Brain Advance Access originally published online on October 8, 2006
Brain 2006 129(12):3209-3223; doi:10.1093/brain/awl239
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Thiorphan, a neutral endopeptidase inhibitor used for diarrhoea, is neuroprotective in newborn mice
1 Inserm, U676 Service de Neurologie Pédiatrique, Paris 2 Université Paris 7, Faculté de Médecine Denis Diderot Service de Neurologie Pédiatrique, Paris 3 Institut Pasteur Service de Neurologie Pédiatrique, Paris 4 AP HP, Hôpital Robert Debré Service de Neurologie Pédiatrique, Paris 5 Laboratoire MERCI UPRESA 2122, Faculté de Médecine et de Pharmacie Rouen, France 6 Department of Psychiatry, Division of Neurochemistry Innsbruck, Austria
Correspondence to: Dr Pierre Gressens, Inserm U 676, Hôpital Robert Debré, 48 Boulevard Sérurier, 75019 Paris, France E-mail: gressens{at}rdebre.inserm.fr
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Excitotoxic damage appears to be a critical factor in the formation of perinatal brain lesions associated with cerebral palsy (CP). When injected into newborn mice, the glutamatergic analogue, ibotenate, produces cortical lesions and white matter cysts that mimic human perinatal brain lesions. Neuropeptides are neuronal activity modulators and could therefore modulate glutamate-induced lesions. However, neuropeptides are rapidly degraded by peptidases. Racecadotril, which is rapidly metabolized to its active metabolite thiorphan, is a neutral endopeptidase (NEP) inhibitor used in clinical practice for diarrhoea with a remarkable safety profile. This study aimed to test the original hypothesis that thiorphan could be neuroprotective against ibotenate-induced lesions in newborn mice. Intraperitoneal administration of thiorphan reduced ibotenate-induced cortical lesions by up to 57% and cortical caspase-3 cleavage by up to 59%. This neuroprotective effect was long-lasting and was still observed when thiorphan was administered 12 h after the insult, showing a remarkable window for therapeutic intervention. Further supporting the neuroprotective effect of pharmacological blockade of NEP, mouse pups with a genetic deletion of NEP displayed a significantly reduced size of the ibotenate-induced cortical grey matter lesion when compared with wild-type animals. Thiorphan effects were mimicked by substance P (SP) and, in a less potent manner, by neurokinin A. Thiorphan effects were inhibited by blockers of NK1 and NK2 receptors. Real-time reverse transcription–polymerase chain reaction, autoradiography and immunohistochemistry confirmed the expression of NK1 and NK2 receptors in the neonatal murine neocortex. These data demonstrate that thiorphan prevents neonatal excitotoxic cortical damage, an effect largely mediated by SP. Thiorphan could represent a promising drug for the prevention of CP, which remains a challenging disease. In a broader context, these results also raise potential implications for the prevention of neurodegenerative diseases involving glutamate-mediated excitotoxic neuronal death.
Key Words: cerebral palsy; glutamate receptor; neuroprotection; neprilysin; NMDA
Abbreviations: CP, cerebral palsy; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; 125I-BH-SP, 125I-Bolton-Hunter substance P; MAPK, mitogen-associated protein kinase; NEP, neutral endopeptidase; NKA, neurokinin A; NKB, neurokinin B; NMDA, N-methyl-D-aspartate; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PK, protein kinase; SP, substance P
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Received April 7, 2006. Revised June 27, 2006. Accepted August 8, 2006.
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Introduction |
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Injury to the perinatal brain is a leading cause of death and disability in children. Of major concern, neurological handicap of perinatal origin is not decreasing in Western countries and has even been shown to increase in some countries (Hagberg and Hagberg, 1996
).
The major brain lesions associated with cerebral palsy (CP) are periventricular leucomalacia mostly occurring in preterm infants and cortico-subcortical lesions mostly observed in term infants. Several pre-conceptional, prenatal and perinatal factors have been implicated in the pathophysiology of brain lesions associated with CP (Dammann et al., 2002). Excess release of glutamate leading to excitotoxicity could represent an important molecular mechanism in the pathophysiology of brain lesions underlying CP. Plausibility of this hypothesis is supported by the observations that injection of glutamate agonists into the striatum, the neocortex or the periventricular white matter of newborn rodents or kittens produces histological lesions mimicking brain damage associated with CP such as neuronal migration disorders, polymicrogyria, cystic periventricular leucomalacia and hypoxic-ischaemic or ischaemic-like cortical and striatal lesions (Innocenti and Berbel, 1991; Barks and Silverstein, 1992; Marret et al., 1995, 1996; Gressens et al., 1997; McDonald et al., 1988; Redecker et al., 1998; Acarin et al., 1999; Follett et al., 2000).
Hypothermia, when introduced very rapidly after birth, has been shown to be neuroprotective in a subset of term human newborns (Eicher et al., 2005; Gluckman et al., 2005). However, the neuroprotection afforded by hypothermia is only partial and, at the present time, there is no pharmacological agent that is useful for the treatment of perinatal brain lesions (Gressens and Spedding, 2004). This has led to a search for more potent alternative therapeutic approaches. Neuropeptides are modulators of neuronal activity and could therefore modulate glutamate-induced brain lesions. This is supported by our evidence that vasoactive intestinal peptide agonists and nociceptin antagonists protect the periventricular white matter against excitotoxic lesions (Gressens et al., 1997; Laudenbach et al., 2001).
