Inhibition of insulin-regulated aminopeptidase confers neuroprotection in a conscious model of ischemic stroke

Stroke is a leading cause of mortality and morbidity with a paucity of effective pharmacological treatments. We have previously identified insulin-regulated aminopeptidase (IRAP) as a potential target for the development of a new class of drugs for the treatment of stroke, as global deletion of this gene in mice significantly protected against ischemic damage. In the current study, we demonstrate that small molecular weight IRAP inhibitors reduce infarct volume and improve neurological outcome in a hypertensive animal model of ischemic stroke. The effects of two structurally distinct IRAP inhibitors (HFI419 or SJM164) were investigated in a model of stroke where the middle cerebral artery was transiently occluded with endothelin-1 in the conscious spontaneously hypertensive rat. IRAP inhibitor was administered into the lateral ventricle at 2 or 6 h after stroke, with subsequent doses delivered at 24, 48 and 70 h post-stroke. Functional outcomes were assessed prior to drug treatment, and on day 1 and 3 post-stroke. Histological analyses and neuroinflammatory cytokine profiling were conducted at 72 and 24 h post-stroke respectively. IRAP inhibitor treatment following stroke significantly reduced infarct volume and improved neurological and motor deficits. These protective effects were maintained even when the therapeutic window was extended to 6 h. Examination of the cellular architecture at 72 h post-stroke demonstrated that IRAP expression was upregulated in CD11b positive cells and activated astrocytes. Furthermore, IRAP inhibitor treatment significantly increased gene expression for interleukin 6 and C–C motif chemokine ligand 2 in the ischemic core. This study provides proof-of-principle that selective inhibition of IRAP activity with two structurally distinct IRAP inhibitors reduces infarct volume and improves functional outcome even when the first dose is administered 6 h post-stroke. This is the first direct evidence that IRAP inhibitors are a class of drug with potential use in the treatment of ischemic stroke.

Ischemic stroke constitutes approximately 80% of all cerebrovascular events and is one of the leading causes of death and disability worldwide 1 .The only effective pharmacological therapeutic intervention is the lysis of the clot with intravenous administration of recombinant tissue plasminogen activator (tPA).However, this is only effective when given within 4-5 h of the ischemic event 2,3 or up to 9 h with favorable computed tomography perfusion imaging 4 .More recently, endovascular thrombectomy with concomitant intravenous or intra-arterial tPA has proven efficacious in improving stroke outcome and reducing disability [5][6][7][8][9] .Despite the efficacy of current stroke therapies, accessibility and administration is time constrained, subject to a strict inclusion criterion and require specialized centers.Furthermore, the therapeutic mechanism is limited to restoring central perfusion which is not without associated risks, including reperfusion injury and hemorrhage.As such, adjunct neuroprotective therapies offer a potentially critical role in acute stroke care firstly by way of preserving the ischemic www.nature.com/scientificreports/penumbra and extending the therapeutic window of the current therapies.Secondly, as a standalone therapy, a pharmacological intervention that could arrest the deleterious processes of the ischemic and inflammatory cascade, as well as promoting neuronal survival and repair would benefit patients who would otherwise be excluded from current best therapies 10 .With this in mind, we have recently identified a role for insulin-regulated aminopeptidase (IRAP) in the pathogenesis of focal ischemic damage 11 .
IRAP is a type II integral membrane protein that belongs to the M1 aminopeptidases that was first identified in muscle and adipose tissue and has strong associations with the insulin-sensitive glucose transporter GLUT4 within specialized vesicles 12 .IRAP is expressed in a number of tissue types and found in specific neuronal populations 13 as well as various immune cells including monocytes 14 , mast cells 15 , and dendritic cells 16 .IRAP contains two functional domains.The N-terminal cytoplasmic domain is a critical regulator of vesicular trafficking and sorting of GLUT4 from endosomes to GLUT4-specialised vesicles 17,18 .The much larger C-terminal catalytic domain contains the substrate recognition motif important for the selective processing of neuropeptides including oxytocin, vasopressin, somatostatin, lys-bradykinin (kallidin) and met-enkephalin as well as a zinc binding motif characteristic of many metallopeptidases 19,20 .Naturally occurring peptide inhibitors of this enzyme such as angiotensin (Ang) IV and LVV-hemorphin 7 enhance learning and memory in the healthy rodent [21][22][23] as well as in several animal models of cognitive impairment [24][25][26][27] .More recently, the effect of enhanced learning and memory elicited, were replicated using the specific small molecular weight non-peptide IRAP inhibitor HFI419 28,29 .
In the phenotypic characterization of global IRAP knockout (KO) mice, we observed that these mice were protected against focal ischemic damage 11 , as well as epileptiform activity 30 , a common post-stroke complication 31 .In the IRAP KO mice, ischemic damage resulting from transient occlusion of the middle cerebral artery (MCA) was 80% less compared to wild type animals.Furthermore, this translated into a concomitant improvement in neurological performance 11 .
The current study aims to obtain proof-of-principle that the inhibition of IRAP activity with either of the two structurally distinct, small molecular weight and specific inhibitors of the enzyme, HFI149 and SJM164, protects against focal ischemic damage in a conscious model of stroke using the spontaneously hypertensive rat (SHR).We provide compelling histological and functional evidence on the benefit of IRAP inhibition following cerebral ischemia and therefore highlight IRAP as a promising new target for the treatment of stroke.

