The major ghrelin forms, acylated ghrelin and des-acylated ghrelin, are novel gastrointestinal hormones. Moreover, emerging evidence indicates that these peptides may have other functions including neuro- and vaso-protection. Here, we investigated whether post-stroke treatment with acylated ghrelin or des-acylated ghrelin could improve functional and histological endpoints of stroke outcome in mice after transient middle cerebral artery occlusion (tMCAo). We found that des-acylated ghrelin (1 mg/kg) improved neurological and functional performance, reduced infarct and swelling, and decreased apoptosis. In addition, it reduced blood-brain barrier (BBB) disruption in vivo and attenuated the hyper-permeability of mouse cerebral microvascular endothelial cells after oxygen glucose deprivation and reoxygenation (OGD + RO). By contrast, acylated ghrelin (1 mg/kg or 5 mg/kg) had no significant effect on these endpoints of stroke outcome. Next we found that des-acylated ghrelin's vasoprotective actions were associated with increased expression of tight junction proteins (occludin and claudin-5), and decreased cell death. Moreover, it attenuated superoxide production, Nox activity and expression of 3-nitrotyrosine. Collectively, these results demonstrate that post-stroke treatment with des-acylated ghrelin, but not acylated ghrelin, protects against ischaemia/reperfusion-induced brain injury and swelling, and BBB disruption, by reducing oxidative and/or nitrosative damage.
Ischaemic stroke is a leading cause of mortality and long-term disability, but treatment options are limited. The major ghrelin forms, acylated ghrelin and des-acylated ghrelin, are novel gastrointestinal hormones. Moreover, recent evidence indicates these peptides may have neuroprotective and vasoprotective actions, and thus may protective actions in ischaemic stroke.
In the present study we tested whether the peptides could mediate protection in a clinically relevant mouse model of ischaemic stroke.
Our findings reveal protective actions of des-acylated ghrelin when administered shortly after induction of reperfusion, which involve a previously unrecognized beneficial effect on BBB integrity. By contrast the acylated form was without effect. Thus, the present study sheds light on the potential of des-acylated ghrelin or longer acting analogues as novels therapeutic agents for ischaemia/reperfusion-induced injury. These findings may prove to be all the more crucial when we consider that the production of this peptide may be reduced after stroke.
Pharmacological treatment of ischaemic stroke mainly relies on the use of recombinant tissue plasminogen activator (rt-PA). However, it is only administered to a small proportion of stroke patients due to its propensity to increase the risk of haemorrhage and oedema if delayed >4.5 h after stroke onset. Also, even if rt-PA-induced or spontaneous restoration of blood flow occurs, reperfusion often exacerbates brain injury. Thus, there is a pressing need for novel agents that protect against not only ischaemia but also reperfusion injury. A burst of reactive oxygen and nitrogen species generation (ROS and RNS; commonly referred to as oxidative/nitrosative stress) is an important mechanism of neuronal cell death after ischaemia and reperfusion [1,2]. Moreover, such mechanisms also contribute to blood–brain barrier (BBB) disruption after ischaemia and reperfusion, as well as dysfunction of arteries upstream of the BBB [3,4]. There is a growing appreciation of the contribution of these vascular changes to brain injury and oedema, and thus the importance of protecting the cerebral endothelium as part of an effective stroke therapy .
Ghrelin is a 28 amino acid peptide hormone produced primarily by the stomach. In the plasma, ghrelin circulates in at least two distinct forms, acylated ghrelin and des-acylated ghrelin. Post-translational addition of a medium-chain fatty acid by the enzyme ghrelin O-acyltransferase (GOAT) produces the acylated form of ghrelin . Acylated ghrelin is a metabolic hormone that stimulates appetite and regulates energy expenditure largely through its actions on the growth hormone secretagogue receptor 1a (GHSR1a) expressed in the hypothalamus and pituitary gland . Des-acylated ghrelin is the most abundant circulating ghrelin form, but it does not bind or activate GHSR1a at physiological concentrations . Recent evidence suggests that it may have opposing metabolic actions to acylated ghrelin . However, to date, a receptor for des-acylated ghrelin has not been identified. Interest in these peptides has intensified over the last decade with the realization that they may have diverse actions beyond their roles in metabolism . Acylated ghrelin signalling in the brain promotes learning and memory, protects against neurodegeneration (e.g. in Parkinson's disease) by activating uncoupling protein 2 (UCP2)-dependent mitochondrial mechanisms, and has antidepressant and anxiolytic effects [7,10]. Furthermore, acylated ghrelin [11–13] and des-acylated ghrelin  reduce brain injury in rat models of cerebral ischaemia and reperfusion when administered before induction of ischaemia, and reduce apoptotic cell death of neurons in vitro [14,15]. Most importantly, however, it remains to be tested whether these peptides are also protective when given after stroke induction, which represents a more clinically relevant scenario. Notably, des-acylated ghrelin may also have beneficial effects on the cerebral endothelium after ischaemia and reperfusion. Indeed, this ghrelin form suppresses superoxide levels in mouse cerebral vessels by inhibiting the Nox/NADPH oxidases , which are major contributors to oxidative-induced BBB disruption after stroke [17,18].