Neuropeptides are subjected to enzymatic proteolysis leading to their inactivation, and an inhibition of this degradation is a potential alternative therapeutic approach. Among the different identified peptidases, neutral endopeptidase (NEP or neprilysin) is the prototypical member of the M13 family of metalloproteases and is widely distributed in various tissues. NEP is involved in the regulation and metabolism of a variety of biologically active peptides such as tachykinins/neurokinins, enkephalins and neurotensin (Erdos and Skidgel, 1989; Roques et al., 1993). Neurokinins have neuroprotective effects against MPTP-induced motor disturbances (Chen et al., 2004) and excitotoxic neuronal cell death in the adult striatum (Sanberg et al., 1993). The neurokinin family contains substance P (SP), neurokinin A (NKA) and neurokinin B (NKB). They act on three specific receptors termed NK1, NK2 and NK3. The rank order of potency for the NK1 receptor is SP 
NKA > NKB, while it is NKA > NKB > SP for the NK2 receptor and NKB > NKA > SP for the NK3 receptor (Regoli et al., 1994; Maggi, 2000).
Different specific inhibitors of NEP have been described (Nawarskas et al., 2001). Interestingly, racecadotril (Tiorfan®), an NEP inhibitor, is used in clinical practice for diarrhoea with a remarkable safety profile (Schwartz, 2000). Racecadotril is rapidly and entirely metabolized to its active metabolite thiorphan.
Building upon this original concept of peptidergic neuroprotection, we made the hypothesis that blockade of NEP with thiorphan would lead to increased levels of neuropeptides, which would in turn protect the newborn brain. To test this hypothesis, we administered thiorphan to newborn mice that were subjected to an excitotoxic insult. In this model, thiorphan proved to prevent excitotoxic neuronal cell death. Further in vivo and in vitro studies revealed the key involvement of SP and NKA in these neuroprotective effects.
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Animals and drugs
All experimental protocols and procedures complied with Inserm guidelines and were conducted in accordance with the Policies on the Use of Animals and Humans in Neuroscience Research (revised and approved by the Society for Neuroscience in January 1995). Pregnant mice were housed in groups and fed with laboratory chow and water ad libitum. Swiss, C57Bl/6 NEP–/– (Lu et al., 1995
) and control C57Bl/6 NEP+/+ mouse pups of both sexes were used for these experiments.
Ibotenate (Tocris, Bristol, UK) was diluted in phosphate-buffered saline (PBS) containing 0.01% acetic acid. MK-801 (Tocris) was diluted in PBS. SP (Biovalley, Marne-la-Vallée, France), NKA (Biovalley), neurotensin (Neosystem, Strasbourg, France), [Lys8-
(CH2-NH)-Lys9] neurotensin (8-13) (JMV-449; Neosystem), and dantrolene (Sigma, St-Quentin Fallavier, France) were diluted in distilled water. NKB (Biovalley) was diluted in PBS containing 0.05% acetic acid. Win-51,708 (Sigma) and L-659,877 (Sigma), U73122
[GenBank]
(Biomol, Plymouth Meeting, PA, USA), PD98059 (New England Biolabs, Beverly, MA, USA), chelerythrine (Sigma) and Z-DEVD-FMK (Alexis, Lausanne, Switzerland) were diluted in PBS containing 0.05% dimethylsulphoxide (DMSO). Thiorphan (Bachem, Bubendorf, Switzerland) was diluted in PBS containing 5% ethanol–chloroform. H89 (Biomol) was diluted in PBS containing 0.05% methanol.
Ibotenate activates N-methyl-D-aspartate (NMDA) and metabotropic glutamatergic receptors but not 
-3-amino-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainate receptors. MK801 is an NMDA receptor antagonist. Win-51,708 is an NK1 receptor antagonist and L-659,877 is an NK2 receptor antagonist. JMV-449 is a synthetic neurotensin agonist. Chelerythrine is a protein kinase (PK) C inhibitor, H89 is a PKA inhibitor, U73122
[GenBank]
is a phospholipase C inhibitor and PD98059 is a mitogen-associated protein kinase (MAPK) kinase inhibitor or Mek-1 inhibitor. Dantrolene is an inhibitor of intracellular calcium release. Z-DEVD-FMK is a caspase-3 and caspase-7 inhibitor.
In most experiments, MK-801, thiorphan, neuropeptides, inhibitors of signal transduction, Z-DEVD-FMK or PBS were administered intracerebrally, concomitantly to ibotenate injection. In the subset of experiments where thiorphan was administered intraperitoneally, thiorphan, MK-801 or PBS were given immediately after the ibotenate injection.
