Raw data obtained from multi-electrode electrophysiological recordings of hippocampal slices

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
Hippocampal slices (350 μm thick) were prepared from Sprague Dawley rats (n=3; 3-4 weeks old; Janvier Laboratories) and cut using a vibratome (Leica VT1200S) in an ice-cold oxygenated, modified ACSF, continuously aerated with 95% O2 and 5% CO2 and containing Glucose 11 mM, NaHCO3 25 mM, NaCl 126 mM, KCl 3.5 mM, NaH2PO4 1.2 mM, MgCl2 1.3 mM and CaCl2 2 mM. Slices were then incubated at RT for at least 1 H in ACSF. Recordings were performed on hippocampal slices using multielectrode arrays (MEA). Slices were continuously perfused with the oxygenated ACSF at the rate of 3 mL/min with a peristaltic pump (MEA chamber volume: ~1 mL). KA (300 nM) and VH-N412 (0.1, 1 and 10 μm) were added to the perfusion solution to assess the effects of VH-N412 compound on KA-induced increase of neuronal firing. Complete solution exchange in the MEA chamber was achieved 20 s after the switch of solutions. The perfusion liquid was continuously pre-heated at 37°C just before reaching the MEA chamber with a heated-perfusion cannula (PH01, MultiChannel Systems, Reutlingen, Germany). The temperature of the MEA chamber was maintained at 37 ± 0.1°C with a heating element located in the MEA amplifier headstage. The spike numbers per second recorded at each electrode were averaged for 30 s slots and normalized to the mean spikes rate value at t = 20-30 min (10 last minutes of KA exposure period). Individual data from independent experiments were then pooled and the mean values of the normalized spike rates (± SEM) were plotted as a function of time (before and after exposure to VH-N412). The control values (KA alone) were averaged from 3 rats, 3 slices and 18 electrodes. The dose response curves from the KA + VH-N412-treated slices were averaged from 3 rats, 4 slices and 25 electrodes.