The aims of the present study were, therefore, to first test the effect of acylated ghrelin and des-acylated ghrelin on outcome following transient middle cerebral artery occlusion (tMCAo) in mice, when administered after induction of cerebral reperfusion. Secondly, we examined whether the peptides can inhibit BBB disruption by measuring their effects on BBB permeability in vivo, and also cerebral endothelial permeability after oxygen glucose deprivation (OGD) and reoxygenation (RO) in vitro.
MATERIALS AND METHODS
The present study followed the ARRIVE Guidelines. All experimental procedures were conducted in accordance with the National Health & Medical Research Council (NHMRC) of Australia guidelines for the care and use of animals for research and were approved by the RMIT University Animal Ethics Committee. A total of 178 male (22 excluded, see below), 8–12 weeks old C57Bl6/J mice were obtained from the Animal Resources Centre (Australia). Mice had access to water and standard chow ab libitum, and were housed under a 12 h light/12 h dark cycle in a specific-pathogen-free (SPF) facility. Mice were randomly assigned to treatment groups by an investigator not performing surgical procedures or post-surgery analyses, and an investigator blinded to the treatment groups performed post-surgery analyses. In total, 22 stroke mice were excluded from the study which occurred when: (1) there was an inadequate (<70%) reduction in regional cerebral blood flow (rCBF) during the ischaemic period or inadequate (>80%) increase within the first 10 min of reperfusion (n=5); (2) technical complications arose during surgery (e.g. loss of >0.2 ml of blood, n=2; the occluding clamp on the common carotid artery was in place for ≥5 min, n=1); or (3) they died or had to be killed (according to clinical severity score) prior to the end of the reperfusion period (n=6 vehicle-treated mice; n=5 acylated ghrelin-treated and n=3 des-acylated ghrelin-treated).
Focal cerebral ischaemia and reperfusion
Mice were anaesthetized between 7 a.m. and 11 a.m. with a mixture of ketamine (150 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.). Body temperature was maintained at 37°C with a heat lamp throughout the procedure and until mice regained consciousness. Focal cerebral ischaemia and reperfusion was performed by transient intraluminal filament-induced middle cerebral artery occlusion (tMCAo) as previously described [19–21]. rCBF in the area of the cortex supplied by the middle cerebral artery (∼2 mm posterior and 5 mm lateral to bregma) was monitored and recorded prior to the induction of cerebral ischaemia, during ischaemia, and during the first 10 min of reperfusion using trans-cranial laser-Doppler flowmetry. For sham surgery, the right external carotid artery and common carotid artery were visualized, but the filament was not inserted. After mice had recovered from anaesthesia, they were housed in individual cages. Mice were monitored hourly for a minimum of 8 h (during 8 a.m. to 6 p.m.) post-surgery and the following morning (7 a.m.) using our monitoring protocol and clinical signs severity scoring system (approved by our ethics committee). At the end of the experiment, mice were killed with an overdose of isoflurane followed by decapitation.
Ghrelin treatment regime
Mice received three injections of vehicle (0.9% saline, i.p.), acylated ghrelin (1 mg/kg or 5 mg/kg, i.p.) or des-acylated ghrelin (1 mg/kg, i.p.). Doses were administered at the time of reperfusion, and at 8 h and 16 h of reperfusion. Doses were calculated from published work showing neuroprotective effects of these peptides in vivo when administered prior to stroke [11–13], and our work showing protective actions of des-acylated ghrelin on cerebral arteries . Acylated ghrelin is known to stimulate food intake, which could in turn improve the well-being and thus influence the recovery of mice. Thus, in a small cohort of mice, we measured food intake overnight for 3 days (baseline) prior to sham or stroke surgery and on the night following surgery.
Neurological deficit scoring and functional impairment test
Neurological assessment was performed 24 h after sham or stroke surgery using a five-point scoring system: 0=normal motor function; 1=flexion of torso and contralateral forelimb when lifted by the tail; 2=circling to the contralateral side when held by the tail on a flat surface with normal posture at rest; 3=leaning on the contralateral side at rest; 4=no spontaneous movement at rest or uncontrolled circling . After neurological assessment and a rest period (10 min), a hanging wire test was performed, as previously described [4,20].
Quantification of cerebral infarct and oedema volumes
Cerebral infarct and oedema volumes were evaluated at 24 h after stroke surgery, as previously described [4,20]. Briefly, brains were coronally sectioned (30 μm thickness; 420 μm apart) and then stained with 0.1% thionin to delineate the infarct. Total infarct volume was quantified using ImageJ image analysis software (NIH), correcting for brain oedema, as previously described [4,20].
Measurement of cell death using TUNEL
The terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling (TUNEL) method was used to quantify cell death in brain sections (10 μm) of mice 24 h after stroke, as per the manufacturer's instructions (In Situ Cell Detection Kit, Roche). Infarcts were located on each section by comparing to corresponding thionin-stained sections. The number of TUNEL-positive cells was then counted in both infarct and peri-infarct regions of the ischaemic hemisphere. The peri-infarct was assessed as any region outside the infarct in the ischaemic hemisphere that displayed TUNEL-positive immunoreactive staining . Three sections were analysed per mouse and the total number of TUNEL-positive cells was averaged.
Expression levels of cleaved caspase-3
Western blotting was used to measure expression levels of the apoptotic protein cleaved caspase-3 in ischaemic/right cerebral hemispheres of mice 24 h after sham or stroke surgery using methods similar to those previously reported [4,20]. Rabbit polyclonal anti-cleaved caspase-3 (1:1000 dilution) and anti-β-actin (1:5000 dilution) primary antibodies were used (both from Cell Signaling Technology). Immunoreactive band intensities were normalized to intensity of corresponding bands for β-actin and expressed as fold-change relative to sham.