Excitotoxic brain lesions
The following protocol was used for ibotenate administration to mouse pups. All ibotenate injections were administered on the fifth post-natal day (P5), as described previously (Marret et al., 1995; Gressens et al., 1997; Laudenbach et al., 2001, 2002; Tahraoui et al., 2001; Husson et al., 2002, 2005). Briefly, after pups were anaesthetized with isoflurane, injections were given under a warming lamp using a 25-gauge needle on a 50 µl Hamilton syringe mounted on a calibrated microdispenser. The needle was inserted 2 mm under the external surface of the scalp skin in the frontoparietal area of the right hemisphere, 1 mm from the midline in the lateral–medial plane and 1.5 mm from the junction between the sagittal and lambdoid sutures in the rostro-caudal plane. Two 1-µl boluses were injected 30 s apart. In all cases, histopathology confirmed that the tip of the needle reached the periventricular white matter. After the injections, the pups were returned to their dams.
Lesion size determination
At different time-points (1, 2, 5, 15 or 30 days) after intracerebral ibotenate injection on P5, the pups were decapitated, and the brains were fixed in 4% formaldehyde for 5 days. After embedding in paraffin, 15-µm sections were cut in the coronal plane, from the frontal to the occipital pole. Every third section was stained with cresyl violet, permitting an accurate and reproducible determination of the maximal sagittal fronto-occipital diameter of the lesion (which is equal to the number of sections where the lesion was present multiplied by 15 µm). This measure was used as an index of the lesion volume (Gressens et al., 1997; Laudenbach et al., 2001; Tahraoui et al., 2001; Husson et al., 2002). Throughout the experiments described below, two investigators blinded to the treatment groups of the animals determined the size of the lesion in each brain. Numbers of brains analysed in each experimental group are given in figures.
To further confirm the correlation between the sagittal diameter and the volume of the lesion, both the sagittal diameter and the total volume of the lesion were measured in a subset of experiments. Brains were serially sectioned as described above and volumes were measured using the Neurolucida software-controlled computer system (MicroBright-Field Europe, Magdeburg, Germany).
Immunohistochemistry for cleaved caspase 3
One, two, three, five, fifteen or thirty days following intracerebral injection of ibotenate plus 10 ng SP or ibotenate plus PBS at P5, Swiss pups were killed, and brains were fixed in formalin and embedded in paraffin. Every third section was stained with cresyl violet for lesion size determination. Sections adjacent to the most affected areas were reacted with anti-cleaved caspase-3 antibody (Cell Signaling, Beverly, MA, USA). Detection of labelled antigens was performed with avidin–biotin horseradish peroxidase kits (Vector, Burlingame, CA, USA) according to manufacturer's instructions. Labelled cells were counted without using a stereological dissector in a 0.25 mm2 area in the neocortical layers at the level of ibotenate-induced lesions. Ten fields from five brains (two fields from two non-adjacent slides per brain taken at the epicentre of the lesion) were studied in each group.
Real-time polymerase chain reaction (PCR) for NK1, NK2 and NK3 receptors
As described previously (Husson et al., 2005; Mesples et al., 2005), neocortex from E14, P0, P5, P10, P20 and P60 mice were rapidly homogenized in guanidium isothiocyanate (GnTC) solution before RNA isolation. DNaseI-treated RNA samples were reverse-transcribed using Iscript cDNA synthesis kit according to manufacturer's procedures (Biorad, Hercules, CA, USA) and then used in real-time reverse transcription–polymerase chain reaction (RT–PCR) experiments. cDNAs encoding the genes of interest were first analysed for secondary structure using M-fold software. Portions of sequences lacking any secondary structure were imported into oligo6 software to design highly stringent primer sets. These primers were blasted in cDNA database to ensure their specificity. For NK1, we chose the following oligonucleotides: 5'-CCCAACAGGACTTACGAGAA-3'OH and 5'-GCCAATCACCAGCAGAGG-3'OH, as sense and anti-sense primers, respectively. PCR amplification resulted in the specific formation of a 78 bp sequence corresponding to the region 1478–1555 of the published sequence of mouse NK1/TACR1 mRNA (NM009313). For NK2, 5'-TCCGATTCTGGCTTGTCAC-3'OH and 5'-GTTGTTTCCGATCTTTCACAC-3'OH were used. The amplified 102 bp sequence corresponded to the 1342–1443 region of the published AF399705
[GenBank]
sequence. For NK3, the primer set 5'-ACTTGCCTTCCCTCAATGTCT-3'OH and 5'-AAATGTTGCTTGGGACCTTAG-3'OH was used. This amplified a unique PCR fragment of 96 bp corresponding to the portion 867–961 of the published NK3/TACR3 gene (NM021382). To standardize the experiments, we designed using the same approach a primer set for the mouse ß2-microglobulin (ß2MG) gene (5-CCGGCTTGTATGCTATC-3'OH and 5-AGTTCAGTATGTTCGGCTTC-3'OH, as sense and anti-sense, respectively) and another set for glyceraldehyde-3-phosphate dehydrogenase (GAPDH: 5'-GGCCTTCCGTGTTCCTAC-3'OH and 5'-TGTCATCATACTTGGCAGGTT-3'OH). These oligonucleotides amplified an 87 pb region encoding the nucleotides 99–185 of the published sequence (X01836
[GenBank]
) and an 80 pb piece spanning the area 1093–1173 of published GAPDH (XM_111622.3) mRNA, respectively. Real-time PCR was set up using Sybergreen-containing Supermix from Biorad, for 50 cycles of a three-step procedure including a 20 s denaturation at 96°C, a 20 s annealing at 60°C, followed by a 20 s extension at 72°C. Amplification specificity was assessed by melting curve and sequencing analyses. Quantification of PCR samples was made using standard curves made from serial dilutions of control samples. Each experiment was run twice, and in both cases, samples were assessed in triplicate. Differences between samples were calculated as the difference between the specific ratios (gene/ß2MG or gene/GAPDH) calculated for each individual sample.