Ethics statement
All animal procedures were performed in accordance with the National Health and Medical Research Council of Australia guidelines with approval from Monash University Animal Ethics Committee.No experiments on humans or human tissue was conducted.

Animals
As this is a proof of principle study, we elected to use only male rats.Male spontaneously hypertensive rats (SHR) (N = 130) purchased from Animal Resource Centre (Western Australia) were 20-24 weeks (350-400 g) at the time of experimentation.The rats were housed individually with access to standard rat chow (Barastoc Ridely, Australia) and water ad libitum.The housing facility followed a 12 h day/night cycle.The study was completed in accordance with ARRIVE guidelines 32 .At the end of the experimental timeline (Supplementary Fig. 1A), animals were anesthetized with ketamine (80 mg/kg) and xylazine (10 mg/kg) and decapitated while heavily anaesthetized.

Surgical procedures
The SHRs were anaesthetized with ketamine (75 mg/kg; Sigma)/xylazine (10 mg/kg; Troy; i.p) for the stereotaxic implantation of a 23-gauge stainless steel guide cannula into the right piriform cortex (coordinates: 0.2 mm anterior, − 4.7 mm lateral relative to bregma, and -7 mm ventral relative to the skull surface) to sit 3 mm dorsal to the right middle cerebral artery [33][34][35] .An additional 23-gauge guide cannula was implanted into the left lateral ventricle (− 0.8 mm anterior, + 1.5 mm lateral relative to bregma and − 3.2 mm ventral relative to the skull surface) for the intracerebroventricular administration of IRAP inhibitor or vehicle control.This was followed by a 5-day recovery period prior to stroke induction.

Stroke induction
Stroke was induced in the conscious SHR by titration of the potent vasoconstrictor endothelin-1 (ET-1) (2 pmol/µl in saline; AusPep) at a rate of 0.2 µl every 30 s, up to 7 µl, through a 30-gauge injector protruding 3 mm beyond the end of the previously implanted guide cannula.Stroke severity was characterized by behavioral indicators described below and ET-1 was infused until a desired level of stroke was achieved.The stroke severity was graded on a pre-determined scale of 1-4.Level 1 graded stroke represents a mild stroke while a level 4 graded stroke represents a severe stroke, based on previously published behavioral criteria 34,36 .Typical changes in behavior include grooming, teeth chatter, contralateral and ipsilateral spinning, clenching or dragging of contralateral forepaw, and jaw flexion or biting with the latter 2 indicative of a grade 4 stroke.Only animals with a stroke grading of level 4 were used for this study (N = 87).Reperfusion begins 20 min following cessation of ET-1 delivery with cerebral blood flow reaching 90% at approximately 60 min followed by a gradual reperfusion lasting 16-22 h 37,38 .Rectal temperature and observations of habitual behaviours were monitored every 30 min for 3 h following stroke.No animals exceeded a stroke grade greater than level 4.

Rotarod assessment
On day 3 post stroke, SHR were placed on the rotarod consisting of a motorized cylindrical assembly of 18 stainless steel rods capable of rotating at a maximum speed of 30 revolutions per minute (30 rpm; Ratek, Australia).Top speed of three attempts was used to determine a final score, to maintain an upright walking position and qualify as a successful completion, an animal had to grip the rotating rods at each speed for 3 s.The rotating speed began at 6 rpm and was raised 1.5 rpm every 3 s until the animal lost an upright walking position or until the maximum 30 rpm was reached.

