Growth Hormone-Releasing Peptide Hexarelin Reduces Neonatal Brain Injury and Alters Akt/Glycogen Synthase Kinase-3 Phosphorylation
http://www.100md.com
《内分泌学杂志》
Department of Obstetrics and Gynecology, Perinatal Center (K.G.B., H.H.), Department of Physiology and Pharmacology (K.G.B., A.-L.L., M.G., C.M., H.H.), and Research Center for Endocrinology and Metabolism, Department of Internal Medicine (J.I.), Sahlgrenska Academy, 405 30 Gteborg, Sweden
Departments of Internal Medicine (R.G., S.D., E.G.) and Biomedical Sciences & Oncology (M.V.), University of Turin, 8-10124 Turin, Italy
Abstract
Hexarelin (HEX) is a peptide GH secretagogue with a potent ability to stimulate GH secretion and recently reported cardioprotective actions. However, its effects in the brain are largely unknown, and the aim of the present study was to examine the potential protective effect of HEX on the central nervous system after injury, as well as on caspase-3, Akt, and extracellular signal-regulated protein kinase (ERK) signaling cascades in a rat model of neonatal hypoxia-ischemia. Hypoxic-ischemic insult was induced by unilateral carotid ligation and hypoxic exposure (7.7% oxygen), and HEX treatment was administered intracerebroventricularly, directly after the insult. Brain damage was quantified at four coronal levels and by regional neuropathological scoring. Brain damage was reduced by 39% in the treatment group, compared with vehicle group, and injury was significantly reduced in the cerebral cortex, hippocampus, and thalamus but not in the striatum. The cerebroprotective effect was accompanied by a significant reduction of caspase-3 activity and an increased phosphorylation of Akt and glycogen synthase kinase-3, whereas ERK was unaffected. In conclusion, we demonstrate for the first time that HEX is neuroprotective in the neonatal setting in vivo and that increased Akt signaling is associated with downstream attenuation of glycogen synthase kinase-3 activity and caspase-dependent cell death.
Introduction
HYPOXIC-ISCHEMIC (HI) brain damage in the perinatal period is a major contributor to neonatal mortality and long-term morbidity with neurological impairments presenting in the survivors (1, 2). Brain damage does not develop acutely during the HI insult but rather evolves over several days (secondary brain injury), opening a window of opportunity for therapeutic intervention (3). Growth factors protect neurons from dying under a wide variety of circumstances in vitro (4, 5, 6, 7). It has also been shown that several trophic factors, including GH and IGF-I, have neuroprotective properties during the secondary phase after HI in vivo (8, 9, 10, 11, 12), although the underlying mechanisms are not yet fully understood. However, it has been demonstrated that activation of phosphatidylinositol-3 kinase (PI3K) pathway and the downstream phosphorylation of the kinase Akt (pAkt) mediate growth factor-induced neuronal survival in vitro (13). pAkt promotes cell survival and can inhibit apoptosis by inactivating several proapoptotic targets, including Bad, glycogen synthase kinase 3 (GSK3), and caspase 9, or by modification of transcription factors (14, 15, 16, 17, 18). GSK3 has been shown to play a role in apoptosis and has been suggested to be a key target of PI3K/Akt survival signaling pathway (16). After HI in the neonatal brain, GSK3 is activated by dephosphorylation and translocated to the nucleus, where it may contribute to the development of brain damage (19). It has been demonstrated that inhibition of GSK3 reduces infarct size in an adult stroke model (20), which lends further support for its important proapoptotic role in brain injury. The mechanisms for induction of apoptosis via GSK3 are not fully clarified, but it has been suggested to cause degradation of -catenin and to inhibit mitochondrial pyruvate dehydrogenase, with subsequent metabolic failure (21, 22). GSK3 also interacts with transcription factors (among them, p53) in both the nucleus and mitochondria and affects cytochrome c release and caspase-3 activity (23, 24). Caspase-dependent mechanisms seem particularly important for cell death in the immature brain (25, 26, 27, 28), because caspase-3 inhibitors reduce neonatal brain injury (29). Moreover, neuroprotection, by both brain derived neurotrophic factor (10) and IGF-I, is linked to a reduction of the activation of caspase-3 (19).
Another kinase pathway activated by growth factors is the MAPK p42/44 ERK pathway. Activation of ERK has been shown to inhibit apoptosis induced by hypoxia (30), growth factor withdrawal (31), hydrogen peroxide (32), and chemotherapeutic agents (33). Furthermore, brain-derived neurotrophic factor neuroprotection in the neonatal rat was shown to be mediated by activation of the MAPK/ERK pathway (34) and IGF-I treatment of neonatal rats after HI activated both Akt and ERK pathways (19).
Hexarelin (HEX) is a six-amino-acid peptide that belongs to a family of synthetic GH secretagogues (GHS) (35) that have a potent ability to release GH from the pituitary and to stimulate food intake (36). Ghrelin is the recently discovered endogenous ligand to the GHS receptor-1a (37, 38). In addition to their neuroendocrine activity, ghrelin and synthetic GHS have been shown to be cardioprotective (39, 40) and to inhibit apoptosis in cardiomyocytes and endothelial cells through activation of ERK and Akt kinases (41).
Based on the cardioprotective effect and activation of survival signaling pathways in cardiomyocytes, we raised the hypothesis that HEX could be neuroprotective. Our aim was to examine the posttreatment effects of HEX on central nervous system injury, caspase-3 activation, and Akt and ERK intracellular signaling in a rat model of neonatal HI.
Materials and Methods
Animal model
Unilateral HI was induced in neonatal rats as previously described (42, 43). Briefly, 7-d-old [postnatal day (PND) 7] Wistar rats were anesthetized with enflurane (3.5% for induction and 1.5% for maintenance) in a mixture of nitrous oxide and oxygen. The left common carotid artery was cut between two prolene sutures (6–0). After anesthesia and surgery, the animals were allowed to recover for 60 min. They were thereafter exposed to 60 min of hypoxia in a humidified chamber at 36 C with 7.7% oxygen in nitrogen. After hypoxia. the pups were returned to and kept with their dams until they were killed as described below. The animal studies were approved by the animal ethics committee in Gteborg.
Intracerebral injection of HEX and vehicle
Animals received intracerebroventricular (icv) injections in a total vol of 5 μl containing either 1 μg HEX (Pharmacia & Upjohn, Inc., Stockholm, Sweden) or vehicle (NaCl, 0.9 mg/ml) starting immediately after HI on PND 7. Injections were aimed at the left ventricle at 1.5 mm lateral to the sagittal suture, 5.7 mm rostral to lambda, and 2.8 mm deep to the skull surface with a 27-gauge needle. Animals were under anesthesia (enflurane, 1.5%), on a snout mask, while injected with a Hamilton syringe connected to a MCA pump, 1 μl/min. Accuracy of injection site with the method was verified using methylene blue staining on control animals (n = 3).
Study protocol I, effect of HEX on brain injury
Immediately after HI, animals were treated with 1 μg HEX (n = 27) or vehicle (n = 27), icv as described above, and this dose was repeated once daily at 24 and 48 h after HI. At PND 10, animals were perfused intracardially with 0.9% NaCl followed by 5% buffered formaldehyde (Histofix; Histolab, Gteborg, Sweden). Brains were removed from the skull and immersion-fixed at 4 C for 24 h and were thereafter dehydrated and embedded in paraffin. Brains were cut into 5-μm coronal sections at four anteroposterior levels from the anterior striatum to the posterior part of hippocampus. Adjacent sections were stained with thionin/acid fuschin (44) and for microtubule-associated protein (MAP-2) (1:1000, 1 h, mouse-anti-MAP-2, clone HM-2; Sigma, St. Louis, MO) (45). Immunoreactivity was visualized using diaminobenzidine (DAB) as described below.
Evaluation of brain damage
Brain damage was evaluated using both area measurements of tissue loss at four anatomical levels and by neuropathological scoring.
Intact neurons (dendrites and soma) express MAP-2 and infarction in gray matter is associated with a distinct loss of MAP-2 immunoreactivity. MAP-2-positive areas in the ipsilateral and contralateral hemispheres were outlined by an observer blinded to the study groups, and calculations were made using the Olympus Micro Image analysis software version 4.0 (Olympus Optical Co., Ltd., Tokyo, Japan). Brain tissue loss was calculated by subtracting the MAP-2-positive area of the ipsilateral hemisphere from the contralateral hemisphere and was expressed as percentage of the contralateral hemisphere as previously described (46).
Regional brain injury was also evaluated by an observer blinded to the study groups, using a neuropathological scoring system where injury in cortex was graded from 0–4 with 0 being no observable injury and 4 being confluent infarction encompassing most of the hemisphere. The damage in the hippocampus, thalamus, and striatum was assessed regarding both hypothrophy (0–3) and observable cell injury/infarction (0–3), resulting in a scoring for each region (0–6) (46).
Rectal temperature
The rectal temperature, which has been shown to correspond to the brain core temperature (47), was measured in HEX-treated (n = 9) and vehicle-treated (n = 8) rats from study protocol I at 1, 3, 6, 12, 24, 36, 48, and 72 h after HI and start of the HEX injections.
Study protocol II, effect of HEX on caspase-3 and pAkt, GSK3, and ERK
Immediately after HI, animals were treated with one injection of HEX (1 μg) (n = 7) or vehicle (n = 6). Animals were killed 24 h after HI at PND 8, and cytosol fractions were prepared for measurement of caspase-3 activity (see below). The cellular localization of pAkt and phosphorylated GSK (pGSK)3 was studied with immunohistochemistry (see below) in sections double-stained with neuronal nuclear protein (NeuN) (n = 3/ group). GH and IGF-I proteins on brain sections from HI animals were also assessed by immunohistochemistry (see below). The effect of HEX or vehicle treatment on pAkt/Akt, pGSK3/GSK3, and IGF-I receptor (IGF-IR) phosphorylation immunoreactivity after HI was evaluated 24 h after HI and drug administration (HEX, n = 7) (vehicle, n = 6) using Western blot (see below); pGSK3 was analyzed in both cytosolic and nuclear fractions.