NK1 binding sites
The heads of untreated P5 pups were rapidly frozen at –30°C in isopentane, then transferred to –80°C until use. Twenty micrometre cryostat sections of forebrains were processed for binding of 125I-Bolton-Hunter substance P (125I-BH-SP; Amersham-Pharmacia Biotech, Saclay, France) as described previously, in Tris buffer including protease inhibitors (Quirion and Dam, 1986; Strigas and Burcher, 1996). Non-specific binding was evaluated in sections co-incubated with 1 µM unlabelled SP (Sigma). Binding localization was visualized after 7–10 days exposure on Kodak Biomax MR films.
Body temperature recording
Oral temperature was recorded in gently restrained P5 Swiss pups using a thermocouple probe. Oral temperature was recorded immediately before and 1, 2, 4, 8 and 24 h after intracerebral injection of ibotenate plus 10 ng SP, ibotenate plus 10 ng NKA, or ibotenate plus PBS.
Seizure activity
P5 Swiss pups were intracerebrally injected with ibotenate and one of the following drugs: 25 µg thiorphan, 10 ng SP or PBS. Seizures were recorded by video during the first 15 min of the 1st, 4th, 8th and 24th hour following the excitotoxic insult. After each period of recording, pups were returned to their dams. Seizures were defined as paroxysmal events characterized by clonic jerks of the contralateral limbs or of the whole body and were quantified by two blinded investigators. As there was no effect of the time elapsed after the insult on seizure occurrence (data not shown), data from the four time-points were pooled in each experimental group.
In vitro neuronal cell death
Two types of primary cultures were used: pure neurons and cultures of neurons plus astrocytes. Primary cultures of neurons were prepared from embryonic mouse brains dissected 14.5 days after conception. Primary cultures of astrocytes were obtained from P2 mouse brains (Laudenbach et al., 2002). The cortex from mouse brains were processed into HBSS–HEPES medium (Hanks Balanced Salt Solution plus Phenol Red plus HEPES buffer, pH 8; Gibco, Cergy Pontoise, France) and treated with trypsin at a concentration of 0.25%. Enzyme-treated tissues were dissociated into single cells by gentle pipetting and suspended into MEM (Modified Eagle Medium; Gibco). Cells were plated at a density of 1.8 millions cells/dish for neuronal cultures (>98% purity by microtubule-associated type-2 immunohistochemistry; data not shown) or 1.2 millions cells/dish for astrocyte cultures (>96% purity by glial fibrillary acidic protein immunohistochemistry; data not shown). For neuronal cultures, cells were re-suspended in Neurobasal plus B27 medium (Gibco) after 2–4 h of incubation in MEM. To inhibit proliferation of non-neuronal cells in neuronal cultures, 5 µM cytosine arabinoside (Sigma) was added 3–6 days after initial incubation. For cultures containing both neurons and astrocytes, neurons were plated on coverslips and cultured in Neurobasal plus B27 medium. Astrocytes that had been in culture separately for 2 weeks (as described above) were plated on the dish containing the coverslip with neurons. This system permitted neurons to be cultured in the presence of astrocytes but supported separate analyses of neurons (on coverslips) and astrocytes (on dishes) at the end of the experiment. The cultures were maintained at 37°C in a humidified 95% air–5% CO2 atmosphere.
Neuronal or neuronal–glial combined cultures were cultured for 10–13 days and then treated for 12 h in the presence of 5 µM thiorphan, 0.1 nM to 10 µM SP, 10 µM SP + one inhibitor of transduction pathway (10 µM H89, 5 µM U73122 [GenBank] , 10 µM chelerythrine, 50 µM PD98059 or 20 µM dantrolene), or medium alone (control). One hour before the end of this 12 h period, 300 µM NMDA was added in cultures. At the end of the 12 h exposure, after replacing the medium, cultures were incubated for an additional 8, 24, 48 or 72 h in the absence of drugs. Neurons were then fixed in 4% paraformaldehyde and stained with bis-benzimide (Hoechst 33452, 10 µg/ml; Sigma), which labels nuclear chromatin. An observer who was blinded to treatment condition counted the nuclei featuring delayed neuronal cell death (e.g. pycnosis or chromatin condensation or fragmentation). Cell counting was done with a fluorescence microscope (UV-2A filter, excitation 370 nm, emission 400 nm; Zeiss, Oberkochen, Germany). Six to nine coverslips were used for each experimental group. The observer examined four to six fields per coverslip, which contained 40–70 neurons. For each field, the ratio between the number of pycnotic nuclei and the total number of nuclei was calculated and used as an index of neuronal death. To take into account variation in cell viability across cultures, this ratio was normalized to the ratio obtained in control cultures (i.e. without any drug), taken as 100% cell death. Two to three plates were used for each experimental condition. Each experiment was performed at least twice.
