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Endocrinology Vol. 146, No. 11 4665-4672
Copyright © 2005 by The Endocrine Society

Growth Hormone-Releasing Peptide Hexarelin Reduces Neonatal Brain Injury and Alters Akt/Glycogen Synthase Kinase-3ß Phosphorylation

Katarina G. Brywe, Anna-Lena Leverin, Malin Gustavsson, Carina Mallard, Riccarda Granata, Silvia Destefanis, Marco Volante, Henrik Hagberg, Ezio Ghigo and Jörgen Isgaard

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 Göteborg, Sweden; and Departments of Internal Medicine (R.G., S.D., E.G.) and Biomedical Sciences & Oncology (M.V.), University of Turin, 8-10124 Turin, Italy

Address all correspondence and requests for reprints to: Dr. Katarina G. Brywe, Perinatal Center, Department of Physiology and Pharmacology, Box 432, 405 30 Göteborg, Sweden. E-mail: katarina.g.brywe{at}medfak.gu.se.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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-3ß, 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-3ß activity and caspase-dependent cell death.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
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 3ß (GSK3ß), and caspase 9, or by modification of transcription factors (14, 15, 16, 17, 18). GSK3ß 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, GSK3ß 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 GSK3ß 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 GSK3ß are not fully clarified, but it has been suggested to cause degradation of ß-catenin and to inhibit mitochondrial pyruvate dehydrogenase, with subsequent metabolic failure (21, 22). GSK3ß 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
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
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 Göteborg.

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, Göteborg, 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, GSK3ß, 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)3ß 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, pGSK3ß/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); pGSK3ß 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 ß-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-GSK3ß(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-GSK3ß(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 ß-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-GSK3ß (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 ß-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 ß-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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
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. 1AGo). 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. 1BGo). 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).



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  		FIG. 1. HEX reduces HI brain injury. A, Area measurement was done at four anteroposterior levels of the brain (MAP-2-stained sections) in HEX- (1 µg) and vehicle-treated animals. Tissue loss was calculated as MAP-2 area deficit in the ipsilateral hemisphere, expressed as percentage of the right undamaged hemisphere. Data are expressed as means ± SEM; vehicle (VEH) (n = 27) and HEX (n = 27) were analyzed by ANOVA test followed by Fisher’s post hoc test; *, P ≤ 0.05; **, P ≤ 0.01. B, Regional morphological analysis using a neuropathological scoring system of brain injury in cerebral cortex, hippocampus, thalamus, and striatum. Values are given as means ± SEM; VEH (n = 27) and HEX (n = 27) were analyzed by ANOVA test followed by Fisher’s post hoc test; *, P ≤ 0.05; ns, Not significant.

 
Study protocol II, HEX increases Akt and pGSK3ß phosphorylation
Double immunostaining demonstrated that pAkt and pGSK3ß colabeled with NeuN, indicating a neuronal localization in the cerebral cortex (Fig. 2Go). Western blot was used to quantify pAkt and pGSK3ß in both nuclear and cytosolic fractions. After HI, pAkt and pGSK3ß immunoreactivity in cytosolic fractions were significantly increased by HEX at 24 h after HI. In the nuclear fraction, pGSK3ß was also increased by HEX-, compared with vehicle-treated animals, after HI (Figs. 3Go and 4Go).



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  		FIG. 2. Neuronal localization of pAkt and pGSK3ß. Double immunolabeling of pAkt (A) and pGSK3ß (D) with NeuN (B and E) shows a neuronal localization of both pAkt and pGSK3ß in cerebral cortex at 24 h after HEX treatment (PND 8) (C and F, overlay graphs).

 


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  		FIG. 3. Phosphorylation of Akt after HI. A, Immunoreactivity of pAkt in cytosolic fractions of cerebral cortex analyzed by Western blots at 24 h after HI and HEX (n = 7) (black bars) or VEH (vehicle) (n = 6) (white bars) treatment in the ipsilateral (I) or contralateral (C) hemisphere. Data are expressed as means ± SEM and analyzed by ANOVA test followed by Fisher’s post hoc test; **, P ≤ 0.01. B, Western blots of pAkt and total Akt in the cytosolic fraction of the ipsilateral (I) and contralateral (C) cerebral cortex at 24 h after HI and HEX or vehicle administration. Each band represents a sample from one animal.