Antibodies
Anti-microtubule-associated protein 2 (MAP-2, clone HM-2) was purchased from Sigma. LaminB (M-20) (sc-6217) and IGF-IR -subunit antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-NeuN (MAB377), Alexa Fluor 488 streptavidin IgG (H+L) conjugate (green) and Alexa Fluor 594 goat antimouse IgG (H+L) were purchased from Chemicon International, Inc. (Temecula, CA). The antibodies against phospho-Akt (Ser473) rabbit polyclonal (no. 9277, no. 9271), Akt rabbit polyclonal (no. 9272), phospho-GSK3(Ser9) rabbit polyclonal (no. 9336), phospho-p44/42 MAPK (pERK) rabbit polyclonal (no. 9101), and p42/44 MAPK (ERK) rabbit polyclonal (no. 9102) were purchased from Cell Signaling Technology, Inc. (Beverly, MA). Phosphotyrosine-PY20 antibody was from BD Transduction Laboratories (Milan, Italy). Anti-GSK3 mouse monoclonal (no. 05–412) was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). An IGF-I goat polyclonal antibody was purchased from Santa Cruz (C-20) and used diluted 1:80 after microwave oven pretreatment (three cycles of 5 min each in EDTA buffer, pH 8.1). GH rabbit polyclonal antibody (DakoCytomation, Carpinteria, CA) was used without antigen retrieval and diluted 1:2000. All secondary antibodies were from Vector Laboratories, Inc. (Burlingame, CA).
Immunohistochemistry
Incubation with primary antibody, anti-phospho-Akt (Ser473) peptide) (no. 9277) 1:50, was performed in PBS, at 4 C, overnight (O/N). Sections were washed in PBS and incubated with biotinylated secondary antibody for 1 h, followed by inhibition of endogenous peroxidase (0.6% H2O2 in methanol, 10 min) and incubation with avidin-biotin enzyme complex (20 μl/ml, 1 h, ABC-Elite; Vector Laboratories). Immunoreactivity was visualized using DAB (0.5 mg/ml) enhanced with nickel sulfate (15 mg/ml). The specificity of antibodies was tested by omission of the primary antibody. Previous studies in neuronal murine tissue have confirmed antibody specificity with preabsorbtion using phospho-Akt Ser 473 peptide (48). For detection of GH and IGF-I protein, a standard immunoperoxidase technique was employed. To reveal the immunohistochemical reaction, a biotin-free detection system (EnVision; DakoCytomation) was employed, and DAB was used as chromogen. Paraffin-embedded tissue from rat pituitary and from rat liver served as positive controls for GH and IGF-I experiments, respectively. Omission of the primary antibodies on parallel sections was used as negative control.
Immunofluorescence
Animals were deeply anesthetized by ip injection of 0.2 ml thiopenthal (50 mg/ml), followed by intracardial perfusion with 0.9% NaCl and then 5% buffered formaldehyde (Histofix; Histolab, Gothenburg, Sweden) after administration of HEX or vehicle. Brains were removed from the skull and immersion-fixed at 4 C for 24 h, dehydrated, embedded in paraffin, and cut into 5-μm coronal sections at the levels of hippocampus and striatum. Before immunohistochemical staining, sections were deparaffinized and boiled in citric acid buffer (0.01 M, pH 6.0; 10 min), and sections were treated with proteinase-K (Boeringer Mannheim, Mannheim, Germany) (10 μg/ml) in PBS for 9 min at room temperature. Nonspecific binding was blocked by incubation with appropriate serum (4%). Sections were incubated with the following antibodies: anti-phospho-Akt (Ser473) rabbit polyclonal (no. 9277) 1:50 in PBS or anti-phospho-GSK3(Ser9), rabbit polyclonal 1:50 in 1% BSA PBS, at 4 C, O/N. After washing, and with washing procedures in between, sections were incubated for 1 h at room temperature for each of the following steps with the subsequent antibodies: biotinylated secondary antibodies, goat antirabbit 1:250 in PBS, Alexa Fluor 488 streptavidin IgG (H+L) conjugate 1:100 (10 μl/ml) (green) in PBS, anti-NeuN 1:200 in PBS, and Alexa Fluor 594 goat antimouse IgG (H+L) conjugate 1.200 (10 μl/ml) (red) in PBS. After washing, sections were mounted using Vectashield mounting medium. Sections were analyzed under an Olympus BX40 fluorescence microscope equipped with an Olympus DP50 cooled digital camera.
IGF-IR immunoprecipitation
Cell lysates from cytosolic fractions of the damaged left hemispheres of HEX- and vehicle-treated HI rats were incubated O/N at 4 C with anti-IGF-IR -antibody (1:500 dilution). Immune complexes were precipitated by adding protein A-Sepharose and incubating for 2 h at 4 C. Pellets were resuspended in 40 μl of reducing Laemmli buffer and the proteins separated by 8% gel SDS-PAGE.
Western blot
Brains were rapidly harvested, hemispheres were split, and cortex dissected out. Each sample was immediately homogenized in ice-cold homogenization buffer (15 mM Tris-HCl, pH 7.6; containing 3 mM EDTA, 1 mM MgCl2, 320 mM sucrose, 1 mM dithiothreitol) protease, and phosphatase inhibitor cocktail was added to final concentrations of 1 and 2%, respectively. The homogenates were centrifuged at 800 x g for 10 min at 4 C for preparation of nuclear fractions. Supernatants were centrifuged at 9200 x g for 15 min at 4 C for preparation of cytosolic fractions. Protein content was quantified using the method presented by Whitaker and Granum (1980) (49), adapted for microplates. Samples were mixed with an equal volume of 3x SDS-PAGE buffer and heated (96 C) for 5 min. One sample containing 20 μg protein from the cytosolic fraction or 10 μg of the nuclear fraction was applied in each well of a Novex (San Diego, CA) precast 8–16% Tris-glycine gel. After electrophoresis, proteins were transferred to a nitrocellulose membrane (Optitran, 0,2 μm; Schleicher & Schuell, Inc., Dassel, Germany). Membranes were blocked in 30 mM Tris-Hcl (pH 7.5), 100 mM NaCl, and 0.1% Tween 20 [Tris-buffered saline with Tween 20 (TBS-T)] containing 5% fat free milk powder. Incubations with the following primary antibodies diluted in TBS-T containing 3% BSA and 9 mM NaN3 for 1 h in room temperature were performed: anti-phospho-Akt (Ser473) (no. 9271) rabbit polyclonal 1:1000, anti-Akt rabbit polyclonal 1:1000, anti-phospho-GSK3 (Ser9) rabbit polyclonal 1:1000, anti-GSK3 mouse monoclonal 1:500, anti-phospho-p44/42 MAPK (pERK) rabbit polyclonal 1:1000, anti-p42/44 MAPK (ERK) rabbit polyclonal 1:1000, and anti-LaminB goat polyclonal 1:200. Membranes from immunoprecipitates were blocked with 1% BSA in TBS with 0.1% Tween for 2 h at room temperature and incubated O/N at 4 C with antibody anti-P-tyrosine (1:500 dilutions). Blots were washed three times with TBS-T and incubated with the appropriate secondary peroxidase conjugate diluted in blocking buffer. Immunoreactive species were visualized using Super Signal Western Dura chemiluminescence substrates (Pierce Biotechnology, Inc., Rockford, IL) and a cooled charge coupled device camera (LAS1000; Fuji Photo Film Co., Ltd., Tokyo, Japan). Immunoreactive bands were quantified using Image Gauge software (version 3.3, Fuji). To standardize quantification between the gels, the same five controls were run on every gel except in experiments with assessment of tyrosine phosphorylation of the IGF-IR -subunit, where samples (two vehicle- and two HEX-treated) were run on each gel. Every sample was analyzed three times, and the average value was used as n = 1. Each protein band on the Western blots was derived from one animal. Each immunoblot was performed using the samples from three to four animals in each experimental group. Membranes were stripped for reprobing with new antibodies by having them incubated in stripping buffer (62.5 mM Tris-Hcl, pH 6.7; 100 mM -mercaptoethanol; and 2% sodium dodecyl sulfate) at 55 C for 30 min.
Fluorometric assay of caspase-3-like activity
Samples of cytosolic fraction were mixed with extraction buffer (50 mM Tris-HCl, pH 7.3; 100 mM NaCl; 5 mM EDTA; 1 mM EGTA; 3 mM NaN3; 1 mM phenylmethylsulfonylfluoride; 1 μg/ml pepstatin; 2.5 μg/ml leupeptin; 10 μg/ml aprotinin 0.2% 3-[(3-cholamidoprophylene) dimethylamonio]propane sulphonic acid; protease inhibitor cocktail (P8340; Sigma) 1%), 1:3, on a microtiter plate (Microflour; Dynatech Laboratories, Inc., Chantilly, VA). After incubation for 15 min at room temperature, 100 μl peptide substrate, 50 μM Ac-Asp-Glu-Val-Asp-aminomethyl coumarine (Ac-DEVD-AMC; Enzyme Systems Products, Livermore, CA) in extraction buffer without inhibitors or 3-[(3-cholamidoprophylene) dimethylamonio]propane sulphonic acid but with 4 mM dithiothreitol were added. Cleavage of the substrate was measured at 37 C using Spectramax Gemini microplate fluorometer (Molecular Devices, Sunnyvale, CA), with an excitation wavelength of 380 nm and emission wavelength of 460 nm. The degradation was followed at 2-min intervals for 2 h, and V-max was calculated from the entire linear part of the curve. Standard curves with AMC in the appropriate buffer were used to express the data in picomoles of AMC (7-amino-4-methyl-coumarin) formed per minute and per milligram of protein.
Statistics
The data are expressed as means ± SEM. The tests used were ANOVA with Fisher’s post hoc test. Values of P < 0.05 were considered to be significant. In all cases, n corresponds to the number of animals.
Results
Study protocol I, effect of HEX on brain injury
Area measurements comparing the lesioned hemisphere with the undamaged hemisphere demonstrated a significant reduction of damage on all four levels of the brain in the HEX-treated animals compared with controls (Fig. 1A). Neuroprotection was most pronounced at the level of anterior hippocampus (–2 mm from bregma), with a 43% reduction of MAP-2 loss (HEX: 25.7 ± 3.5% vs. vehicle: 45.2 ± 4.6%; P < 0.01). When using a neuropathological scoring system, brain injury was reduced in the cerebral cortex (HEX: 1.9 ± 0.2 vs. vehicle: 2.6 ± 0.2; P < 0.05), hippocampus (HEX: 2.4 ± 0.3 vs. vehicle: 3.6 ± 0.3; P < 0.05), and thalamus (HEX: 1.7 ± 0.2 vs. vehicle: 2.6 ± 0.3; P < 0.05) but not in the striatum (Fig. 1B). There were no differences in body temperature, mortality, or body weight (data not shown) between the two groups. Moreover, there were no significant differences in brain injury between male and female animals (data not shown).
Study protocol II, HEX increases Akt and pGSK3 phosphorylation
Double immunostaining demonstrated that pAkt and pGSK3 colabeled with NeuN, indicating a neuronal localization in the cerebral cortex (Fig. 2). Western blot was used to quantify pAkt and pGSK3 in both nuclear and cytosolic fractions. After HI, pAkt and pGSK3 immunoreactivity in cytosolic fractions were significantly increased by HEX at 24 h after HI. In the nuclear fraction, pGSK3 was also increased by HEX-, compared with vehicle-treated animals, after HI (Figs. 3 and 4).