In vitro determination of SP degradation
Hydrolysis of SP was measured by monitoring its extra-cellular metabolism rate in neuronal cultures 5 min, 1, 4 or 8 h after the addition of 10 µM SP, in the presence and absence of 5 µM thiorphan. At the end of the incubation period, 2 ml of culture medium was withdrawn, acidified with HCl (0.1 N final concentration) and lyophilized for 24 h at –110°C. The samples were then re-suspended in 200 µl pure water and centrifuged (4500 g for 15 min at 4°C) and supernatants (90 µl) were submitted to HPLC. Native SP and its metabolites were isolated and quantified by RP-HPLC coupled to a spectrophotometer detector (C-18 LUNA column, Phenomenex, AIT-France; Surveyor LC system and ChromQuest analyser, Thermo-Electron). Elution with a 25 min linear gradient from 0.1% TFA in water to 0.1% TFA in acetonitrile, at a 1 ml/min flow rate, separated the two SP metabolites (Rt: 4.2 and 6.4 min) and the intact SP (Rt: 17.9 min). Their identities and relative quantities (peak area) were checked by monitoring and the column outflow was plotted at 224 nm, 254 nm and at spectrum max. The relative SP recovery for each sample was calculated as follows: area under the peak for native SP/area under the peak for the three SP related compounds (native and the two SP metabolites).
Statistical analyses
To study the effect of each treatment most of the data were analysed with a Student's t-test or a one-way ANOVA (analysis of variance). In ANOVA analyses, when a main effect of treatment group was found to be significant, Bonferroni's, Dunnett's or Kruskal–Wallis multiple comparison tests were performed (GraphPad Prism version 4.01 for Windows, GraphPad Software, San Diego, CA, USA). In the subset of experiments where lesion size or density of cleaved caspase-3 labelled cells was evaluated at different time-points after ibotenate injection or where thiorphan was administered at different time-points following ibotenate, results were studied using a two-way ANOVA with Treatment and Age (time elapsed after ibotenate injection) as between-subject factors. When an effect of Treatment or Age or their interaction was found to be significant, we conducted pair-wise comparisons between treatment groups at each age.
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Thiorphan protects the cortical grey matter against an excitotoxic challenge
Pups injected with intracerebral ibotenate plus PBS on P5 developed cortical lesions and periventricular white matter cysts when analysed on P10 (Fig. 1A). The cortical lesion was typically characterized by neuronal loss in all neocortical layers and almost complete disappearance of neuronal cell bodies along the axis of excitotoxin injection. Both cortical grey matter and white matter lesions induced by ibotenate were abolished by co-intracerebral injection of MK-801, a specific NMDA receptor antagonist (Figs 1B and 2A and B).
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Intracerebral co-injection of ibotenate and thiorphan induced a dose-dependent reduction (up to 51% with 25 µg thiorphan) of the cortical grey matter lesion size but did not affect white matter lesions (Figs 1C and 2A and B). This neuroprotective effect of intracerebral thiorphan against excitotoxic cortical grey matter lesions was replicated by intraperitoneal injection of thiorphan (Fig. 2C and D). When intraperitoneal injection of thiorphan followed the excitotoxic challenge with some delay, neuroprotection was a function of time. Protection of the cortical grey matter was observed in groups receiving thiorphan within the first 12 h after ibotenate administration (Fig. 2C and D). As described previously (Husson et al., 2004
), intraperitoneal administration of MK-801 immediately after ibotenate challenge only partially protected against excitotoxic brain damage (Figs 1D and 2C and D). Interestingly, level of neuroprotection of the cortical grey matter afforded by intraperitoneal injection of thiorphan was very similar to the level of neuroprotection produced by intraperitoneal MK-801 injection (Figs 1D and 2C and D).
Further supporting the neuroprotective effect of pharmacological blockade of NEP, mouse pups with a genetic deletion of NEP (NEP–/–) displayed a significantly reduced size of the cortical grey matter lesion induced by ibotenate on P5 when compared with wild-type NEP+/+ animals (Figs 1E and F and 2E and F). Intracerebral injection of thiorphan protected grey matter against excitotoxic damage in NEP+/+ animals but not in NEP–/– pups (Fig. 2E and F). Of note, lesion sizes in C57Bl/6 NEP+/+ and NEP–/– animals were smaller than those observed in Swiss animals (compare Fig. 2A and B with Fig. 2E and F). As described previously in a similar model (Laudenbach et al., 2002), these variations are most likely attributable to differences in the genetic background of the animals.
In this first set of experiments, measurement of the maximal sagittal fronto-occipital diameter of the lesion or of the volume of the lesion showed very comparable results (Fig. 2). Accordingly, in the following sets of experiments, measurement of the maximal sagittal fronto-occipital diameter of the lesion was used as an index of the volume of the lesion.
SP and NKA mimic neuroprotective effects of thiorphan
Among the targets of NEP, we tested the potential neuroprotective effects of tachykinins (SP, NKA and NKB) and of neurotensin (neurotensin itself and JMV-449, a synthetic neurotensin analogue).