Reverse transcription (RT)–PCR was used to measure GHSR1a mRNA expression levels in: (1) ischaemic/right cerebral hemispheres of control mice (received no surgical intervention), and of mice 24 h after sham or stroke surgery (vehicle and acylated ghrelin-treated mice). It is known that des-acyl ghrelin does not bind to nor activate GHSR1a [8,22]. Thus, any protective actions of des-acyl ghrelin after stroke will not mediated through GHSR1a. As such, we did not assess GHSR1a mRNA expression levels in brains from des-acylated ghrelin-treated mice; (2) middle cerebral arteries of sham and stroke mice; and pituitary of control mice (positive control); and (3) bEnd.3 cells cultured under normoxic or OGD + RO conditions (see below), as previously described . QuantiFast SYBR® Green primers were used (GHSR1a, catalogue number QT00138439; 18S, catalogue number QT02448075). Data were normalized to ribosomal 18S and represented relative to expression levels in either control mice (brain) or pituitary gland (cerebral arteries and bEnd.3 cells) using the 2−ΔΔCT method.
Assessment of blood–brain barrier disruption in vivo
A 4 ml/kg amount of 2% Evan's Blue was injected into the tail vein of mice 21 h after sham or stroke surgery. After 3 h, mice were killed by inhalation of isofluorane and transcardially perfused with 0.9% saline. The ischaemic/right and contralateral/left hemispheres were then homogenized in N,N-dimethylformamide, incubated overnight at 55°C and centrifuged. Supernatants were collected and absorbance was read in triplicate, alongside a standard curve of 0–15000 ng/ml Evan's Blue, using a BMG Clariostar plate reader (620 nm excitation, 680 nm emission). The amount of Evan's Blue per ischaemic/right hemispheres (micrograms) was expressed as a ratio of levels in contralateral/left hemispheres.
Oxygen glucose deprivation and reoxygenation of bEnd.3 cells
Mouse microvascular cerebral endothelial cells (bEnd.3 cells; ATCC CRL-2299) were grown in culture medium [Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS)] at 37°C in a humidified 5% CO2 atmosphere. Cells were passaged every 3–4 days. Culture medium was changed after 24 h of passaging and every 2 days thereafter. Experiments were performed with cells from passages 24 to 34.
bEnd.3 cells were washed twice with OGD medium composed of glucose- and serum-free DMEM media (Invitrogen) pre-equilibrated for 5 min with 100% N2. Cells were then transferred to a humidified hypoxia chamber linked to a digital oxygen controller (0.3% oxygen, Biospherix) filled with 95% N2, 5% CO2 and left to incubate for 1 h at 37°C. Cells were then washed twice with glucose-containing DMEM (oxygenated with air), and incubated for a further 23 h in glucose-containing DMEM (no serum) in a humidified normoxic incubator (5% CO2 atmosphere) at 37°C. For normoxic controls, bEnd.3 cultures were incubated for the same duration of time as the OGD + RO cells in glucose-containing serum-free medium (equilibrated with air) at 37°C (5% CO2 atmosphere).
Measurement of paracellular permeability in bEnd.3 cells after OGD + RO
To measure endothelial cell paracellular permeability, the passage of FITC–dextran (70 kDa) across bEnd.3 cells monolayers was measured after exposure to normoxic conditions and OGD + RO. Cells were treated with vehicle (0.9% saline), acylated ghrelin or des-acylated ghrelin (10 nmol/l) at the time of RO (post-treatment protocol). Moreover, we tested the effect of des-acylated ghrelin when administered 24 h prior to OGD (pre-treatment protocol). A concentration of 10 nmol/l was selected based on our published work showing protective actions of des-acylated ghrelin on the cerebral vasculature .
bEnd.3 cells were seeded at a density of 5×104 cells/well in DMEM (10% FBS) on to the upper ‘apical’ chamber of transwell inserts (0.4 μm average pore size, ThinCerts™) fitted into 24-well culture plates. Following normoxic or OGD + RO exposure, inserts were transferred into new wells of 24-well culture plates containing pre-warmed Krebs/HEPES buffer (pH 7.4). FITC–dextran (1 mg/ml) was then added to the upper chamber and left to incubate for 60 min at 37°C. Inserts were then removed from the wells and samples were taken from the lower chamber and transferred into a 96-well black plate. Fluorescence intensity was then measured using the BMG Clariostar plate reader with excitation at 492 nm and emission at 518 nm. Fluorescence intensity was calculated as fold-change relative to normoxic controls.
Measurement of tight junction protein expression in bEnd.3 cells after OGD + RO
Western blotting was used to measure expression levels of the tight junction proteins occludin, claudin-5 and zonula occludens 1 (ZO-1), in bEnd.3 cells exposed to normoxic conditions or OGD + RO, using methods similar to those previously reported . Cells were treated with either vehicle (0.9% saline) or des-acylated ghrelin (10 nmol/l) at the time of RO. Mouse monoclonal anti-occludin and anti-claudin-5, rabbit polyclonal anti-ZO-1 (Invitrogen; 1:1000 dilution for all antibodies) and anti-β-actin (1:5000 dilution, Cell Signaling Technology) primary antibodies were used. Immunoreactive band intensities were normalized to intensity of corresponding bands for β-actin and expressed as fold-change relative to normoxic controls.