HEX had no effect on ERK phosphorylation
Western blot was also used to quantify pERK. HEX did not alter total ERK or pERK immunoexpression at 24 h after HI (data not shown).
HEX reduced caspase-3-like activity after HI
A fluorometric assay was used to quantify caspase-3-like activity. HEX reduced caspase-3-like activity (by 56%) in the cerebral cortex 24 h after HI (P < 0.01) but did not affect the activity in the contralateral hemisphere (Fig. 5).
Immunohistochemical expression of GH and IGF-I
To investigate the possible activation of the GH/IGF-I axis by HEX, immunohistochemistry was performed on brain sections 24 h after HI in rats treated with HEX or vehicle. GH antibody specificity was demonstrated by intense reactivity in single cells of rat pituitary (Fig. 6A). However, staining for GH was totally negative or only weakly positive on sections of HI brains from both HEX- and vehicle-treated animals (Fig. 6, C and E). IGF-I immunoreactivity was found in rat liver cells (Fig. 6B) and cerebral neurons in both contralateral (Fig. 6D) and ipsilateral, perilesional areas (Fig. 6F), without significant difference in intensity between HEX- and vehicle-treated rats.
Phosphorylation of the IGF-IR
To determine whether the neuroprotective effects of HEX were mediated by increased signaling through the IGF-IR, tyrosine phosphorylation of the IGF-IR -subunit in cytosolic fractions from the left damaged hemisphere of HI rats was analyzed by Western blot. As shown in Fig. 7, there was a significant increase in phosphorylation of the IGF-IR at 24 h after injury in HEX-treated rats compared with vehicle controls.
Discussion
The main findings in the present study are: that HEX reduces brain injury in an in vivo model of HI, and that the protection is accompanied by phosphorylation of Akt and GSK3, indicating possible involvement of the PI3K pathway.
HEX significantly reduced injury evaluated both by measuring tissue loss at four levels of the brain and by regional neuropathological scoring. Significant protection was seen in cortex, hippocampus, and thalamus but not in the striatum. The spatial distribution of protection correlates with previously reported GH effects in the brain, given that GH did not offer protection in the striatum and that localization of GH receptor/binding protein immunoreactivity is lacking in the striatum (50, 51, 11). This would suggest that the protective effect of HEX could either, in part, be mediated by induction of GH or that HEX and GH share common pathways for cellular protection. The latter alternative is more probable because there was no detectable immunostaining of neuronal GH on brain sections from either HEX- or vehicle-treated HI rats.
So far, no studies regarding possible neuroprotective effects of GHS after HI have been published. However, a recent study of administration of another GHS, GH-releasing peptide 6, to adult rats under physiological conditions has shown increased IGF-I mRNA levels in hypothalamus, cerebellum, and hippocampus but not in cortex (52). Thus, it could be hypothesized that the neuroprotective effects reported in the present study were mediated by increased expression of IGF-I. To address this possibility, we performed immunohistochemistry on brain sections from HEX- and vehicle-treated HI rats. We could not detect any significant increase in IGF-I immunoreactivity in HEX-treated rats compared with controls. However, it is important to point out that immunohistochemistry was only performed on brain sections 24 h after HI, and an induction of IGF-I at other time points cannot categorically be ruled out. On the other hand, if IGF-I was an important mediator of HEX effects, a reduction of brain injury in striatum would have been expected because IGF-IRs are present in the striatum (53, 54), and icv administration of IGF-I does indeed protect the striatum after HI in neonatal rats (19). Furthermore, the present study indicates that HEX activated the PI3K pathway in the central nervous system after HI but did not affect ERK phosphorylation, in agreement with the study by Frago et al. (52). This is in contrast to IGF-I, which has been demonstrated to activate both the PI3K (19) and the ERK pathways after icv administration after HI (unpublished observations). The finding that HEX increased phosphorylation of the IGF-IR is interesting and intriguing. In the absence of an obvious induction of IGF-I, it is possible to speculate that the increased phosphorylation may be due to transactivation of the IGF-IR by HEX or an endogenous factor. It has previously been shown that G protein-coupled receptor agonists such as angiotensin-II, thrombin, and endothelin-1 can stimulate the phosphorylation of IGF-IR and/or Akt (55, 56); although, to our knowledge, this has not previously been shown for GHS.
In summary, results from our laboratory would suggest that the neuroprotective effect of HEX seen in this experimental model is not primarily mediated by an induction of the GH/IGF-I axis, although an increased signaling through the IGF-IR may contribute to the reduction of brain injury. More studies are certainly needed to elucidate a possible link between these endocrine systems and their neuroprotective effects.
Synthetic GHS and the endogenous ligand ghrelin have been found to have cardioprotective properties in several in vivo studies (39, 40), although the molecular mechanisms remain largely unknown so far. Two recent in vitro studies on cardiomyocytes and endothelial cells have addressed mechanistical aspects and suggest that the antiapoptotic effects of GHS are mediated by activation of both Akt and ERK kinases (41) and by regulating the activity of caspase-3 and expression of bax and bcl-2 (57). These findings are largely in agreement with the mechanisms proposed in the present study, although, as has been previously discussed, we could not detect any activation of ERK. We suggest that this difference could be due to tissue-specific properties and different experimental conditions.
Another finding in the present study was that GSK3 phosphorylation was increased after HEX treatment both in cytosol and nucleus in post-HI animals, which is the expected response after activation of the PI3K pathway (58, 59). To our knowledge, phosphorylation of GSK3 by GHS has not previously been reported, but such a response could partly explain the neuroprotective effect of HEX because GSK3 is strongly proapoptotic (60), and inhibitors of GSK3 reduce infarction after ischemia in adult animals (20) and improve neuronal survival in vitro (61). Activation of GSK3 has been linked to triggering of caspase-dependent apoptosis (23), but GSK3 inhibitors given in adult stroke models did not affect caspase activation (20), implicating a role also for caspase-independent mechanisms. However, caspase activation is considered to be of greater importance in the immature brain compared with the adult (25, 26, 28, 62). Hence, the HEX-induced reduction of caspase-3-like activity after HI offers additional support for the involvement of caspases in our experimental model. Moreover, studies on juvenile and adult rat cerebellum have shown HEX-induced reduction of caspase-3 and -9 activity (61), and HEX attenuated apoptosis and caspase-3-like activity in rat cardiomyocytes (55).
In conclusion, we demonstrate, for the first time, that HEX is markedly neuroprotective in an in vivo model of HI. Moreover, we provide evidence that the neuroprotective effect of HEX is, at least partly, mediated through the PI3-kinase pathway, including activation of Akt and inhibition of GSK3 through phosphorylation. The present findings suggest a possible role for GHS as neuroprotective agents, which may be of future clinical significance because no available medical therapy against HI brain injury exists today. However, further studies in other experimental models are mandatory to confirm the neuroprotective effects of GHS.
Footnotes
This work was supported by grants from the Swedish Medical Research Council (09455), the Willhelm and Martina Lundgren Foundation, and the Gteborg Medical Society and by grants to researchers in the public health service from the Swedish government (LUA).
Abbreviations: DAB, Diaminobenzidine; GHS, GH secretagogue(s); GSK3, glycogen synthase kinase 3; HEX, hexarelin; HI, hypoxia-ischemia; icv, intracerebroventricular(ly); IGF-IR, IGF-I receptor; MAP-2, microtubule-associated protein 2; NeuN, neuronal nuclear protein; O/N, overnight; p (prefix), phosphorylated; PI3K, phosphatidylinositol-3 kinase; PND, postnatal day; TBS-T, Tris-buffered saline with Tween 20.