Intracerebral co-injection of SP or NKA on P5 induced a dose-dependent neuroprotection of the cortical grey matter but had no effect on white matter lesions (Fig. 3A and B). Although the maximal neuroprotective effect was comparable between the two neuropeptides (43% reduction of lesion size for SP and 39% for NKA), SP exhibited a higher and broader potency when compared with NKA (Fig. 3A and B).
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Intracerebral co-injection of NKB, neurotensin or JMV-449 had no detectable effect on ibotenate-induced lesions (Fig. 2C and D). These negative results strongly suggest that NKB and neurotensin, two substrates of NEP, are not involved in the thiorphan-induced neuroprotection observed in the present model.
SP affords long-term neuroprotection and prevents ibotenate-induced cell death
The study of the cortical grey matter lesion size at different time-points (between 1 and 30 days) after ibotenate injection revealed that intracerebral co-injection of SP induced a neuroprotection that was significant as early as 2 days after the insult and which lasted over the 30-day period of examination (Fig. 4A). The neuroprotective effects of SP on ibotenate-induced cortical grey matter lesions in P5 pups were confirmed by immunohistochemistry for cleaved caspase 3 (Fig. 4B and D). Blockade of ibotenate-induced caspase-3 activation by Z-DEVD-FMK confirmed the specificity of the results (Fig. 4B).
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50–60% of the lesion in the coronal plane. Bar = 20 µm.NK1 and NK2 tachykinin receptors mediate neuroprotective effects
In Swiss pups, neuroprotective effects of thiorphan on cortical grey matter lesions were inhibited by co-treatment with Win-51,708 (an NK1 receptor antagonist) and with L-659,877 (an NK2 receptor antagonist) (Fig. 5A). In the absence of thiorphan administration, receptor antagonists had no detectable effect on ibotenate-induced lesions (Fig. 5A). Similarly, neuroprotective effects of the genetic deletion of NEP were blocked by Win-51,708 and L-659,877 (Fig. 5B). In Swiss pups, neuroprotective effects of SP were blocked by Win-51,708 but not by L-659,877 (Fig. 5C) while neuroprotective effects of NKA were blocked by L-659,877 but not by Win-51,708 (Fig. 5D).
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Real-time PCR showed detectable levels of NK1 and NK2 receptor mRNA between E14 and P60, with a peak at P5 for NK2 receptor and at P10 for NK1 receptor (Fig. 6A). In contrast, mRNA for NK3 receptor was undetectable at E14 and was present at very low levels from P0 on (Fig. 6A). In addition, specific binding of 125I-BH-SP was observed in P5 brains. Moderate densities of binding site were detected in cortical grey matter but no specific binding was observed in the underlying white matter (Fig. 6B).
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PLC–PKC pathway mediates SP-induced neuroprotective effects
Neuroprotective effects of SP on cortical grey matter lesions were reversed by co-treatment with U731221 (a PLC inhibitor), chelerythrine (a PKC inhibitor) or PD98059 (a MAPK inhibitor) but not with H89 (a PKA inhibitor) or dantrolene (an inhibitor of intracellular calcium release) (Fig. 7). In the absence of SP administration, U731221, chelerythrine and PD98059 had no detectable effect on ibotenate-induced cortical grey matter lesions (Fig. 7). The selection of inhibitors and the doses used were based on previous studies (Husson et al., 2002
, 2005).
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SP and NKA have no significant effect on body temperature
Intracerebral injection of 10 ng SP or of 1 µg NKA plus ibotenate had no detectable effect on body temperature when compared with that of pups injected with ibotenate plus PBS (Fig. 8A).
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Thiorphan and neurokinins do not affect ibotenate-induced mortality and epileptic manifestations
Overall mortality was low, with death seen in <4% of the animals injected with ibotenate. No significant difference was observed in a test of contingency (Fisher's exact test) when each treatment group was compared with the animals injected with ibotenate plus PBS. Epileptic manifestations including clonic or tonic seizures and apnoeas were observed in all ibotenate-treated animals. Treatment with thiorphan, SP, NKA or NKB did not induce any detectable difference in severity and frequency of seizures (frequency of seizures was quantitatively assessed during a 15-min period, once every hour during the first 6 h following excitotoxin injection; severity of seizures was qualitatively assessed according to the same schedule) when compared with controls (Fig. 8B).
Thiorphan protects cultured neurons against an excitotoxic challenge and prevents SP degradation
In order to further confirm the neuroprotective effects of thiorphan and SP against excitotoxic neuronal death, we used a well-established model of primary neuronal cultures exposed to NMDA. Fluorescent microscopy after staining with bis-benzimide clearly discriminated between normal neuronal nuclei and pycnotic nuclei. In this in vitro model, exposure to 300 µM NMDA for 8 h induces a significant increase of neuronal cell death (up to a 45% increase when compared with spontaneous neuronal cell death observed in untreated neuronal cultures).
When compared with neuronal cultures exposed to 300 µM NMDA only, co-incubation with thiorphan induced a significant, dose-dependant reduction and long-lasting reduction of the number per unit area of pycnotic nuclei (Fig. 9A and D).