Immunofluorescence for ZO-1 and 3-nitrotyrosine in bEnd.3 cells after OGD + RO
bEnd.3 cells were seeded on to glass coverslips in culture medium (DMEM with 10% FBS). At 2 days post-confluence, cells were exposed to normoxic conditions or OGD + RO [treated with either vehicle (0.9% saline) or des-acylated ghrelin (10 nmol/l) at the time of RO], fixed with 4% paraformaldehyde, washed with 0.01 mol/l PBS (3×10 min), and then incubated with 10% goat serum (30 min). Immunofluorescence was then performed using polyclonal rabbit anti-ZO-1 (1:300 dilution, Zymed Laboratories) or polyclonal rabbit anti-3-nitrotyrosine (1:350 dilution, Abcam) primary antibodies, and goat anti-rabbit AlexaFluor 594-conjugated (ZO-1; 1:500 dilution, Invitrogen) or AlexaFluor 488-conjugated (3-nitrotyrosine; 1:350 dilution, Invitrogen) secondary antibodies. Cells were counterstained with Hoechst 33342 solution (1:500 dilution, Sigma). For 3-nitrotyrosine, five fields of view were randomly selected per coverslip and semi-quantitative analysis of fluorescence intensity was performed as previously described , and appropriate negative and positive controls were performed. Values were then expressed as fold-change relative to normoxic controls.
Measurement of cell viability of bEnd.3 cells and cleaved caspase-3 expression after OGD + RO
Cell viability of bEnd.3 cells (exposed to normoxic or OGD + RO conditions) was assessed using the MTS assay (Promega). Cells were treated with either vehicle (0.9% saline) or des-acylated ghrelin (10 nmol/l) at the time of RO. Absorbance was measured detected at 490 nm using a BMG Clariostar plate reader. Mean absorbance of cells exposed to OGD + RO were expressed as fold-change relative to the absorbance of normoxic cells. Western blotting was used to measure expression levels of cleaved caspase-3 in bEnd.3 cells exposed to normoxic conditions or OGD + RO, using methods similar to those previously reported .
Measurement of superoxide production by bEnd.3 cells after OGD + RO
Basal and angiotensin II-stimulated superoxide production by bEnd.3 cells (exposed to normoxic or OGD + RO conditions) was assessed using either L-012 (100 μmol/l) enhanced chemiluminescence or ethidium/oxyethidium fluorescence (dihydroethidium; 10 μmol/l), as previously described [4,25]. Cells exposed to OGD + RO were treated with either vehicle or des-acyl ghrelin (10 nmol/l) at the time of RO. We have previously demonstrated that superoxide dismutase (SOD) or Tempol abolishes the L-012 chemiluminescence signal detected from vascular preparations, implying that L-012 detects superoxide and not other ROS . To confirm that superoxide was the major ROS detected by dihydroethidium, bEnd.3 cells were treated with PEG–SOD (500 units/ml) at the time of RO. For L-012 experiments, superoxide counts were measured for 60 min and background counts were then subtracted. Values were then expressed as percentage relative to normoxic controls.
Data and statistical analysis
All results are presented as means ± S.E.M. and P<0.05 was considered statistically significant. Statistical analyses were performed using Prism version 6 (GraphPad Software). Statistical comparisons between treatment groups were performed using either Student's unpaired t test or one-way ANOVA with Dunnett's multiple comparisons post-hoc test. Mann–Whitney U test was used for non-parametric data.
Drugs and their sources
Drugs and their sources are: acylated ghrelin (SC1357; Polypeptide Laboratories); des-acylated ghrelin (SC1483; Polypeptide Laboratories) and all other drugs (Sigma). All ghrelin-related peptides were dissolved in 0.9% saline. All other drugs were dissolved in either Krebs/HEPES buffer or distilled water.
Degree of hypoperfusion, mortality and food intake
Following insertion of the monofilament, rCBF was reduced by ∼75% and increased upon removal to a similar extent in groups (P>0.05, Figure 1A). In sham-operated mice, rCBF remained at ∼100% for the duration of the monitoring period (Figure 1A). Mortality during the reperfusion period was 6/48 (12%) of vehicle-treated mice, 5/44 (11%) of acylated ghrelin-treated mice, and 3/38 (8%) of des-acylated ghrelin-treated mice. Overnight food intake was reduced in vehicle-treated stroke mice relative to baseline and sham-operated mice (Table 1, P<0.05, one-way ANOVA with Dunnett's post-hoc test). Treatment with acylated ghrelin had no effect on food intake in mice after stroke (Table 1, P>0.05 compared with vehicle-treated stroke mice, unpaired t test), but increased food intake in sham-operated mice relative to baseline (Table 1, P<0.05, unpaired t test).