References
Volpe JJ 2001 Neurology of the newborn. Philadelphia: WB Saunders
Cowan F, Rutherford M, Groenendaal F, Eken P, Mercuri E, Bydder GM, Meiners LC, Dubowitz LM, de Vries LS 2003 Origin and timing of brain lesions in term infants with neonatal encephalopathy. Lancet 361:736–742
Wyatt JS, Edwards AD, Azzopardi D, Reynolds EO 1989 Magnetic resonance and near infrared spectroscopy for investigation of perinatal hypoxic-ischaemic brain injury. Arch Dis Child 64:953–963
Russell JW, Windebank AJ, Schenone A, Feldman EL 1998 Insulin-like growth factor-I prevents apoptosis in neurons after nerve growth factor withdrawal. J Neurobiol 36:455–467
Delaney CL, Cheng HL, Feldman EL 1999 Insulin-like growth factor-I prevents caspase-mediated apoptosis in Schwann cells. J Neurobiol 41:540–548
Takadera T, Matsuda I, Ohyashiki T 1999 Apoptotic cell death and caspase-3 activation induced by N-methyl-D-aspartate receptor antagonists and their prevention by insulin-like growth factor I. J Neurochem 73:548–556
van Golen CM, Castle VP, Feldman EL 2000 IGF-I receptor activation and BCL-2 overexpression prevent early apoptotic events in human neuroblastoma. Cell Death Differ 7:654–665
Johnston BM, Mallard C, Williams C, Gluckman P 1996 Insulin-like growth factor-1 is a potent neuronal rescue agent after hypoxic-ischemic injury in fetal lambs. J Clin Invest 97:300–308
Gustafson K, Hagberg H, Bengtsson B, Brantsing C, Isgaard J 1999 Possible protective role of growth hormone in hypoxia-ischemia in neonatal rats. Pediatr Res 45:318–323
Han BH, D’Costa A, Back SA, Parsadanian M, Patel S, Shah AR, Gidday JM, Srinivasan A, Deshmukh M, Holtzman DM 2000 BDNF blocks caspase-3 activation in neonatal hypoxia-ischemia. Neurobiol Dis 7:38–53
Scheepens A, Sirimanne ES, Breier BH, Clark RG, Gluckman PD, Williams CE 2001 Growth hormone as a neuronal rescue factor during recovery from CNS injury. Neuroscience 104:677–687
Sizonenko S, Sirimanne ES, Williams C, Gluckman P 2001 Neuroprotective effects of the N-terminal tripeptide of IGF1, glycine-proline-glutamate, in the immature rat brain after hypoxic-ischemic injury. Brain Res 922:42–50
Kulik G, Klippel A, Weber MJ 1997 Antiapoptotic signalling by the insulin-like growth factor I receptor, phosphatidylinositol 3-kinase, and Akt. Mol Cell Biol 17:1595–1606
Datta SR, Dudek H, Tao X, Masters S, Fu H, Gotoh Y, Greenberg ME 1997 Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell 91:231–241
Cardone MH, Roy N, Stennicke HR, Salvesen GS, Franke TF, Stanbridge E, Frisch S, Reed JC 1998 Regulation of cell death protease caspase-9 by phosphorylation. Science 282:1318–1321
Pap M, Cooper GM 1998 Role of glycogen synthase kinase-3 in the phosphatidylinositol 3-kinase/Akt cell survival pathway. J Biol Chem 273:19929–19932
Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS, Anderson MJ, Arden KC, Blenis J, Greenberg ME 1999 Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 96:857–868
Romashkowa JA, Makarov SS 1999 NF-B is a target for Akt in anti-apoptotic PDGF signaling. Nature 401:86–90
Brywe KG, Mallard C, Gustavsson M, Hedtjrn M, Leverin A-L, Wang X, Blomgren K, Isgaard J, Hagberg H 2005 IGF-I neuroprotection in the immature brain after hypoxia-ischemia, involvement of Akt and GSK3 Eur J Neurosci 21:1489–1502
Kelly S, Heng Z, Guo SH, Danye C 2003 Treatment with the GSK3B inhibitor Chir98025 decreases infarct size following MCAo. Stroke 34:301
Hoshi M, Takashima A, Noguchi K, Murayama M, Sato M, Kondo S, Saitoh Y, Ishiguro K, Hoshino T, Imahori K 1996 Regulation of mitochondrial pyruvate dehydrogenase activity by protein kinase I/glycogen synthase kinase 3 in brain. Proc Natl Acad Sci USA 93:2719–2723
Zhang Z, Hartmann H, Do VM, Abramowski D, Sturchler-Pierrat C, Staufenbiel M, Sommer B, van de Wetering M, Clevers H, Saftig P, De Strooper B, He X, Yankner BA 1998 Destabilization of -catenin by mutations in presenilin-1 potentiates neuronal apoptosis. Nature 395:698–702
Watcharasit P, Bijur GN, Zmijewski JW, Song, L, Zmijewska A, Chen X, Johnson GV, Jope RS 2002 Direct, activating interaction between glycogen synthase kinase-3 and p53 after DNA damage. Proc Natl Acad Sci USA 99:7951–7955
Watcharasit P, Bijur GN, Song L, Zhu J, Chen X, Jope RS 2003 Glycogen synthase kinase-3 (GSK3) binds to and promotes the actions of p53. J Biol Chem 278:48872–48879
Cheng Y, Deshmukh M, D’Costa A, Demaro JA, Gidday JM, Shah A, Sun Y, Jacquin MF, Johnson EM, Holtzman DM 1998 Caspase inhibitor affords neuroprotection with delayed administration in a rat model of neonatal hypoxic-ischemic brain injury. J Clin Invest 101:1992–1999
Hu BR, Liu CL, Ouyang YB, Blomgren K, Siesjo BK 2000 Involvement of caspase-3 in cell death after hypoxia-ischemia declines during brain maturation. J Cereb Blood Flow Metab 20:1294–1300
Zhu C, Wang X, Hagberg H, Blomgren K 2000 Correlation between caspase-3 activation and three different markers of DNA damage in neonatal cerebral hypoxia-ischemia. J Neurochem 75:819–829
Wang X, Karlsson JO, Zhu C, Bahr BA, Hagberg H, Blomgren K 2001 Caspase-3 activation after neonatal rat cerebral hypoxia-ischemia. Biol Neonate 79:172–179
Han BH, Xu D, Choi J, Han Y, Xanthoudakis S, Roy S, Tam J, Vaillancourt J, Colucci J, Siman R, Giroux A, Robertson GS, Zamboni R, Nicholson DW, Holtzman DM 2002 Selective, reversible caspase-3 inhibitor is neuroprotective and reveals distinct pathways of cell death after neonatal hypoxic-ischemic brain injury. J Biol Chem 277:30128–30136
Buckley S, Driscoll B, Weinberg L, Anderson K, Warburton D 1999 ERK activation protects against DNA damage and apoptosis in hyperoxic rat AEC2. Am J Physiol 277:159–166
Erhardt P, Schremser EJ, Cooper GM 1999 B-Raf inhibits programmed cell death downstream of cytochrome c release from mitochondria by activating the MEK/ERK pathway. Mol Cell Biol 19:5308–5315
Wang X, Martindale JL, Liu Y, Holbrook NJ 1998 The cellular response to oxidative stress: influences of mitogen-activated protein kinases signalling pathways on cell survival. Biochem J 333:291–300
Anderson CNG, Tolkovsky AM 1999 A role for MAPK/ERK in sympathetic neuron survival: protection against a p53-dependent, JNK-independent induction of apoptosis by cytosine arabinoside. J Neurosci 19:664–673
Han BH, Holtzman DM 2000 BDNF protects the neonatal brain from hypoxic-ischemic injury in vivo via the ERK pathway. J Neurosci 20:5775–5781
Ghigo E, Arvat E, Muccioli G, Camanni F 1997 Growth hormone-releasing peptides. Eur J Endocrinol 136:445–460
Nagaya N, Kangawa K 2003 Ghrelin, a novel growth hormone-releasing peptide, in the treatment of chronic heart failure. Regul Pept 114:71–77
Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K 1999 Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 402:656–660
Nakazato M, Murakami N, Date Y, Kojima M, Matsuo H, Kangawa K, Matsukura S 2001 A role for ghrelin in the central regulation of feeding. Nature 409:194–198
Locatelli V, Rossoni G, Schweiger F, Torsello A, De Gennaro Colonna V, Bernareggi M, Deghenghi R, Muller EE, Berti F 1999 Growth hormone independent cardioprotective effects of hexarelin in the rat. Endocrinology 140:4024–4031
Tivesten , Bollano E, Caidahl K, Kujacic V, Sun XY, Hedner T, Hjalmarson , Bengtsson B-, Isgaard J 2000 The growth hormone secretagogue hexarelin improves cardiac function in rats after experimental myocardial infarction. Endocrinology 141:60–66
Baldanzi G, Filigheddu N, Cutrupi S, Catapano F, Bonissoni S, Fubini A, Malan D, Baj G, Granata R, Broglio F, Papotti M, Surico N, Bussolino F, Isgaard J, Deghenghi R, Sinigaglia F, Prat M, Muccioli G, Ghigo E, Graziani A 2002 Ghrelin and des-acyl ghrelin inhibit cell death in cardiomyocytes and endothelial cells through ERK1/2 and PI 3-kinase/AKT. J Cell Biol 159:1029–1037
Rice JE, Vanucci RC, Brierley JB 1981 The influence of immaturity on hypoxic-ischemic brain damage in the rat. Ann Neurol 9:131–141
Hagberg H, Bona E, Gilland E, Puka Sunvall M 1997 Hypoxia-ischemia model in 7 day old rat: possibilities and shortcomings. Acta Paediatr Suppl 442:85–88
Mallard EC, Williams CE, Gunn AJ, Gunning MI, Gluckman PD 1993 Frequent episodes of brief ischemia sensitize the fetal sheep brain to neuronal loss and induce striatal injury. Pediatr Res 33:61–65
Gilland E, Bona E, Hagberg H 1998 Temporal changes of regional glucose use, blood flow, and microtubule-associated protein 2 immunostaining after hypoxia-ischemia in the immature rat brain. J Cereb Blood Flow Metab 18:222–228
Hedtjrn M, Leverin A-L, Eriksson K, Blomgren K, Mallard C, Hagberg H 2002 Interleukin-18 involvement in hypoxic-ischemic brain injury. J Neurosci 22:5910–5919
Thoresen M, Bagenholm R, Loberg BR, Apricena F, Kjellmer I 1996 Posthypoxic cooling of neonatal rats provides protection against brain injury. Arch Dis Child 74:F3–F9
Noshita N, Lewen A, Sugawara T, Chan PH 2002 Akt phosphorylation and neuronal survival after traumatic brain injury in mice. Neurobiol Dis 9:294–304
Whitaker JG, Granum PE 1980 An absolute method for protein determination based on a difference in absorbance at 235 and 280 nm. Anal Biochem 109:156–159
Lobie PE, García-Aragón J, Lincoln DT, Barnard R, Wilcox JN, Waters MJ 1993 Localization and ontogeny of growth hormone receptor gene expression in the central nervous system. Dev Brain Res 74:225–233
Scheepens A, Sirimanne E, Beilharz E, Breier BH, Waters MJ, Gluckman PD, Williams CE 1999 Alterations in the neural growth hormone axis following hypoxic-ischemic brain injury. Brain Res Mol Brain Res 68:88–100
Frago LM, Paneda C, Dickson SL, Hewson AK, Argente J, Chowen JA 2002 Growth hormone (GH) and GH-releasing peptide-6 increase brain insulin-like growth factor-I expression and activate intracellular signaling pathways involved in neuroprotection. Endocrinology 143:4113–4122
Bondy C, Werner H, Roberts Jr CT, LeRoith D 1992 Cellular pattern of type-I insulin-like growth factor gene expression during maturation of the rat brain: comparison with insulin-like growth factors I and II. Neuroscience 46:909–923
Guan J, Williams C, Gunning M, Mallard C, Gluckman P 1993 The effects of IGF-1 treatment after hypoxic-ischemic brain injury in adult rats. J Cereb Blood Flow Metab 13:609–616
Sumitomo M, Milowsky MI, Shen R, Navarro D, Dai J, Asano T, Hayakawa M, Nanus DM 2001 Neutral endopeptidase inhibits neuropeptide-mediated transactivation of the insulin-like growth factor receptor-Akt cell survival pathway. Cancer Res 61:3294–3298
Zahradka P, Litchie B, Storie B, Helwer G 2004 Transactivation of the insulin-like growth factor-I receptor by angiotensin II mediates downstream signaling from the angiotensin II type 1 receptor to phosphatidylinositol 3-kinase. Endocrinology 145:2978–2987
Pang JJ, Xu RK, Xu XB, Cao JM, Ni C, Zhu WL, Asotra K, Chen MC, Chen C 2004 Hexarelin protects rat cardiomyocytes from angiotensin II-induced apoptosis in vitro. Am J Physiol Heart Circ Physiol 286:H1063–H1069
Cross DA, Alessi DR, Cohen P, Andjelkovich M, Hemmings BA 1995 Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 378:785–789
Moule SK, Welsh GI, Edgell NJ, Foulstone EJ, Proud CG, Denton RM 1997 Regulation of protein kinase B and glycogen synthase kinase-3 by insulin and B-adrenergic agonists in rat epididymal fat cells. J Biol Chem 272:7713–7719
Eldar-Finkelman H 2002 Glycogen synthase kinase 3: an emerging therapeutic target. Trends Mol Med 8:126–132
Cross DA, Culbert A, Chalmers K, Facci L, Skaper S, Reith AD 2001 Selective small-molecule inhibitors of glycogen synthase kinase-3 activity protect primary neurones from death. J Neurochem 77:94–102
Gill R, Soriano M, Blomgren K, Hagberg H, Wybrecht R, Miss M-T, Hoefer S, Adam G, Niederhauser O, Kemp JA, Loetscher H 2002 Role of caspase-3 activation in cerebral ischemia-induced neurodegeneration in adult and neonatal brain. J Cereb Blood Flow Metab 22:420–430(Katarina G. Brywe, Anna-L)
Departments of Internal Medicine (R.G., S.D., E.G.) and Biomedical Sciences & Oncology (M.V.), University of Turin, 8-10124 Turin, Italy
Abstract
Hexarelin (HEX) is a peptide GH secretagogue with a potent ability to stimulate GH secretion and recently reported cardioprotective actions. However, its effects in the brain are largely unknown, and the aim of the present study was to examine the potential protective effect of HEX on the central nervous system after injury, as well as on caspase-3, Akt, and extracellular signal-regulated protein kinase (ERK) signaling cascades in a rat model of neonatal hypoxia-ischemia. Hypoxic-ischemic insult was induced by unilateral carotid ligation and hypoxic exposure (7.7% oxygen), and HEX treatment was administered intracerebroventricularly, directly after the insult. Brain damage was quantified at four coronal levels and by regional neuropathological scoring. Brain damage was reduced by 39% in the treatment group, compared with vehicle group, and injury was significantly reduced in the cerebral cortex, hippocampus, and thalamus but not in the striatum. The cerebroprotective effect was accompanied by a significant reduction of caspase-3 activity and an increased phosphorylation of Akt and glycogen synthase kinase-3, whereas ERK was unaffected. In conclusion, we demonstrate for the first time that HEX is neuroprotective in the neonatal setting in vivo and that increased Akt signaling is associated with downstream attenuation of glycogen synthase kinase-3 activity and caspase-dependent cell death.