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As shown in Fig. 9E, an important part of the 10 µM SP was proteolysed by the cultured neurons expressing ectopeptidase activities, even if SP was exposed only for a few minutes (21% of the exogenous SP hydrolysed). Adding 5 µM thiorphan to the medium significantly prevented SP breakdown by the cultured neurons even if SP was exposed for an 8 h culture period (89–92% of SP relative recovery).
SP protects cultured neurons against an excitotoxic challenge through a PLC–PKC pathway
When compared with neuronal cultures exposed to 300 µM NMDA only, incubation with 10 µM SP mimicked the neuroprotective effects of thiorphan and induced a significant reduction (24% reduction versus NMDA alone) of the number per unit area of pycnotic nuclei, while lower concentrations of SP had no detectable effects (Fig. 10A). In neuronal–glial co-cultures, a significant neuroprotective effect against NMDA-induced cell death was already observed with concentrations of SP as low as 0.1 µM (19% reduction versus NMDA alone) and was also observed with 10 µM SP (25% reduction versus NMDA alone) (Fig. 10A). Consistent with the in vivo findings, these neuroprotective effects of SP were abolished by co-incubation with Win-51,708 (Fig. 10A) and inhibitors of PLC and PKC pathways (Fig. 10B).
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P < 0.05, **, |
Discussion |
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The most salient feature of the present study is the demonstration that thiorphan potently protects the developing brain from ibotenate-induced insults. Thiorphan is particularly efficient in preventing excitotoxic neuronal loss. In contrast, thiorphan has no significant effect on ibotenate-induced white matter lesions. Neuroprotective effects of thiorphan are mimicked by SP and NKA. These neuroprotective effects are blocked by NK1 and NK2 receptor antagonists as well as by PLC–PKC pathway inhibitors.
Neuroprotection conferred by thiorphan and neurokinins on excitotoxic injury in the developing brain: comparison of our findings with those of others
To our knowledge, this is the first original report demonstrating a neuroprotective effect of thiorphan or of a NEP inhibitor.
Several studies have shown neuroprotective effects of tachykinins or SP against a variety of insults including MPTP-induced motor disturbances (Chen et al., 2004), excitotoxic neuronal cell death in the adult striatum (Sanberg et al., 1993) and basal forebrain (Calvo et al., 1996; Wenk et al., 1997), apoptotic cell death of auditory neurons (Lallemend et al., 2003) and neurotoxic effects of the ß-amyloid protein on hippocampal cells in vitro (Yankner et al., 1990) and in vivo (Kowall et al., 1991). In contrast, adult mice with disruption of the preprotachykinin A gene, which encodes SP and NKA, are resistant to kainate-induced necrosis and apoptosis of hippocampal neurons (Liu et al., 1999).
Mechanistic basis for the neuroprotection conferred by thiorphan on excitotoxic cortical plate lesions in newborn mice
As previously mentioned, thiorphan binds and inhibits NEP with a high affinity. It was also shown to bind with a lower affinity to NEP2 (Voisin et al., 2004), a recently identified member of the M13 subfamily of metalloproteases. We did not observe, however, any significant protective effect of thiorphan in mice deleted for NEP. In addition, thiorphan induced a neuroprotection of the cortical plate in NEP+/+ control mice, which was of comparable amplitude to the neuroprotection observed in untreated NEP–/– mice. Altogether, these data support the hypothesis that, in the present model, thiorphan works mainly through a specific inhibition of NEP. Of note, neuroprotection afforded by thiorphan in control NEP+/+ C57Bl/6 mice was less important than thiorphan-induced neuroprotection in Swiss mice, suggesting that the genetic background can modulate the impact of NEP inhibition on excitotoxic brain lesions in newborn mice.
NEP is involved in the metabolism of a variety of peptides such as tachykinins (SP, NKA and NKB), enkephalins, NT, atrial natriuretic factor, bradykinin, gastrin and the chemotactic peptide (Erdos and Skidgel, 1989; Roques et al., 1993). Although we did not test the potential neuroprotective effects of all the substrates of NEP, the present study showed that thiorphan increases the half life of SP, suggesting that this neuropeptide mediates thiorphan-induced neuroprotection. Accordingly, we showed that SP and NKA mimicked thiorphan neuroprotective effects in our model. Interestingly, SP exhibited a potency at least 10-fold higher than NKA.
The observed inhibitory effects of NK1 and NK2 receptor antagonists on ibotenate-induced damage support the involvement of both receptor subtypes in thiorphan-induced neuroprotection, of NK1 receptors in SP-induced neuroprotection and of NK2 receptors in NKA-induced neuroprotection. The detection of both NK1 and NK2 receptors by different techniques (real-time PCR, autoradiography and/or immunohistochemistry) in the P5 neocortex further supports our pharmacological data.
The observed distribution of specific binding of 125I-BH-SP in the neocortical grey matter but not in the underlying white matter is in agreement with the neuroprotective effects of thiorphan and SP restricted to the grey matter. Immunohistochemistry for NK1 receptor confirmed the expression of receptors in the cortical grey matter but not in the underlying white matter (data not shown).