Effect of acylated ghrelin and des-acylated ghrelin on stroke outcomes
|Overnight food intake (g)|
|Experimental group||Baseline||24 h after surgery||n|
|Overnight food intake (g)|
|Experimental group||Baseline||24 h after surgery||n|
Des-acylated ghrelin improves stroke outcomes
Post-stroke treatment of mice with 1 mg/kg acylated ghrelin had no significant effect on neurological deficit scores, hanging grip times, or infarct and oedema volume relative to vehicle-treated mice (Figures 1B–1E). Similarly, mice treated with a higher dose of acylated ghrelin (5 mg/kg) displayed no improvement in neurological deficit, hanging grip times (5 mg/kg acylated ghrelin: 29±6 compared with vehicle: 30±9 s, P>0.05, unpaired t test, n=9), or infarct and oedema volumes (Figures 1F and 1G). By contrast, post-stroke treatment with 1 mg/kg des-acylated ghrelin significantly improved neurological score and hanging grip time, and reduced both infarct and oedema volume by >50% (Figures 1B–1E, P<0.05).
Des-acylated ghrelin reduces apoptosis after stroke
In vehicle-treated mice, there was an abundance of TUNEL-positive cells in both peri-infarct (149±36) and infarct brain regions (4814±704, Figure 2A). Post-stroke treatment with 1 mg/kg acylated ghrelin had no significant effect on the number of TUNEL-positive cells in both peri-infarct (111±16) and infarct (4187±468) regions relative to vehicle-treated mice. However, in mice treated with 1 mg/kg des-acylated ghrelin, there were ∼70% fewer TUNEL-positive cells in peri-infarct regions (46±9, P<0.05), and there was also a trend for less TUNEL-positive cells in infarct regions (2720±706 TUNEL-positive cells, P=0.08). Virtually no TUNEL-positive cells were observed in the contralateral hemisphere of brains from stroke mice (Figure 2A).
Effect of acylated ghrelin and des-acylated ghrelin on apoptosis after stroke
Western blot analysis indicated that expression of cleaved caspase-3 (17 kDa subunit) was significantly increased in ischaemic hemispheres of vehicle-treated mice relative to sham mice (P<0.05, Figure 2B). Consistent with our infarct and TUNEL data, expression levels of cleaved caspase-3 in ischaemic hemispheres of acylated ghrelin-treated mice were comparable to that of vehicle-treated mice (P<0.05, Figure 2B). By contrast, expression levels in ischaemic hemispheres of des-acylated ghrelin-treated mice (1 mg/kg) were significantly lower than in vehicle-treated mice (P<0.05, Figure 2C) and comparable to levels in sham mice (P>0.05). Expression levels of the 19 kDa subunit of cleaved caspase-3 were similar between sham (0.35±0.04, relative intensity to β-actin), vehicle-treated (0.44±0.04) and des-acylated ghrelin-treated mice (0.36±0.05; P>0.05).
Effect of stroke on GHSR1a mRNA expression
Using RT–PCR, we next tested whether acylated ghrelin's lack of effect on stroke outcome relates to down-regulation of its receptor, GHSR1a, in the ischaemic hemisphere. Expression levels of GHSR1a mRNA in the brain were similar between control and sham-operated mice, indicating that anaesthesia/surgery does not affect GHSR1a expression (Figure 3A). In vehicle-treated stroke mice, GHSR1a mRNA expression was significantly lower relative to control and sham mice (P<0.05), whereas levels were not decreased in mice treated with acylated ghrelin (1 mg/kg). Thus, post-stroke treatment with acylated ghrelin prevents down-regulation of its receptor in the ischaemic brain.
Effect of stroke on GHSR1a mRNA expression
We have previously reported that although GHSR1a mRNA is expressed at low levels in mouse cerebral arteries, it is not expressed at the protein level . Similarly, we found here that GHSR1a mRNA expression in cerebral arteries of sham-operated mice was very low [∼22-fold lower relative to pituitary (positive control); P<0.05, Figure 3B], and expression levels were negligible in cerebral arteries of stroke mice (∼14-fold lower relative to sham-operated, P<0.05). Similarly, GHSR1a mRNA expression was negligible in bEnd.3 cells cultured under either normoxic or OGD conditions (∼2200-fold lower relative to pituitary, P<0.05, results not shown). Thus, acylated ghrelin is unlikely to mediate direct effects on the mouse cerebral endothelium due to the lack of GHSR1a.
Des-acylated ghrelin reduces BBB disruption
There was marked extravasation of Evan's Blue into ischaemic hemispheres of vehicle-treated mice, indicative of increased BBB permeability and thus BBB disruption (Figure 4A, P<0.05). Post-stroke treatment of mice with acylated ghrelin (1 mg/kg) had no significant effect on Evans's Blue leakage relative to vehicle-treated mice, whereas des-acylated ghrelin (1 mg/kg) reduced leakage by ∼80% (Figure 4A, P<0.05).
Effect of acylated ghrelin and des-acylated ghrelin on BBB disruption
Acylated ghrelin (10 nmol/l) had no effect on paracellular hyper-permeability of bEnd.3 exposed to OGD + RO (Figure 4B). By contrast, des-acylated ghrelin (10 nmol/l) attenuated hyper-permeability of bEnd.3 cells. Specifically, post-treatment (i.e. at the time of RO) decreased the passage of FITC–dextran across bEnd.3 monolayers by ∼30% relative to vehicle-treated cells (Figure 4B, P<0.05). Similarly, pre-treatment (i.e. 24 h before OGD) decreased the passage of FITC–dextran [normoxic: 5237±142, OGD + RO vehicle-treated: 20 409±109, OGD + RO des-acylated ghrelin-treated: 15 072±189 relative fluorescent units (RFU), P<0.05, n=6].
