with LabVIEW software (National Instruments, Austin, TX, USA) that also automatically recorded the behavioral data. The learning procedure included 3 habituation days followed by 5 days of training, each daily session being composed of 12 trials/odor presentations corresponding to 3 clockwise laps/blocks of 4 trials. Two parameters were examined: the inter-trial interval (ITI) and the percentage of correct responses. The ITI was calculated as the time between the response to an odor presentation in one testing chamber and the response in the next chamber. The evolution of the ITI reflects the fact that, after a response to an odor, the animal must learn to backtrack to the testing chamber and to run to the next chamber waiting for the opening of the entrance door for the next trial. This constitutes a procedural aspect of the task. The percentage of correct responses was the ratio of the number of correct responses to the total number of odor presentations per session. This percentage was used to evaluate the effectiveness of the association between an odor and its reinforcement, a process that pertains to the hippocampus-dependent declarative memory subcategory.

Open field test

As we described previously (Girard et al., 2016, 2014), the open field test was used to assess both exploratory behavior and spontaneous locomotor activity 4 weeks after SE and VH-N412 treatment. The mice were tested using an open field square made of white plastic with 50 x 50 cm surface area and 45 cm-high walls. Testing was carried out in a dimly illuminated room (40 lux). Monitoring was done by an automated tracking system using a video camera mounted above the apparatus (Viewpoint VideoTrack version 3.0, Lyon, France). The field was divided virtually into 2 regions of interest: a central area (20 x 20 cm) and a peripheral area. The animals were placed in the center of the field and 2 behavioral parameters were registered during 2 consecutive 5 min sessions: i) the percentage of time spent in the central part versus total time, ii) the total traveled distance. The square was cleaned prior to the test and between each animal with 70% ethanol.