In vitro data confirmed the capability of SP to prevent excitotoxic neuronal cell death through NK1 receptor activation. Interestingly, potency of SP to protect cultured neurons was 100-fold higher when neurons were co-cultured with astrocytes as compared with neurons cultured alone. This observation suggests an important cross-talk between astrocytes and neurons to mediate SP-induced neuroprotective effects. Further studies will be necessary to address the underlying mechanisms of these cell–cell interactions.
Receptors for tachykinins are seven transmembrane domain receptors coupled to Gq-type proteins (Macdonald et al., 1996). Tachykinin receptors can activate several transduction pathways (Almeida et al., 2004). In terms of NK1 receptor-mediated neuroprotection, a previous study has shown that SP prevents auditory neuron apoptosis through PKC activation, calcium mobilization and MAPK pathway activation (Lallemend et al., 2003). Similarly, in the present study, both in vivo and in vitro, inhibitors of PLC (U73122
[GenBank]
), PKC (chelerythrine) and MAPK pathway (PD98059) completely reversed the neuroprotective effects of SP, supporting a key role of the PLC–PKC and MAPK pathways in SP-induced neuroprotection. In contrast, an inhibitor of intracellular calcium release (dantrolene) did not significantly affect SP-induced neuroprotection. The reason for this last discrepancy could be related to differences in NK1 receptor coupling between adult auditory neurons and developing neocortical neurons.
Hypothermia has been shown to be neuroprotective in several models of perinatal brain lesions while hyperthermia is neurotoxic (Thoresen, 2000). Nemeroff et al. (1979) reported that intracisternal SP induces hyperthermia in adult mice while Richter and Oehme (1982) reported that intraperitoneal SP induces hypothermia in adult rats. In the present study, intracerebral SP or NKA did not significantly affect body temperature of newborn mice, suggesting that, in the present model, neuroprotective effects of these neuropeptides are not mediated by a hypothermic effect.
Potential implications for human newborns at risk of developing CP
Neocortical neuronal cell death induced by ibotenate in P5 mouse pups mimics hypoxic–ischaemic grey matter lesions observed in some human term or near-term neonates (Johnston, 2005). Excitotoxicity does not mimic all aspects of hypoxic–ischaemic injury. However, it was previously shown that MK-801, a specific NMDA receptor inhibitor, prevents hypoxic–ischaemic neuronal brain lesions in newborn rats (Olney et al., 1989) and that sensitivity of the developing rat brain to hypoxic–ischaemic damage parallels sensitivity to NMDA neurotoxicity (Ikonomidou et al., 1989).
In this well-defined mouse model, as a candidate neuroprotective drug, thiorphan displayed several promising characteristics: (i) it induced a significant neuroprotection against excitotoxic neuronal death when administered by a systemic route; (ii) this neuroprotection was long lasting; (iii) a significant neuroprotection was still observed when thiorphan was administered up to 12 h after the insult, a critical issue for any potential intervention in clinical practice; (iv) no side-effect was observed. In particular, we did not observe any exacerbation of ibotenate-induced epileptic manifestations even if Liu et al. (1999) reported that adult mice with disruption of the preprotachykinin A gene are resistant to kainate-induced seizures. This discrepancy might be related to the age of animals (newborn versus adult mice) and/or to the nature of the insult (kainate-induced hippocampal lesion versus ibotenate-induced cortical lesion).
The fact that thiorphan and SP did not modify the frequency and severity of ibotenate-induced seizures within the first 6 h following the insult strongly argues against a direct effect of thiorphan or SP on NMDA receptors. Thiorphan-mediated SP protection against proteolysis induces NK1/NK2 receptor activation and their activated transduction pathways impede on the excitotoxic cascade induced by NMDA receptor over-activation (Fig. 11). The fact that thiorphan and SP do not act directly on NMDA receptors is reassuring since it was previously shown that blocking NMDA receptors during rodent neonatal development leads to a massive apoptotic neuronal cell death (Ikonomidou et al., 1999).
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Further supporting the potential interest of thiorphan as a candidate drug for hypoxic–ischaemic human neonates, racecadotril (Tiorfan®), the precursor drug of thiorphan, is used in clinical practice for diarrhoea, including in infants and children, with a remarkable safety profile (Schwartz, 2000
). However, before considering extrapolation of these data to humans, current working hypotheses must be validated in other models of perinatal brain lesions. By definition, animal models are artificial and simplistic, as they extract a small number of processes from the complex situation encountered in clinical practice. This simplification inevitably introduces biases, and efforts must be made to reconstruct a more complex picture by combining data from different models.
In conclusion, the present study showed that thiorphan potently protects neocortical neurons from excitotoxic cell death in newborn mice. These findings suggest that thiorphan therapy may be particularly promising to limit grey matter injury evident in a subset of human term newborns at risk of developing CP.
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Footnotes |
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*These authors have contributed equally to this work.
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Acknowledgements |
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We are grateful to Patrick Robberecht and William Rostène for helpful discussion and to Leslie Schwendimann for excellent technical assistance. This work was supported by the Inserm, Université Paris 7, APETREIMC and Fondation Grace de Monaco.
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