Effect of des-acylated ghrelin on tight junction protein expression in bEnd.3 cells
Western blot analysis showed that expression of occludin and ZO-1 were reduced in vehicle-treated cells after OGD + RO relative to normoxic (P<0.05, Figures 5A and 5B). The decline in occludin levels was virtually prevented by des-acylated ghrelin (10 nmol/l; post-treatment, P<0.05, Figure 5A); however, it had no significant effect on ZO-1 expression (Figure 5B). Expression of claudin-5 was unchanged in vehicle-treated cells after OGD + RO (P>0.05, Figure 5C). Treatment with des-acylated ghrelin significantly increased expression levels (P<0.05, Figure 5C).
Effect of des-acylated ghrelin on endothelial tight junction protein expression, cell viability and caspase-3 expression after OGD + RO
In accordance with our Western blot data, immunofluorescence staining for ZO-1 appeared to be less intense in vehicle-treated cells. Also, ZO-1 immunoreactivity was more discontinuous along the cell membrane than normoxic controls, which might suggest redistribution of this protein (Figure 5D). In cells treated with des-acylated ghrelin, ZO-1 immunoreactivity was also less intense; however, there appeared to be a more continuous alignment of ZO-1 to the cell membrane.
Effect of des-acylated ghrelin on viability and caspase-3 expression in bEnd.3 cells
After exposure to OGD + RO, the viability of vehicle-treated bEnd.3 cells was decreased by >20% relative to normoxic controls (Figure 5E, P<0.05). Treatment of cells with des-acylated ghrelin significantly increased cell viability relative to vehicle-treated cells (P<0.05).
Western blot analysis revealed a significant increase in expression levels of cleaved caspase-3 (17 kDa subunit) in vehicle-treated and des-acylated ghrelin-treated cells exposed to OGD + RO (P<0.05, Figure 5F), and levels were comparable between vehicle- and des-acylated ghrelin-treated cells. The 19 kDa subunit was undetectable in either normoxic or OGD + RO cells.
Des-acylated ghrelin attenuates superoxide levels, Nox activity and 3-nitrotyrosine in bEnd.3 cells
After exposure to OGD + RO, basal superoxide production by bEnd.3 cells was ∼2.5-fold greater than levels generated by normoxic cells, as measured by L-012 enhanced chemiluminescence (P<0.05, Figure 6A). However, in des-acylated ghrelin-treated cells, superoxide production was decreased by ∼40% (P<0.05). We also found that ethidium/oxyethidium fluorescence appeared to be less intense in cells treated with des-acylated ghrelin relative to vehicle-treated (Figure 6B). PEG–SOD also appeared to significantly reduce fluorescence intensity, confirming that superoxide was the major ROS detected by dihydroethidium.
Effect of des-acylated ghrelin on endothelial superoxide levels and Nox activity after OGD + RO
Angiotensin II is known to increase superoxide production by cerebral vessels by activating the Nox/NADPH oxidases (Nox1- and Nox2-containing isoforms) and is commonly used to assess Nox activity in vessel preparations and vascular cells [16,27,28]. Acylated ghrelin (0.1–10 nmol/l) had no significant effect on Nox activity, which is consistent with our findings in intact mouse cerebral vessels  (Figure 6C). Similar to its inhibitory effect on Nox activity in intact mouse cerebral arteries , des-acylated ghrelin (1–10 nmol/l) markedly reduced angiotensin II-stimulated superoxide generation (∼50%), and thus Nox activity, in bEnd.3 cells exposed to normoxic conditions (Figure 6D, P<0.05). After exposure to OGD + RO, angiotensin II-stimulated superoxide generation was increased ∼2-fold in vehicle-treated bEnd.3 cells relative to normoxic controls (P<0.05, Figure 6E). Moreover, treatment with des-acylated ghrelin prevented this increase in Nox activity such that superoxide levels were comparable to levels in normoxic controls (P>0.05).
Using immunofluorescence and semi-quantitative analysis, we found that 3-nitrotyrosine expression (marker of protein nitration by peroxynitrite) was increased ∼2-fold in bEnd.3 cells after OGD + RO (P<0.05, Figure 7). Importantly, treatment with des-acylated ghrelin significantly reduced expression levels (P<0.05, Figure 7).
Effect of des-acylated ghrelin on endothelial 3-nitrotyrosine expression after OGD + RO
Our data demonstrate that the des-acylated form of ghrelin improves functional and neurological deficit, and reduces the degree of injury, apoptosis and swelling when administered to mice shortly after induction of reperfusion. By contrast, at the two doses we tested, acylated ghrelin fails to improve these end-point measures of stroke outcome. The present study also reveals that the beneficial effect of des-acylated ghrelin relates, at least in part, to its ability to protect against BBB disruption. Moreover, this effect occurs in association with reduced ROS/RNS production and Nox activity, inferring des-acylated ghrelin enhances BBB integrity by reducing oxidative/nitrosative damage.