Systolic blood pressure measurement
A small subset from each treatment group (n = 3-5) were used to determine if stroke or drug treatment influenced blood pressure regulation.Systolic blood pressure was measured via a non-invasive, tail-cuff pressure analysis system (Model MC400, Hatteras Instruments) where the SHR were placed in a restrainer on a heated platform.The SHR were habituated to the restrainer 1 day before the first systolic blood pressure measurement.Systolic blood pressure was measured 5 days prior to stroke and 70 h post-stroke with the procedure taking 5-10 min to complete.Each single measure represents an average of 5 systolic measurements.

Infarct and forebrain volume quantification
At 72 h after the induction of cerebral ischemia, SHR were anesthetized with ketamine (75 mg/kg; Sigma)/xylazine (10 mg/kg; Troy; i.p) and transcardially perfused with phosphate buffered saline (10 mM sodium phosphate and 150 mM sodium chloride; pH 7.4).The brains were removed, snap frozen in liquid nitrogen and sectioned in a cryostat at 16 µm thickness in the coronal plane at the following 8 predetermined levels: + 2.10 mm, + 1.18 mm, + 0.74 mm, − 0.10 mm, − 1.06, − 1.82 mm, − 2.70 mm and − 3.88 mm relative to bregma.The sections were then imaged and the infarct volume was calculated using the ballistic light method 42 .Using ImageJ, the total infarct volume was quantified by integrating the area of infarct at each level with the distance between each level 43 .Correction for oedema was calculated as previously described 42 .The same sections were used to determine hemispheric and total brain volumes as a marker of forebrain oedema.The hemispheric volumes were calculated as a percentage change (PC) in which the ipsilateral hemisphere (IH) was compared to the contralateral hemisphere (CH) using the formula: PC = ((IH/CH)*100) − 100.

Immunohistochemistry
Coronal sections taken at + 0.74 mm relative to bregma were post-fixed for 15 min in 4% paraformaldehyde followed by three washes in phosphate buffered saline (PBS) for 10 min each.The sections were then incubated in blocking solution (0.3% Triton-X-100 and 10% normal donkey serum (NDS) prepared in PBS) for 1 h at room temperature.This was followed by an overnight incubation with one of the following antibodies: NeuN for neurons (1:500 dilution, Invitrogen), glial fibrillary acidic protein (GFAP) for activated astrocytes (1:500 dilution, Invitrogen), CD11b (1:500 dilution, Serotec), GLUT-1 for cerebral microvessels (1:500 dilution, Millipore) or IRAP (1:500 dilution, Invitrogen) at 4 °C.All primary antibodies were diluted in 0.3% (v/v) Triton-X-100 and 3% (v/v) NDS prepared in PBS.Slides were washed three times with 0.1% (v/v) Tween-20 prepared in PBS before www.nature.com/scientificreports/being incubated with an Alexa-Fluor® fluorescently conjugated secondary antibody 1:500 (Alexa-Fluor® 488 or 568) for 1 h at room temperature.All secondary antibodies were diluted in 0.3% (v/v) Triton X-100 and 3% (v/v) NDS prepared in PBS.Slides were washed three times with 0.1% (v/v) Tween-20 prepared in PBS and then coverslipped with DAKO Fluorescence Mounting Medium.The regions imaged include the cortical and striatal ischemic core, representing infarcted tissue, and the cortical penumbra which represents mildly damaged but salvageable tissue surrounding the ischemic core (Supplementary Fig. 1B).In addition, the respective matched contralateral regions were imaged.For each brain section, three images were captured within each brain region.Single images for animals treated with an IRAP inhibitor beginning at 2 h post stroke was captured using a Zeiss LSM700 inverted confocal microscope (Carl Zeiss Microscopy GmbH, Jena, Germany).A 0.56 mm 2 field of view was used and NeuN, GFAP and CD11b immuno-positive cells were counted using Image J software and cell counts were averaged over the 3 images per brain region for each animal.For animals treated with an IRAP inhibitor beginning at 6 h post stroke, images were captured using the Leica® Aperio Scanscope AT Turbo slide scanner where a 1 mm 2 field of view was used to count NeuN, GFAP and CD11b immuno-positive cells.