Introduction
HYPOXIC-ISCHEMIC (HI) brain damage in the perinatal period is a major contributor to neonatal mortality and long-term morbidity with neurological impairments presenting in the survivors (1, 2). Brain damage does not develop acutely during the HI insult but rather evolves over several days (secondary brain injury), opening a window of opportunity for therapeutic intervention (3). Growth factors protect neurons from dying under a wide variety of circumstances in vitro (4, 5, 6, 7). It has also been shown that several trophic factors, including GH and IGF-I, have neuroprotective properties during the secondary phase after HI in vivo (8, 9, 10, 11, 12), although the underlying mechanisms are not yet fully understood. However, it has been demonstrated that activation of phosphatidylinositol-3 kinase (PI3K) pathway and the downstream phosphorylation of the kinase Akt (pAkt) mediate growth factor-induced neuronal survival in vitro (13). pAkt promotes cell survival and can inhibit apoptosis by inactivating several proapoptotic targets, including Bad, glycogen synthase kinase 3 (GSK3), and caspase 9, or by modification of transcription factors (14, 15, 16, 17, 18). GSK3 has been shown to play a role in apoptosis and has been suggested to be a key target of PI3K/Akt survival signaling pathway (16). After HI in the neonatal brain, GSK3 is activated by dephosphorylation and translocated to the nucleus, where it may contribute to the development of brain damage (19). It has been demonstrated that inhibition of GSK3 reduces infarct size in an adult stroke model (20), which lends further support for its important proapoptotic role in brain injury. The mechanisms for induction of apoptosis via GSK3 are not fully clarified, but it has been suggested to cause degradation of -catenin and to inhibit mitochondrial pyruvate dehydrogenase, with subsequent metabolic failure (21, 22). GSK3 also interacts with transcription factors (among them, p53) in both the nucleus and mitochondria and affects cytochrome c release and caspase-3 activity (23, 24). Caspase-dependent mechanisms seem particularly important for cell death in the immature brain (25, 26, 27, 28), because caspase-3 inhibitors reduce neonatal brain injury (29). Moreover, neuroprotection, by both brain derived neurotrophic factor (10) and IGF-I, is linked to a reduction of the activation of caspase-3 (19).
Another kinase pathway activated by growth factors is the MAPK p42/44 ERK pathway. Activation of ERK has been shown to inhibit apoptosis induced by hypoxia (30), growth factor withdrawal (31), hydrogen peroxide (32), and chemotherapeutic agents (33). Furthermore, brain-derived neurotrophic factor neuroprotection in the neonatal rat was shown to be mediated by activation of the MAPK/ERK pathway (34) and IGF-I treatment of neonatal rats after HI activated both Akt and ERK pathways (19).
Hexarelin (HEX) is a six-amino-acid peptide that belongs to a family of synthetic GH secretagogues (GHS) (35) that have a potent ability to release GH from the pituitary and to stimulate food intake (36). Ghrelin is the recently discovered endogenous ligand to the GHS receptor-1a (37, 38). In addition to their neuroendocrine activity, ghrelin and synthetic GHS have been shown to be cardioprotective (39, 40) and to inhibit apoptosis in cardiomyocytes and endothelial cells through activation of ERK and Akt kinases (41).
Based on the cardioprotective effect and activation of survival signaling pathways in cardiomyocytes, we raised the hypothesis that HEX could be neuroprotective. Our aim was to examine the posttreatment effects of HEX on central nervous system injury, caspase-3 activation, and Akt and ERK intracellular signaling in a rat model of neonatal HI.
Materials and Methods
Animal model
Unilateral HI was induced in neonatal rats as previously described (42, 43). Briefly, 7-d-old [postnatal day (PND) 7] Wistar rats were anesthetized with enflurane (3.5% for induction and 1.5% for maintenance) in a mixture of nitrous oxide and oxygen. The left common carotid artery was cut between two prolene sutures (6–0). After anesthesia and surgery, the animals were allowed to recover for 60 min. They were thereafter exposed to 60 min of hypoxia in a humidified chamber at 36 C with 7.7% oxygen in nitrogen. After hypoxia. the pups were returned to and kept with their dams until they were killed as described below. The animal studies were approved by the animal ethics committee in Gteborg.
Intracerebral injection of HEX and vehicle
Animals received intracerebroventricular (icv) injections in a total vol of 5 μl containing either 1 μg HEX (Pharmacia & Upjohn, Inc., Stockholm, Sweden) or vehicle (NaCl, 0.9 mg/ml) starting immediately after HI on PND 7. Injections were aimed at the left ventricle at 1.5 mm lateral to the sagittal suture, 5.7 mm rostral to lambda, and 2.8 mm deep to the skull surface with a 27-gauge needle. Animals were under anesthesia (enflurane, 1.5%), on a snout mask, while injected with a Hamilton syringe connected to a MCA pump, 1 μl/min. Accuracy of injection site with the method was verified using methylene blue staining on control animals (n = 3).
Study protocol I, effect of HEX on brain injury
Immediately after HI, animals were treated with 1 μg HEX (n = 27) or vehicle (n = 27), icv as described above, and this dose was repeated once daily at 24 and 48 h after HI. At PND 10, animals were perfused intracardially with 0.9% NaCl followed by 5% buffered formaldehyde (Histofix; Histolab, Gteborg, Sweden). Brains were removed from the skull and immersion-fixed at 4 C for 24 h and were thereafter dehydrated and embedded in paraffin. Brains were cut into 5-μm coronal sections at four anteroposterior levels from the anterior striatum to the posterior part of hippocampus. Adjacent sections were stained with thionin/acid fuschin (44) and for microtubule-associated protein (MAP-2) (1:1000, 1 h, mouse-anti-MAP-2, clone HM-2; Sigma, St. Louis, MO) (45). Immunoreactivity was visualized using diaminobenzidine (DAB) as described below.
Evaluation of brain damage
Brain damage was evaluated using both area measurements of tissue loss at four anatomical levels and by neuropathological scoring.
Intact neurons (dendrites and soma) express MAP-2 and infarction in gray matter is associated with a distinct loss of MAP-2 immunoreactivity. MAP-2-positive areas in the ipsilateral and contralateral hemispheres were outlined by an observer blinded to the study groups, and calculations were made using the Olympus Micro Image analysis software version 4.0 (Olympus Optical Co., Ltd., Tokyo, Japan). Brain tissue loss was calculated by subtracting the MAP-2-positive area of the ipsilateral hemisphere from the contralateral hemisphere and was expressed as percentage of the contralateral hemisphere as previously described (46).
Regional brain injury was also evaluated by an observer blinded to the study groups, using a neuropathological scoring system where injury in cortex was graded from 0–4 with 0 being no observable injury and 4 being confluent infarction encompassing most of the hemisphere. The damage in the hippocampus, thalamus, and striatum was assessed regarding both hypothrophy (0–3) and observable cell injury/infarction (0–3), resulting in a scoring for each region (0–6) (46).
Rectal temperature
The rectal temperature, which has been shown to correspond to the brain core temperature (47), was measured in HEX-treated (n = 9) and vehicle-treated (n = 8) rats from study protocol I at 1, 3, 6, 12, 24, 36, 48, and 72 h after HI and start of the HEX injections.
Study protocol II, effect of HEX on caspase-3 and pAkt, GSK3, and ERK
Immediately after HI, animals were treated with one injection of HEX (1 μg) (n = 7) or vehicle (n = 6). Animals were killed 24 h after HI at PND 8, and cytosol fractions were prepared for measurement of caspase-3 activity (see below). The cellular localization of pAkt and phosphorylated GSK (pGSK)3 was studied with immunohistochemistry (see below) in sections double-stained with neuronal nuclear protein (NeuN) (n = 3/ group). GH and IGF-I proteins on brain sections from HI animals were also assessed by immunohistochemistry (see below). The effect of HEX or vehicle treatment on pAkt/Akt, pGSK3/GSK3, and IGF-I receptor (IGF-IR) phosphorylation immunoreactivity after HI was evaluated 24 h after HI and drug administration (HEX, n = 7) (vehicle, n = 6) using Western blot (see below); pGSK3 was analyzed in both cytosolic and nuclear fractions.
Antibodies
Anti-microtubule-associated protein 2 (MAP-2, clone HM-2) was purchased from Sigma. LaminB (M-20) (sc-6217) and IGF-IR -subunit antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-NeuN (MAB377), Alexa Fluor 488 streptavidin IgG (H+L) conjugate (green) and Alexa Fluor 594 goat antimouse IgG (H+L) were purchased from Chemicon International, Inc. (Temecula, CA). The antibodies against phospho-Akt (Ser473) rabbit polyclonal (no. 9277, no. 9271), Akt rabbit polyclonal (no. 9272), phospho-GSK3(Ser9) rabbit polyclonal (no. 9336), phospho-p44/42 MAPK (pERK) rabbit polyclonal (no. 9101), and p42/44 MAPK (ERK) rabbit polyclonal (no. 9102) were purchased from Cell Signaling Technology, Inc. (Beverly, MA). Phosphotyrosine-PY20 antibody was from BD Transduction Laboratories (Milan, Italy). Anti-GSK3 mouse monoclonal (no. 05–412) was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). An IGF-I goat polyclonal antibody was purchased from Santa Cruz (C-20) and used diluted 1:80 after microwave oven pretreatment (three cycles of 5 min each in EDTA buffer, pH 8.1). GH rabbit polyclonal antibody (DakoCytomation, Carpinteria, CA) was used without antigen retrieval and diluted 1:2000. All secondary antibodies were from Vector Laboratories, Inc. (Burlingame, CA).