When administered prior to or at the immediate onset of ischaemia, acylated ghrelin reduces brain injury in rat models of ischaemia and reperfusion through anti-apoptotic and anti-inflammatory mechanisms [11–13]. By contrast, we have shown here that treatment with 1 mg/kg acylated ghrelin after induction of reperfusion failed to improve several end-point measures of stroke outcome, with no significant reduction in infarct, swelling or apoptosis (TUNEL or cleaved caspase-3 expression), and no improvement in neurological or motor performance. Similarly, even a 5-fold higher dose of acylated ghrelin had no beneficial effect on these measures of post-stroke outcome. Interestingly, the neuroprotective effects of acylated ghrelin in a mouse model of Parkinson's disease are dependent on metabolic status. Specifically, acylated ghrelin was neuroprotective when administered to fasted mice, whereas it had no effect during ab libitum feeding . Here, we studied mice that had been fed ab libitum prior to stroke surgery. However, food intake was markedly reduced after stroke induction (∼90%), which is consistent with the tMCAo model of stroke . Thus, although the mice were fed ab libitum prior to stroke surgeries, there were in fact in a relatively ‘fasted state’ during the treatment regime. Notably, of the studies showing the protective actions of acylated ghrelin when administered pre-stroke, only one study detailed that rats were fasted overnight prior to surgery and acylated ghrelin administration , whereas the remaining studies did not describe whether they were fasted or fed ab libitum [11–13].
Although GHSR1a mRNA expression levels were decreased in ischaemic hemispheres of vehicle-treated mice, levels in mice treated with acylated ghrelin were comparable to sham-operated mice, showing that post-stroke treatment up-regulates GHSR1a. Consistent with this, other studies have shown that peripheral administration of acylated ghrelin up-regulates GHSR1a expression [12,32]. However, the mechanisms underpinning this effect are unknown. Nevertheless, our data indicate that down-regulation of acylated ghrelin's receptor does not account for its lack of effect on stroke outcome. Importantly, acylated ghrelin's appetite- stimulating properties may be altered after stroke, actions that are largely mediated through the hypothalamus. Specifically, we found that acylated ghrelin had no effect on food intake by stroke mice during the treatment period, whereas it increased intake in sham controls. This finding raises the possibility of a generalized ‘resistance’ to acylated ghrelin in the acute period after stroke, which may extend to extra-hypothalamic brain regions involved in infarct development. An additional and plausible explanation for the apparent time-dependence of acylated ghrelin's protective effects could relate to its ability to more effectively prevent/inhibit mechanisms of ischaemia-related injury than those caused by reperfusion (see discussion below). Irrespective of the reasons, our findings suggest that the timing of administration may be critical for acylated ghrelin's neuroprotective effects, casting doubts on its suitability for development as an acute stroke therapy.
Des-acylated ghrelin is structurally identical with the acylated form apart from the absence of a medium-chain fatty acid at serine position 3, which is essential for activity at GHSR1a . Despite this, des-acylated ghrelin often produces distinct biological effects to that of the acylated form. For example, we have recently shown that it exerts protective effects on cerebral arteries (e.g. suppressing Nox oxidase activity), whereas acylated ghrelin has no effect . In contrast with acylated ghrelin, post-stroke administration of des-acylated ghrelin was protective, with a marked reduction in both infarct and swelling at 24 h, and an improvement in neurological and motor performance. In rodents, the vast majority (∼70–80%) of the infarct volume development takes place during the first 24 h . However, we cannot exclude the possibility that des-acylated ghrelin slowed the evolution of ischaemic damage rather than reducing final infarct volume. Next we found evidence that this beneficial effect of des-acylated ghrelin was related to the degree of apoptosis in the ischaemic hemisphere. Specifically, des-acylated ghrelin reduced the number of TUNEL-positive cells in peri-infarct regions, and prevented the increase in cleaved caspase-3 expression. Des-acylated ghrelin is known to directly protect neurons against ischaemia-like conditions in vitro through anti-apoptotic mechanisms , thus it is likely that direct neuroprotective actions contribute des-acylated ghrelin's beneficial effect on infarct volume in vivo.
There is a growing appreciation of the importance of protecting the cerebral endothelium as part of an effective stroke therapy. Thus, we next tested the effects of the peptides on BBB disruption in vivo and in vitro. Consistent with its lack of effect of stroke outcomes, acylated ghrelin had no beneficial effect on BBB permeability in vivo or the hyper-permeability of cerebral microvascular endothelial cells exposed to OGD + RO in vitro. This may relate, at least in part, to the lack of GHSR1a in the cerebral vasculature  and cerebral microvascular endothelial cells. Thus, it is highly improbable that acylated ghrelin modulates end-point measures of BBB disruption such as tight junction protein expression, cell viability, and apoptosis. Hence, we did not examine the effect of acylated ghrelin on these end-point measures in bEnd.3 cells exposed to OGD + RO. Des-acylated ghrelin on the other hand strongly protected against BBB hyper-permeability and thus BBB disruption in vivo. Moreover, it attenuated the hyper-permeability of cerebral microvascular endothelial cells when administered either before OGD or at the time of RO, inferring that direct effects on the cerebral endothelium contribute to its beneficial actions on BBB integrity in vivo. In accordance with the permeability data, des-acylated ghrelin prevented the loss of the tight junction protein occludin, increased levels of claudin-5, and appeared to reduce of ZO-1 redistribution from the cell membrane to the cytosol. Des-acylated ghrelin also inhibited the death of cerebral endothelial cells exposed to OGD + RO; however, this occurred independently of a reduction in cleaved caspase-3 expression. The lack of effect of des-acylated ghrelin on cleaved caspase-3 expression is clearly in contrast with our earlier data showing that it markedly reduces expression levels in the ischaemic hemispheres of mice after stroke. It is conceivable that apoptotic pathways triggered in whole brain after ischaemia and reperfusion differ from those of cerebral microvascular endothelial cells. Thus, we presume that des-acylated ghrelin reduces death of bEnd.3 cells through caspase-3-independent anti-apoptotic pathways and/or necrotic cell death. Taken together, these data suggest that des-acylated ghrelin protects against BBB disruption by preventing the loss of tight junction integrity and by inhibiting endothelial cell death.