Real-time PCR (RT-qPCR)
As treatment effects were evident as early as 24 h post stroke, in a subgroup of SHRs, we examined 24 h mRNA expression of inflammatory chemokines and cytokines with RT-qPCR.Rats treated with either vehicle or 0.1 nmol HFI419 2 h post-stroke, were anesthetized with ketamine (75 mg/kg; Sigma)/xylazine (10 mg/kg; Troy; i.p), decapitated and the whole brain removed.The brain was dissected into striatal core (whole striatum), cortical core (ventral cortex) and cortical penumbra (dorsal cortex) as well as the respective contralateral unlesioned matched regions.25 mg of RNA was extracted from each dissected brain region and stored at -80 °C using Trizol reagent (Life Technologies) according to the manufacturer's protocol.The quantity and quality of the RNAs were determined using A260/A280 readings by NanoPhotometer (Implen).For RT-PCR, 2 mg of RNA was reverse transcribed into cDNA using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems).The synthesized cDNA was used as a template for PCR reactions using TaqMan Fast Advanced Master Mix (Applied Biosystems).The TaqMan probes have the following identification number: 45S (Rn03928990_ gl), GFAP (Rn00566603_m1), CD11b (Rn00709342_m1), TNF-⍺ (Rn00562055_m1), IL6 (Rn01410330_m1), BDNF (Rn01484924_m1), IL-1b (Rn00580432_m1), CCL2 (Rn00580555_m1), iNOS (Rn00561646_m1), TGF-β (Rn00572010_m1) and eNOS (Rn02132634_s1).Relative quantification of gene expression was performed by comparative threshold (CT) calculated by the 2 −ΔΔCT method 44 .Changes in mRNA expression levels were calculated following normalization to the house-keeping gene 45S.

Wire myograph
Wire myograph was used to investigate blood vessel contractility in response to increasing doses of IRAP inhibitor.A subgroup of naïve SHR (n = 5) were anesthetized with ketamine (75 mg/kg; Sigma)/xylazine (10 mg/kg; Troy; i.p), decapitated and the whole brain removed.Fresh basilar arteries were dissected and placed in a cold carbogen-bubbled (95% O 2 , 5% CO 2 ) solution of Krebs-bicarbonate (KB; composition in mM NaCl 118, KCl 4.5, MgSO 4 0.45, KH 2 PO 4 1.03, NaHCO 3 25, glucose 11.1, CaCl 2 2.5).Arteries were cut into 2 mm segments and mounted in a Mulvany-Halpern myograph (Danish Myo Technology A/S) via two 40 µm wires.Arteries were equilibrated at 37 °C for 30 min, washed with KB and then allowed to rest for 10 min before tensioning to 5 mN.Arteries were maximally contracted with high potassium salt solution (KPPS; composition containing 122.5 mM KCl as an equimolar replacement of NaCl with KCl), washed out with KB and then 30 min later sub-maximally contracted to 50% of maximal KPPS contraction with the thromboxane A2 receptor agonist U46619 (1 × 10 -9 -1 × 10 -7 ).To ensure the viability of the endothelium, 10 µM of acetylcholine was added to assess relaxation.Preparations with a relaxation percentage of 65 or higher of maximal contraction were used.
Once the contractions were stable, either HFI419 (1 × 10 -12 -1 × 10 −6 M) or vehicle (time control) was delivered in cumulative concentrations.To ensure maximal relaxation could be achieved, sodium nitroprusside (SNP; 1 × 10 -5 M) was added to each preparation at the conclusion of the experiment.

IRAP inhibitor treatment following ischemic stroke reduced neurological deficit
The neurological deficit score was assessed at 24 and 70 h post-stroke as a broad measure of neurological function where a higher score indicates a worse outcome.Neurological deficits were significantly reduced in all IRAP inhibitor treatment groups regardless of whether the treatment began at 2 or 6 h post-stroke.It is worth noting that the treatment effect was evident at 24 h after stroke, following just a single dose of either SJM164 or HFI419 and was maintained up to 70 h post-stroke (Fig. 1D).