 


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  		FIG. 4. Phosphorylation of GSK3ß after HI. A and C, Densities of cytosolic pGSK3ß (A) and nuclear pGSK3ß (C) protein bands on Western blots 24 h after HI and treatment with HEX (n = 6) (black bars) or vehicle (n = 7) (white bars) in the ipsilateral (I) and contralateral (C) hemispheres. Data are expressed as means ± SEM; statistical analysis was done with ANOVA test followed by Fisher’s post hoc test; *, P ≤ 0.05; ***, P ≤ 0.001. B and D, Western blots of pGSK3ß and total GSK3 in the cytosolic (B) and nuclear (D) fractions 24 h after HEX and HI. Each band represents a sample from one animal.

 
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. 5Go).



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  		FIG. 5. HEX reduces caspase-3-like activity. Caspase-3-like activity (DEVD cleavage of the amino acids Asp-Glu-Val-Asp) 24 h after HI exposure and HEX (black bars, n = 7) or vehicle (white bars, n = 6) treatment in the HI ipsilateral (I) hemisphere and contralateral (C) hemisphere. Data are expressed as means ± SEM; **, P ≤ 0.01.

 
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. 6AGo). However, staining for GH was totally negative or only weakly positive on sections of HI brains from both HEX- and vehicle-treated animals (Fig. 6Go, C and E). IGF-I immunoreactivity was found in rat liver cells (Fig. 6BGo) and cerebral neurons in both contralateral (Fig. 6DGo) and ipsilateral, perilesional areas (Fig. 6FGo), without significant difference in intensity between HEX- and vehicle-treated rats.



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  		FIG. 6. Immunohistochemical staining of GH and IGF-I in cortex after HI. GH antibody specificity is demonstrated by intense reactivity in single cells of rat pituitary (A). Negative immunohistochemical results are observed in cerebral neurons in perilesional areas (C and E). IGF-I immunoreactivity is found in rat liver cells (B) and cerebral neurons in both contralateral (D) and ipsilateral, perilesional areas (F), without significant difference in intensity. C, E, and F, Perilesional areas with the ischemic lesion represented at the left bottom. A–F, Immunoperoxidase; A, B, and E, x400; C, D, and F, x200

 
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 ß-subunit in cytosolic fractions from the left damaged hemisphere of HI rats was analyzed by Western blot. As shown in Fig. 7Go, there was a significant increase in phosphorylation of the IGF-IR at 24 h after injury in HEX-treated rats compared with vehicle controls.



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  		FIG. 7. HEX induces the phosphorylation of IGF-IR. Cell lysates in cytosolic fractions from the damaged left hemisphere of cerebral cortex at 24 h after HI and HEX treatment were immunoprecipitated and analyzed by phosphotyrosine (P-Tyr) immunoblotting at 24 h after vehicle control (n = 8) or HEX (n = 8) treatment, as described in Materials and Methods. A, The band intensities, normalized to IGF-IR, were quantified by densitometric analysis and reported as percent of vehicle control. Results are the means ± SEM from four independent experiments (P ≤ 0.05). B, IGF-IR phosphorylation evaluated by Western blotting. Bands are from two different, and representative gels of a total of four, independent experiments, where protein samples from two vehicle- and two HEX-treated rats were run on the same gel. Equal loading of immunoprecipitated proteins (IP) was confirmed by reprobing immunoblots with IGF-IR ß-subunit antibody.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
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 GSK3ß, 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 GSK3ß 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 GSK3ß by GHS has not previously been reported, but such a response could partly explain the neuroprotective effect of HEX because GSK3ß is strongly proapoptotic (60), and inhibitors of GSK3ß reduce infarction after ischemia in adult animals (20) and improve neuronal survival in vitro (61). Activation of GSK3ß has been linked to triggering of caspase-dependent apoptosis (23), but GSK3ß 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 GSK3ß 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 Göteborg Medical Society and by grants to researchers in the public health service from the Swedish government (LUA).

First Published Online August 4, 2005

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.

Received April 4, 2005.

Accepted for publication July 29, 2005.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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