During early reperfusion, excess ROS/RNS formation by cerebral endothelial cells (and other cell types) triggers a number of downstream pathways that can directly mediate BBB disruption. Moreover, the Nox/NADPH oxidases are a significant source of ROS during reperfusion injury [4,17,18,21,34]. Several lines of evidence indicate that des-acylated ghrelin may protect against BBB disruption by targeting these mechanisms. First, des-acylated ghrelin has powerful ROS-limiting properties in peripheral endothelial cells  and cerebral vessels . Indeed, we have shown that des-acylated ghrelin suppresses superoxide generation by cerebral arteries from ‘non-stroke’ mice via inhibition of the Nox oxidases. Similarly, we have shown here that des-acylated ghrelin inhibited Nox activity in cerebral microvascular endothelial cells cultured under normoxic conditions. Secondly, des-acylated ghrelin attenuated OGD/RO-induced elevations in basal superoxide production by cerebral microvascular endothelial cells. Also, this was associated with reduced Nox activity, indicating an inhibitory action on the Nox oxidases. Lastly, des-acylated ghrelin decreased expression levels of 3-nitrotyrosine after OGD + RO, indicative of reduced peroxynitrite formation, the downstream product of the reaction between superoxide and NO. Taken together, these findings imply that the beneficial effects of des-acylated on BBB integrity and thus stroke outcome (in particular oedema) involve a reduction in ROS/RNS generation by the cerebral endothelium as a result of Nox inhibition. Our previously published work  and data from the present study indicate that, unlike the des-acylated form, acylated ghrelin does not modulate ROS levels in cerebral vessels or inhibit cerebral vascular Nox oxidases. As such, this could be one possible reason why this ghrelin form is ineffective when administered during reperfusion, whereas des-acylated ghrelin strongly protects. Notably, Nox oxidases are also a major source of pathological ROS in endothelial cells of arteries upstream of the BBB, resident brain cells (e.g. neurons and microglia) and infiltrating leucocytes after stroke [4,36]. Des-acylated ghrelin could therefore improve stroke outcome by inhibiting the Nox oxidases in these cell types.
In summary, the present study has revealed protective actions of des-acylated ghrelin when administered after initiating post-ischaemic reperfusion, which involve a previously unrecognized beneficial effect on BBB integrity. Notably, we have shown previously that des-acylated ghrelin is a potent stimulator of vasodilator NO in cerebral vessels , and thus might also improve stroke outcome by supporting perfusion of the ischaemic brain. Thus, taken together with its known direct neuroprotective actions , the present study sheds light on the potential of des-acylated ghrelin (or longer-acting analogues, e.g. AZP-531–currently in clinical trials for the treatment of obesity/Type 2 diabetes) as a novel therapeutic agent that can target both neuronal and vascular mechanisms of ischaemia/reperfusion-induced injury. Lastly, our findings may prove to be all the more crucial when we consider that its production may be reduced after stroke and in patients with stroke risk factors .
Jacqueline Ku acquired, analysed and interpreted data relating to: (1) neurological and functional tests; (2) infarct and oedema volumes; (3) TUNEL quantification; (4) BBB disruption in vivo and in vitro; (5) Western blotting for occludin and ZO-1; and (6) cell death and superoxide production/Nox activity. Mohammadali Taher acquired, analysed and interpreted data relating to: (1) TUNEL experiments; (2) PCR for GHSR1a in the brain; and (3) immunofluorescence for ZO-1 and 3-nitrotyrosine. Kai Yee Chin acquired, analysed and interpreted data relating to: (1) Western blotting analysis of caspase-3 expression in brain and bEnd.3 cells; and (2) occludin in bEnd.3 cells. Tom Barsby acquired, analysed and interpreted data relating to PCR for GHSR1a in cerebral vessels and bEnd.3 cells. Victoria Austin performed sham and stroke surgeries, and harvested tissue for associated experiments. Jacqueline Ku, Mohammadali Taher, Kai Yee Chin, Tom Barsby and Victoria Austin contributed to discussion, drafting of the article and revision/approval of the final version. Connie Wong, Zane Andrews and Sarah Spencer contributed to the conception of the project, and revised/approved the final version of the article. Alyson Miller conceived and designed the study, performed stroke and sham surgeries, analysed and interpreted data, and drafted, revised and approved the final version of the article.
This work was supported by the National Health and Medical Research Council of Australia [grant number APP1068442]; an Australian Postgraduate Award (to J.K.); Australian Research Council Future Fellowships [grant number FT 100109666 ZBA (to S.J.S.)]; and the RMIT Vice-Chancellor's Senior Fellowships (to A.A.M. and S.J.S.).
Dulbecco's modified Eagle's medium
growth hormone secretagogue receptor 1a
oxygen glucose deprivation
regional cerebral blood flow
reactive nitrogen species
reactive oxygen species
recombinant tissue plasminogen activator
transient middle cerebral artery occlusion
terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling
zonula occludens 1