IRAP inhibitor treatment following ischemic stroke improved motor deficits
In addition to neurological deficit, at 24 and 70 h following stroke, motor deficits were determined by measuring the number of contralateral hindlimb foot faults using the tapered ledge beam test and a rotarod assessment.Treatment with 1 nmol HFI419 beginning at 6 h post stroke significantly improved motor performance on the rotarod compared to the vehicle group as assessed at 70 h post-stroke (4.33 ± 0.64 vehicle vs 6.71 ± 0.62 1 nmol HFI419, P < 0.05, Fig. 1E).Treatment with an IRAP inhibitor beginning at 2 h post stroke significantly reduced the percentage of foot faults measured at 24 h after the ischaemic insult (0.1 nmol HFI41 18.70 ± 4.20%; 1 nmol HFI419 22.61 ± 7.65%; 1 nmol SJM164 31 ± 5.47%, P < 0.001) compared to vehicle control treatment (71.72 ± 7.91%; Fig. 1F).Additionally, the treatment effect was not diminished when the first dose was delayed to 6 h post-stroke, such that animals receiving HFI419 still performed better than the vehicle group (15.40 ± 5.06% vs 41.85 ± 11.22% respectively, P < 0.001).Remarkably, similar to the neurological deficit assessment, a treatment effect was evident as early as 24 h post stroke following only a single dose of IRAP inhibitor that was delivered at either 2 or 6 h after the onset of ischemia.Furthermore, this neuroprotection persisted such that at 70 h poststroke all groups receiving either SMJ164 or HFI419 performed better than the vehicle treated control animals, regardless of when treatment was commenced.

IRAP inhibitor treatment enhanced neuronal survival in the cortical ischaemic core
Treatment with an IRAP inhibitor reduced infarct volume and consequently improved neurological and motor function.However, to determine whether additional neuronal survival within the striatal or cortical core also contributed to improved functional outcome, surviving neurons at 72 h post-stroke were visualised and counted after immunostaining with the nuclear marker of neurones, NeuN (Fig. 2A-C).Stroke resulted in a significantly reduced number of NeuN positive cells in the striatum and cortical core regions.Treatment with either 0.1 or 1 nmol HFI419 beginning at 2 h post-stroke attenuated this loss of NeuN positive staining in the infarcted cortex (375.33 ± 43.62 cells per 0.54 mm 2 , P < 0.0001 and 263.67 ± 24.18 cells per 0.54 mm 2 , P < 0.05, respectively) compared to vehicle treated controls (141.43 ± 12.82 cells per 0.54 mm 2 , Fig. 2B).Within the cortical penumbra, there was a significant reduction of NeuN positive staining in the vehicle control compared to sham however we did not detect any difference with any IRAP inhibitor treatment group compared to vehicle (Fig. 2C).When delaying the first dose of HFI419 to 6 h post-stroke, despite the reduction of infarct volume with treatment, there was no evidence of additional neuronal preservation within any brain region compared to the vehicle treated animals (Supplementary Fig. 2A-C).

IRAP expression was upregulated in CD11b positive cells and astrocytes in the injured ischaemic brain
CD11b positive cells in the ischaemic regions were found to express IRAP at 72 h post-stroke, as evident by colocalisation with IRAP immunostaining (Fig. 3A), unlike in quiescent brain where there is minimal CD11b/IRAP colocalization.Interestingly, IRAP inhibitor treatment beginning at 2 h post-stroke significantly increased CD11b within the striatal and cortical core with 0.1 nmol HFI419 (striatum; 122.2G-H).There was no difference in CD11b within the cortical penumbra between vehicle or inhibitor treated groups (Fig. 2I).In addition, a small population of activated astrocytes were shown to express IRAP following stroke as demonstrated by GFAP and IRAP colocalization (Fig. 3B).At 72 h following stroke, the critically injured ischaemic core demonstrated a reduced GFAP cell count compared to sham however animals treated with 0.1 nmol of HFI419 beginning at 2 h post-stroke demonstrated a greater number of activated astrocytes within the cortical core (178.62 ± 49.62 cells per 0.54 mm 2 , P < 0.01) compared to the vehicle control (21.81 ± 10.78 cells per 0.54 mm 2 , Fig. 2E).GFAP positive cell counts in the striatal core and cortical penumbra did not differ between treatment groups (Fig. 2D and F).When the first dose of HFI419 was delayed to 6 h post stroke, treatment significantly increased CD11b counts in the cortical penumbra (Supplementary Fig. 2I) however  www.nature.com/scientificreports/no differences were detected in the number of GFAP positive cells between the drug treated and vehicle treated groups in any brain region (Supplementary Fig. 2D-F).