Immunohistochemistry
Incubation with primary antibody, anti-phospho-Akt (Ser473) peptide) (no. 9277) 1:50, was performed in PBS, at 4 C, overnight (O/N). Sections were washed in PBS and incubated with biotinylated secondary antibody for 1 h, followed by inhibition of endogenous peroxidase (0.6% H2O2 in methanol, 10 min) and incubation with avidin-biotin enzyme complex (20 μl/ml, 1 h, ABC-Elite; Vector Laboratories). Immunoreactivity was visualized using DAB (0.5 mg/ml) enhanced with nickel sulfate (15 mg/ml). The specificity of antibodies was tested by omission of the primary antibody. Previous studies in neuronal murine tissue have confirmed antibody specificity with preabsorbtion using phospho-Akt Ser 473 peptide (48). For detection of GH and IGF-I protein, a standard immunoperoxidase technique was employed. To reveal the immunohistochemical reaction, a biotin-free detection system (EnVision; DakoCytomation) was employed, and DAB was used as chromogen. Paraffin-embedded tissue from rat pituitary and from rat liver served as positive controls for GH and IGF-I experiments, respectively. Omission of the primary antibodies on parallel sections was used as negative control.
Immunofluorescence
Animals were deeply anesthetized by ip injection of 0.2 ml thiopenthal (50 mg/ml), followed by intracardial perfusion with 0.9% NaCl and then 5% buffered formaldehyde (Histofix; Histolab, Gothenburg, Sweden) after administration of HEX or vehicle. Brains were removed from the skull and immersion-fixed at 4 C for 24 h, dehydrated, embedded in paraffin, and cut into 5-μm coronal sections at the levels of hippocampus and striatum. Before immunohistochemical staining, sections were deparaffinized and boiled in citric acid buffer (0.01 M, pH 6.0; 10 min), and sections were treated with proteinase-K (Boeringer Mannheim, Mannheim, Germany) (10 μg/ml) in PBS for 9 min at room temperature. Nonspecific binding was blocked by incubation with appropriate serum (4%). Sections were incubated with the following antibodies: anti-phospho-Akt (Ser473) rabbit polyclonal (no. 9277) 1:50 in PBS or anti-phospho-GSK3(Ser9), rabbit polyclonal 1:50 in 1% BSA PBS, at 4 C, O/N. After washing, and with washing procedures in between, sections were incubated for 1 h at room temperature for each of the following steps with the subsequent antibodies: biotinylated secondary antibodies, goat antirabbit 1:250 in PBS, Alexa Fluor 488 streptavidin IgG (H+L) conjugate 1:100 (10 μl/ml) (green) in PBS, anti-NeuN 1:200 in PBS, and Alexa Fluor 594 goat antimouse IgG (H+L) conjugate 1.200 (10 μl/ml) (red) in PBS. After washing, sections were mounted using Vectashield mounting medium. Sections were analyzed under an Olympus BX40 fluorescence microscope equipped with an Olympus DP50 cooled digital camera.
IGF-IR immunoprecipitation
Cell lysates from cytosolic fractions of the damaged left hemispheres of HEX- and vehicle-treated HI rats were incubated O/N at 4 C with anti-IGF-IR -antibody (1:500 dilution). Immune complexes were precipitated by adding protein A-Sepharose and incubating for 2 h at 4 C. Pellets were resuspended in 40 μl of reducing Laemmli buffer and the proteins separated by 8% gel SDS-PAGE.
Western blot
Brains were rapidly harvested, hemispheres were split, and cortex dissected out. Each sample was immediately homogenized in ice-cold homogenization buffer (15 mM Tris-HCl, pH 7.6; containing 3 mM EDTA, 1 mM MgCl2, 320 mM sucrose, 1 mM dithiothreitol) protease, and phosphatase inhibitor cocktail was added to final concentrations of 1 and 2%, respectively. The homogenates were centrifuged at 800 x g for 10 min at 4 C for preparation of nuclear fractions. Supernatants were centrifuged at 9200 x g for 15 min at 4 C for preparation of cytosolic fractions. Protein content was quantified using the method presented by Whitaker and Granum (1980) (49), adapted for microplates. Samples were mixed with an equal volume of 3x SDS-PAGE buffer and heated (96 C) for 5 min. One sample containing 20 μg protein from the cytosolic fraction or 10 μg of the nuclear fraction was applied in each well of a Novex (San Diego, CA) precast 8–16% Tris-glycine gel. After electrophoresis, proteins were transferred to a nitrocellulose membrane (Optitran, 0,2 μm; Schleicher & Schuell, Inc., Dassel, Germany). Membranes were blocked in 30 mM Tris-Hcl (pH 7.5), 100 mM NaCl, and 0.1% Tween 20 [Tris-buffered saline with Tween 20 (TBS-T)] containing 5% fat free milk powder. Incubations with the following primary antibodies diluted in TBS-T containing 3% BSA and 9 mM NaN3 for 1 h in room temperature were performed: anti-phospho-Akt (Ser473) (no. 9271) rabbit polyclonal 1:1000, anti-Akt rabbit polyclonal 1:1000, anti-phospho-GSK3 (Ser9) rabbit polyclonal 1:1000, anti-GSK3 mouse monoclonal 1:500, anti-phospho-p44/42 MAPK (pERK) rabbit polyclonal 1:1000, anti-p42/44 MAPK (ERK) rabbit polyclonal 1:1000, and anti-LaminB goat polyclonal 1:200. Membranes from immunoprecipitates were blocked with 1% BSA in TBS with 0.1% Tween for 2 h at room temperature and incubated O/N at 4 C with antibody anti-P-tyrosine (1:500 dilutions). Blots were washed three times with TBS-T and incubated with the appropriate secondary peroxidase conjugate diluted in blocking buffer. Immunoreactive species were visualized using Super Signal Western Dura chemiluminescence substrates (Pierce Biotechnology, Inc., Rockford, IL) and a cooled charge coupled device camera (LAS1000; Fuji Photo Film Co., Ltd., Tokyo, Japan). Immunoreactive bands were quantified using Image Gauge software (version 3.3, Fuji). To standardize quantification between the gels, the same five controls were run on every gel except in experiments with assessment of tyrosine phosphorylation of the IGF-IR -subunit, where samples (two vehicle- and two HEX-treated) were run on each gel. Every sample was analyzed three times, and the average value was used as n = 1. Each protein band on the Western blots was derived from one animal. Each immunoblot was performed using the samples from three to four animals in each experimental group. Membranes were stripped for reprobing with new antibodies by having them incubated in stripping buffer (62.5 mM Tris-Hcl, pH 6.7; 100 mM -mercaptoethanol; and 2% sodium dodecyl sulfate) at 55 C for 30 min.
Fluorometric assay of caspase-3-like activity
Samples of cytosolic fraction were mixed with extraction buffer (50 mM Tris-HCl, pH 7.3; 100 mM NaCl; 5 mM EDTA; 1 mM EGTA; 3 mM NaN3; 1 mM phenylmethylsulfonylfluoride; 1 μg/ml pepstatin; 2.5 μg/ml leupeptin; 10 μg/ml aprotinin 0.2% 3-[(3-cholamidoprophylene) dimethylamonio]propane sulphonic acid; protease inhibitor cocktail (P8340; Sigma) 1%), 1:3, on a microtiter plate (Microflour; Dynatech Laboratories, Inc., Chantilly, VA). After incubation for 15 min at room temperature, 100 μl peptide substrate, 50 μM Ac-Asp-Glu-Val-Asp-aminomethyl coumarine (Ac-DEVD-AMC; Enzyme Systems Products, Livermore, CA) in extraction buffer without inhibitors or 3-[(3-cholamidoprophylene) dimethylamonio]propane sulphonic acid but with 4 mM dithiothreitol were added. Cleavage of the substrate was measured at 37 C using Spectramax Gemini microplate fluorometer (Molecular Devices, Sunnyvale, CA), with an excitation wavelength of 380 nm and emission wavelength of 460 nm. The degradation was followed at 2-min intervals for 2 h, and V-max was calculated from the entire linear part of the curve. Standard curves with AMC in the appropriate buffer were used to express the data in picomoles of AMC (7-amino-4-methyl-coumarin) formed per minute and per milligram of protein.
Statistics
The data are expressed as means ± SEM. The tests used were ANOVA with Fisher’s post hoc test. Values of P < 0.05 were considered to be significant. In all cases, n corresponds to the number of animals.
Results
Study protocol I, effect of HEX on brain injury
Area measurements comparing the lesioned hemisphere with the undamaged hemisphere demonstrated a significant reduction of damage on all four levels of the brain in the HEX-treated animals compared with controls (Fig. 1A). Neuroprotection was most pronounced at the level of anterior hippocampus (–2 mm from bregma), with a 43% reduction of MAP-2 loss (HEX: 25.7 ± 3.5% vs. vehicle: 45.2 ± 4.6%; P < 0.01). When using a neuropathological scoring system, brain injury was reduced in the cerebral cortex (HEX: 1.9 ± 0.2 vs. vehicle: 2.6 ± 0.2; P < 0.05), hippocampus (HEX: 2.4 ± 0.3 vs. vehicle: 3.6 ± 0.3; P < 0.05), and thalamus (HEX: 1.7 ± 0.2 vs. vehicle: 2.6 ± 0.3; P < 0.05) but not in the striatum (Fig. 1B). There were no differences in body temperature, mortality, or body weight (data not shown) between the two groups. Moreover, there were no significant differences in brain injury between male and female animals (data not shown).
Study protocol II, HEX increases Akt and pGSK3 phosphorylation
Double immunostaining demonstrated that pAkt and pGSK3 colabeled with NeuN, indicating a neuronal localization in the cerebral cortex (Fig. 2). Western blot was used to quantify pAkt and pGSK3 in both nuclear and cytosolic fractions. After HI, pAkt and pGSK3 immunoreactivity in cytosolic fractions were significantly increased by HEX at 24 h after HI. In the nuclear fraction, pGSK3 was also increased by HEX-, compared with vehicle-treated animals, after HI (Figs. 3 and 4).
HEX had no effect on ERK phosphorylation
Western blot was also used to quantify pERK. HEX did not alter total ERK or pERK immunoexpression at 24 h after HI (data not shown).
HEX reduced caspase-3-like activity after HI
A fluorometric assay was used to quantify caspase-3-like activity. HEX reduced caspase-3-like activity (by 56%) in the cerebral cortex 24 h after HI (P < 0.01) but did not affect the activity in the contralateral hemisphere (Fig. 5).
Immunohistochemical expression of GH and IGF-I
To investigate the possible activation of the GH/IGF-I axis by HEX, immunohistochemistry was performed on brain sections 24 h after HI in rats treated with HEX or vehicle. GH antibody specificity was demonstrated by intense reactivity in single cells of rat pituitary (Fig. 6A). However, staining for GH was totally negative or only weakly positive on sections of HI brains from both HEX- and vehicle-treated animals (Fig. 6, C and E). IGF-I immunoreactivity was found in rat liver cells (Fig. 6B) and cerebral neurons in both contralateral (Fig. 6D) and ipsilateral, perilesional areas (Fig. 6F), without significant difference in intensity between HEX- and vehicle-treated rats.
Phosphorylation of the IGF-IR
To determine whether the neuroprotective effects of HEX were mediated by increased signaling through the IGF-IR, tyrosine phosphorylation of the IGF-IR -subunit in cytosolic fractions from the left damaged hemisphere of HI rats was analyzed by Western blot. As shown in Fig. 7, there was a significant increase in phosphorylation of the IGF-IR at 24 h after injury in HEX-treated rats compared with vehicle controls.
Discussion
The main findings in the present study are: that HEX reduces brain injury in an in vivo model of HI, and that the protection is accompanied by phosphorylation of Akt and GSK3, indicating possible involvement of the PI3K pathway.
HEX significantly reduced injury evaluated both by measuring tissue loss at four levels of the brain and by regional neuropathological scoring. Significant protection was seen in cortex, hippocampus, and thalamus but not in the striatum. The spatial distribution of protection correlates with previously reported GH effects in the brain, given that GH did not offer protection in the striatum and that localization of GH receptor/binding protein immunoreactivity is lacking in the striatum (50, 51, 11). This would suggest that the protective effect of HEX could either, in part, be mediated by induction of GH or that HEX and GH share common pathways for cellular protection. The latter alternative is more probable because there was no detectable immunostaining of neuronal GH on brain sections from either HEX- or vehicle-treated HI rats.
So far, no studies regarding possible neuroprotective effects of GHS after HI have been published. However, a recent study of administration of another GHS, GH-releasing peptide 6, to adult rats under physiological conditions has shown increased IGF-I mRNA levels in hypothalamus, cerebellum, and hippocampus but not in cortex (52). Thus, it could be hypothesized that the neuroprotective effects reported in the present study were mediated by increased expression of IGF-I. To address this possibility, we performed immunohistochemistry on brain sections from HEX- and vehicle-treated HI rats. We could not detect any significant increase in IGF-I immunoreactivity in HEX-treated rats compared with controls. However, it is important to point out that immunohistochemistry was only performed on brain sections 24 h after HI, and an induction of IGF-I at other time points cannot categorically be ruled out. On the other hand, if IGF-I was an important mediator of HEX effects, a reduction of brain injury in striatum would have been expected because IGF-IRs are present in the striatum (53, 54), and icv administration of IGF-I does indeed protect the striatum after HI in neonatal rats (19). Furthermore, the present study indicates that HEX activated the PI3K pathway in the central nervous system after HI but did not affect ERK phosphorylation, in agreement with the study by Frago et al. (52). This is in contrast to IGF-I, which has been demonstrated to activate both the PI3K (19) and the ERK pathways after icv administration after HI (unpublished observations). The finding that HEX increased phosphorylation of the IGF-IR is interesting and intriguing. In the absence of an obvious induction of IGF-I, it is possible to speculate that the increased phosphorylation may be due to transactivation of the IGF-IR by HEX or an endogenous factor. It has previously been shown that G protein-coupled receptor agonists such as angiotensin-II, thrombin, and endothelin-1 can stimulate the phosphorylation of IGF-IR and/or Akt (55, 56); although, to our knowledge, this has not previously been shown for GHS.
In summary, results from our laboratory would suggest that the neuroprotective effect of HEX seen in this experimental model is not primarily mediated by an induction of the GH/IGF-I axis, although an increased signaling through the IGF-IR may contribute to the reduction of brain injury. More studies are certainly needed to elucidate a possible link between these endocrine systems and their neuroprotective effects.
Synthetic GHS and the endogenous ligand ghrelin have been found to have cardioprotective properties in several in vivo studies (39, 40), although the molecular mechanisms remain largely unknown so far. Two recent in vitro studies on cardiomyocytes and endothelial cells have addressed mechanistical aspects and suggest that the antiapoptotic effects of GHS are mediated by activation of both Akt and ERK kinases (41) and by regulating the activity of caspase-3 and expression of bax and bcl-2 (57). These findings are largely in agreement with the mechanisms proposed in the present study, although, as has been previously discussed, we could not detect any activation of ERK. We suggest that this difference could be due to tissue-specific properties and different experimental conditions.
Another finding in the present study was that GSK3 phosphorylation was increased after HEX treatment both in cytosol and nucleus in post-HI animals, which is the expected response after activation of the PI3K pathway (58, 59). To our knowledge, phosphorylation of GSK3 by GHS has not previously been reported, but such a response could partly explain the neuroprotective effect of HEX because GSK3 is strongly proapoptotic (60), and inhibitors of GSK3 reduce infarction after ischemia in adult animals (20) and improve neuronal survival in vitro (61). Activation of GSK3 has been linked to triggering of caspase-dependent apoptosis (23), but GSK3 inhibitors given in adult stroke models did not affect caspase activation (20), implicating a role also for caspase-independent mechanisms. However, caspase activation is considered to be of greater importance in the immature brain compared with the adult (25, 26, 28, 62). Hence, the HEX-induced reduction of caspase-3-like activity after HI offers additional support for the involvement of caspases in our experimental model. Moreover, studies on juvenile and adult rat cerebellum have shown HEX-induced reduction of caspase-3 and -9 activity (61), and HEX attenuated apoptosis and caspase-3-like activity in rat cardiomyocytes (55).
In conclusion, we demonstrate, for the first time, that HEX is markedly neuroprotective in an in vivo model of HI. Moreover, we provide evidence that the neuroprotective effect of HEX is, at least partly, mediated through the PI3-kinase pathway, including activation of Akt and inhibition of GSK3 through phosphorylation. The present findings suggest a possible role for GHS as neuroprotective agents, which may be of future clinical significance because no available medical therapy against HI brain injury exists today. However, further studies in other experimental models are mandatory to confirm the neuroprotective effects of GHS.
Footnotes
This work was supported by grants from the Swedish Medical Research Council (09455), the Willhelm and Martina Lundgren Foundation, and the Gteborg Medical Society and by grants to researchers in the public health service from the Swedish government (LUA).
Abbreviations: DAB, Diaminobenzidine; GHS, GH secretagogue(s); GSK3, glycogen synthase kinase 3; HEX, hexarelin; HI, hypoxia-ischemia; icv, intracerebroventricular(ly); IGF-IR, IGF-I receptor; MAP-2, microtubule-associated protein 2; NeuN, neuronal nuclear protein; O/N, overnight; p (prefix), phosphorylated; PI3K, phosphatidylinositol-3 kinase; PND, postnatal day; TBS-T, Tris-buffered saline with Tween 20.
References
Volpe JJ 2001 Neurology of the newborn. Philadelphia: WB Saunders
Cowan F, Rutherford M, Groenendaal F, Eken P, Mercuri E, Bydder GM, Meiners LC, Dubowitz LM, de Vries LS 2003 Origin and timing of brain lesions in term infants with neonatal encephalopathy. Lancet 361:736–742
Wyatt JS, Edwards AD, Azzopardi D, Reynolds EO 1989 Magnetic resonance and near infrared spectroscopy for investigation of perinatal hypoxic-ischaemic brain injury. Arch Dis Child 64:953–963
Russell JW, Windebank AJ, Schenone A, Feldman EL 1998 Insulin-like growth factor-I prevents apoptosis in neurons after nerve growth factor withdrawal. J Neurobiol 36:455–467
Delaney CL, Cheng HL, Feldman EL 1999 Insulin-like growth factor-I prevents caspase-mediated apoptosis in Schwann cells. J Neurobiol 41:540–548
Takadera T, Matsuda I, Ohyashiki T 1999 Apoptotic cell death and caspase-3 activation induced by N-methyl-D-aspartate receptor antagonists and their prevention by insulin-like growth factor I. J Neurochem 73:548–556
van Golen CM, Castle VP, Feldman EL 2000 IGF-I receptor activation and BCL-2 overexpression prevent early apoptotic events in human neuroblastoma. Cell Death Differ 7:654–665
Johnston BM, Mallard C, Williams C, Gluckman P 1996 Insulin-like growth factor-1 is a potent neuronal rescue agent after hypoxic-ischemic injury in fetal lambs. J Clin Invest 97:300–308
Gustafson K, Hagberg H, Bengtsson B, Brantsing C, Isgaard J 1999 Possible protective role of growth hormone in hypoxia-ischemia in neonatal rats. Pediatr Res 45:318–323
Han BH, D’Costa A, Back SA, Parsadanian M, Patel S, Shah AR, Gidday JM, Srinivasan A, Deshmukh M, Holtzman DM 2000 BDNF blocks caspase-3 activation in neonatal hypoxia-ischemia. Neurobiol Dis 7:38–53
Scheepens A, Sirimanne ES, Breier BH, Clark RG, Gluckman PD, Williams CE 2001 Growth hormone as a neuronal rescue factor during recovery from CNS injury. Neuroscience 104:677–687
Sizonenko S, Sirimanne ES, Williams C, Gluckman P 2001 Neuroprotective effects of the N-terminal tripeptide of IGF1, glycine-proline-glutamate, in the immature rat brain after hypoxic-ischemic injury. Brain Res 922:42–50
Kulik G, Klippel A, Weber MJ 1997 Antiapoptotic signalling by the insulin-like growth factor I receptor, phosphatidylinositol 3-kinase, and Akt. Mol Cell Biol 17:1595–1606
Datta SR, Dudek H, Tao X, Masters S, Fu H, Gotoh Y, Greenberg ME 1997 Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell 91:231–241
Cardone MH, Roy N, Stennicke HR, Salvesen GS, Franke TF, Stanbridge E, Frisch S, Reed JC 1998 Regulation of cell death protease caspase-9 by phosphorylation. Science 282:1318–1321
Pap M, Cooper GM 1998 Role of glycogen synthase kinase-3 in the phosphatidylinositol 3-kinase/Akt cell survival pathway. J Biol Chem 273:19929–19932
Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS, Anderson MJ, Arden KC, Blenis J, Greenberg ME 1999 Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 96:857–868
Romashkowa JA, Makarov SS 1999 NF-B is a target for Akt in anti-apoptotic PDGF signaling. Nature 401:86–90
Brywe KG, Mallard C, Gustavsson M, Hedtjrn M, Leverin A-L, Wang X, Blomgren K, Isgaard J, Hagberg H 2005 IGF-I neuroprotection in the immature brain after hypoxia-ischemia, involvement of Akt and GSK3 Eur J Neurosci 21:1489–1502
Kelly S, Heng Z, Guo SH, Danye C 2003 Treatment with the GSK3B inhibitor Chir98025 decreases infarct size following MCAo. Stroke 34:301
Hoshi M, Takashima A, Noguchi K, Murayama M, Sato M, Kondo S, Saitoh Y, Ishiguro K, Hoshino T, Imahori K 1996 Regulation of mitochondrial pyruvate dehydrogenase activity by protein kinase I/glycogen synthase kinase 3 in brain. Proc Natl Acad Sci USA 93:2719–2723
Zhang Z, Hartmann H, Do VM, Abramowski D, Sturchler-Pierrat C, Staufenbiel M, Sommer B, van de Wetering M, Clevers H, Saftig P, De Strooper B, He X, Yankner BA 1998 Destabilization of -catenin by mutations in presenilin-1 potentiates neuronal apoptosis. Nature 395:698–702
Watcharasit P, Bijur GN, Zmijewski JW, Song, L, Zmijewska A, Chen X, Johnson GV, Jope RS 2002 Direct, activating interaction between glycogen synthase kinase-3 and p53 after DNA damage. Proc Natl Acad Sci USA 99:7951–7955
Watcharasit P, Bijur GN, Song L, Zhu J, Chen X, Jope RS 2003 Glycogen synthase kinase-3 (GSK3) binds to and promotes the actions of p53. J Biol Chem 278:48872–48879
Cheng Y, Deshmukh M, D’Costa A, Demaro JA, Gidday JM, Shah A, Sun Y, Jacquin MF, Johnson EM, Holtzman DM 1998 Caspase inhibitor affords neuroprotection with delayed administration in a rat model of neonatal hypoxic-ischemic brain injury. J Clin Invest 101:1992–1999
Hu BR, Liu CL, Ouyang YB, Blomgren K, Siesjo BK 2000 Involvement of caspase-3 in cell death after hypoxia-ischemia declines during brain maturation. J Cereb Blood Flow Metab 20:1294–1300
Zhu C, Wang X, Hagberg H, Blomgren K 2000 Correlation between caspase-3 activation and three different markers of DNA damage in neonatal cerebral hypoxia-ischemia. J Neurochem 75:819–829
Wang X, Karlsson JO, Zhu C, Bahr BA, Hagberg H, Blomgren K 2001 Caspase-3 activation after neonatal rat cerebral hypoxia-ischemia. Biol Neonate 79:172–179
Han BH, Xu D, Choi J, Han Y, Xanthoudakis S, Roy S, Tam J, Vaillancourt J, Colucci J, Siman R, Giroux A, Robertson GS, Zamboni R, Nicholson DW, Holtzman DM 2002 Selective, reversible caspase-3 inhibitor is neuroprotective and reveals distinct pathways of cell death after neonatal hypoxic-ischemic brain injury. J Biol Chem 277:30128–30136
Buckley S, Driscoll B, Weinberg L, Anderson K, Warburton D 1999 ERK activation protects against DNA damage and apoptosis in hyperoxic rat AEC2. Am J Physiol 277:159–166
Erhardt P, Schremser EJ, Cooper GM 1999 B-Raf inhibits programmed cell death downstream of cytochrome c release from mitochondria by activating the MEK/ERK pathway. Mol Cell Biol 19:5308–5315
Wang X, Martindale JL, Liu Y, Holbrook NJ 1998 The cellular response to oxidative stress: influences of mitogen-activated protein kinases signalling pathways on cell survival. Biochem J 333:291–300
Anderson CNG, Tolkovsky AM 1999 A role for MAPK/ERK in sympathetic neuron survival: protection against a p53-dependent, JNK-independent induction of apoptosis by cytosine arabinoside. J Neurosci 19:664–673
Han BH, Holtzman DM 2000 BDNF protects the neonatal brain from hypoxic-ischemic injury in vivo via the ERK pathway. J Neurosci 20:5775–5781
Ghigo E, Arvat E, Muccioli G, Camanni F 1997 Growth hormone-releasing peptides. Eur J Endocrinol 136:445–460
Nagaya N, Kangawa K 2003 Ghrelin, a novel growth hormone-releasing peptide, in the treatment of chronic heart failure. Regul Pept 114:71–77
Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K 1999 Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 402:656–660
Nakazato M, Murakami N, Date Y, Kojima M, Matsuo H, Kangawa K, Matsukura S 2001 A role for ghrelin in the central regulation of feeding. Nature 409:194–198
Locatelli V, Rossoni G, Schweiger F, Torsello A, De Gennaro Colonna V, Bernareggi M, Deghenghi R, Muller EE, Berti F 1999 Growth hormone independent cardioprotective effects of hexarelin in the rat. Endocrinology 140:4024–4031
Tivesten , Bollano E, Caidahl K, Kujacic V, Sun XY, Hedner T, Hjalmarson , Bengtsson B-, Isgaard J 2000 The growth hormone secretagogue hexarelin improves cardiac function in rats after experimental myocardial infarction. Endocrinology 141:60–66
Baldanzi G, Filigheddu N, Cutrupi S, Catapano F, Bonissoni S, Fubini A, Malan D, Baj G, Granata R, Broglio F, Papotti M, Surico N, Bussolino F, Isgaard J, Deghenghi R, Sinigaglia F, Prat M, Muccioli G, Ghigo E, Graziani A 2002 Ghrelin and des-acyl ghrelin inhibit cell death in cardiomyocytes and endothelial cells through ERK1/2 and PI 3-kinase/AKT. J Cell Biol 159:1029–1037
Rice JE, Vanucci RC, Brierley JB 1981 The influence of immaturity on hypoxic-ischemic brain damage in the rat. Ann Neurol 9:131–141
Hagberg H, Bona E, Gilland E, Puka Sunvall M 1997 Hypoxia-ischemia model in 7 day old rat: possibilities and shortcomings. Acta Paediatr Suppl 442:85–88
Mallard EC, Williams CE, Gunn AJ, Gunning MI, Gluckman PD 1993 Frequent episodes of brief ischemia sensitize the fetal sheep brain to neuronal loss and induce striatal injury. Pediatr Res 33:61–65
Gilland E, Bona E, Hagberg H 1998 Temporal changes of regional glucose use, blood flow, and microtubule-associated protein 2 immunostaining after hypoxia-ischemia in the immature rat brain. J Cereb Blood Flow Metab 18:222–228
Hedtjrn M, Leverin A-L, Eriksson K, Blomgren K, Mallard C, Hagberg H 2002 Interleukin-18 involvement in hypoxic-ischemic brain injury. J Neurosci 22:5910–5919
Thoresen M, Bagenholm R, Loberg BR, Apricena F, Kjellmer I 1996 Posthypoxic cooling of neonatal rats provides protection against brain injury. Arch Dis Child 74:F3–F9
Noshita N, Lewen A, Sugawara T, Chan PH 2002 Akt phosphorylation and neuronal survival after traumatic brain injury in mice. Neurobiol Dis 9:294–304
Whitaker JG, Granum PE 1980 An absolute method for protein determination based on a difference in absorbance at 235 and 280 nm. Anal Biochem 109:156–159
Lobie PE, García-Aragón J, Lincoln DT, Barnard R, Wilcox JN, Waters MJ 1993 Localization and ontogeny of growth hormone receptor gene expression in the central nervous system. Dev Brain Res 74:225–233
Scheepens A, Sirimanne E, Beilharz E, Breier BH, Waters MJ, Gluckman PD, Williams CE 1999 Alterations in the neural growth hormone axis following hypoxic-ischemic brain injury. Brain Res Mol Brain Res 68:88–100
Frago LM, Paneda C, Dickson SL, Hewson AK, Argente J, Chowen JA 2002 Growth hormone (GH) and GH-releasing peptide-6 increase brain insulin-like growth factor-I expression and activate intracellular signaling pathways involved in neuroprotection. Endocrinology 143:4113–4122
Bondy C, Werner H, Roberts Jr CT, LeRoith D 1992 Cellular pattern of type-I insulin-like growth factor gene expression during maturation of the rat brain: comparison with insulin-like growth factors I and II. Neuroscience 46:909–923
Guan J, Williams C, Gunning M, Mallard C, Gluckman P 1993 The effects of IGF-1 treatment after hypoxic-ischemic brain injury in adult rats. J Cereb Blood Flow Metab 13:609–616
Sumitomo M, Milowsky MI, Shen R, Navarro D, Dai J, Asano T, Hayakawa M, Nanus DM 2001 Neutral endopeptidase inhibits neuropeptide-mediated transactivation of the insulin-like growth factor receptor-Akt cell survival pathway. Cancer Res 61:3294–3298
Zahradka P, Litchie B, Storie B, Helwer G 2004 Transactivation of the insulin-like growth factor-I receptor by angiotensin II mediates downstream signaling from the angiotensin II type 1 receptor to phosphatidylinositol 3-kinase. Endocrinology 145:2978–2987
Pang JJ, Xu RK, Xu XB, Cao JM, Ni C, Zhu WL, Asotra K, Chen MC, Chen C 2004 Hexarelin protects rat cardiomyocytes from angiotensin II-induced apoptosis in vitro. Am J Physiol Heart Circ Physiol 286:H1063–H1069
Cross DA, Alessi DR, Cohen P, Andjelkovich M, Hemmings BA 1995 Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 378:785–789
Moule SK, Welsh GI, Edgell NJ, Foulstone EJ, Proud CG, Denton RM 1997 Regulation of protein kinase B and glycogen synthase kinase-3 by insulin and B-adrenergic agonists in rat epididymal fat cells. J Biol Chem 272:7713–7719
Eldar-Finkelman H 2002 Glycogen synthase kinase 3: an emerging therapeutic target. Trends Mol Med 8:126–132
Cross DA, Culbert A, Chalmers K, Facci L, Skaper S, Reith AD 2001 Selective small-molecule inhibitors of glycogen synthase kinase-3 activity protect primary neurones from death. J Neurochem 77:94–102
Gill R, Soriano M, Blomgren K, Hagberg H, Wybrecht R, Miss M-T, Hoefer S, Adam G, Niederhauser O, Kemp JA, Loetscher H 2002 Role of caspase-3 activation in cerebral ischemia-induced neurodegeneration in adult and neonatal brain. J Cereb Blood Flow Metab 22:420–430(Katarina G. Brywe, Anna-L)