Treatment with HFI419 2 h post stroke upregulated IL-6 and CCL2
IRAP was upregulated in activated astrocytes and CD11b positive immune cells in the post-ischaemic brain.
To further delineate the neuroinflammatory role of IRAP post-stroke, cytokine and chemokine profiles were examined at 24 h post-stroke (Supplementary Table 1).In contrast to our immunohistochemical analysis of the brains at 72 h post stroke, at 24 h post stroke, the expression of CD11b and GFAP genes were upregulated in the striatal and cortical core but not in the penumbra in the ischaemic hemisphere of all groups (Fig. 4A-F).Furthermore, the gene expression of the inflammatory cytokines, TNF⍺ and IL-6, as well as the chemokine CCL2, were upregulated in the striatal and cortical ischaemic core (Fig. 4G-O).Treatment with HFI419 potentiated the upregulation of IL-6 in the cortical and striatal core (Fig. 4J-K) and upregulated CCL2 in the cortical penumbra (Fig. 4O) compared to vehicle control.When brain tissue was assessed at 72 h post stroke, there was no treatment effect evident on any of the cytokine or chemokine of interest (Supplementary Table 2).

Treatment with HFI419 attenuated stroke-induced increases in forebrain volume
Vasopressin is a physiological substrate of IRAP 45 and can exacerbate cerebral oedema 46 .To assess if IRAP inhibition post-stroke influenced cerebral oedema, forebrain volumes were assessed.At 72 h following stroke, total forebrain volume of the vehicle treated group (1276.00± 51.02 mm 3 ) was significantly greater than sham (966.50 ± 49.20 mm 3 ; P < 0.001; Fig. 5A).Treatment with 0.1 nmol HFI419 significantly attenuated the strokeinduced increase in forebrain volume (1043 ± 40.38 mm 3 vs vehicle, P < 0.01).Surprisingly, treatment with 1 nmol HFI419 did not show the same level of protection against oedema as the animals receiving the lower dose of 0.1 nmol HFI419 (Fig. 5B).No effect of oedema was evident in animals treated 6 h post-stroke (Fig. 5C).

Neuroprotection mediated by IRAP inhibition was independent of regulation of systolic blood pressure, vascular reactivity or microvascular density
The SHR is a hypertensive animal model and to determine if any neuroprotective effect of IRAP inhibition was secondary to hypertension management, systolic blood pressure was assessed via tail cuff prior to stroke and at 72 h post-stroke.Stroke did not alter blood pressure of the rats (Fig. 6A).Furthermore, central administration of HFI419 post-stroke had no apparent blood pressure lowering effects (Fig. 6A).In addition, in vitro analysis of vascular reactivity in a pre-constricted isolated basilar artery for a naïve SHR using a wire myograph, indicated that increasing doses of HFI419 failed to elicit any vascular relaxation responses (Fig. 6B).Finally, immunohistochemistry visualising the cerebral blood vessel marker GLUT 1 within the cortex and striatum was performed to determine if improvements with HFI419 treatment following stroke was as a result of angiogenesis.In all treatment groups, no differences in GLUT 1 were detected within the cortex and striatum suggesting that there was no effect of stroke or drug treatment on cerebral microvascular density (Fig. 6C-E).

Discussion
The major findings of the current study are that treatment with an IRAP inhibitor 2 h after stroke protected against ischemic damage resulting in improved functional outcomes.We also found that this neuroprotection was not diminished even when the first administration of the IRAP inhibitor was delayed to 6 h post stroke.To date, apart from fibrinolytic therapies, all drug candidates for the treatment of stroke have demonstrated poor clinical translation, despite their proven efficacy in preclinical models 47,48 .This highlights an ongoing need for novel therapies to be developed to improve stroke care either as an adjuvant therapy to current clinical practice or as a standalone monotherapy.We have previously shown that global IRAP gene deletion was neuroprotective following transient occlusion of the MCA in mice 11 congruous with an earlier report demonstrating that intracarotid administration of angiotensin IV (Ang IV), a competitive albeit non-selective inhibitor of IRAP 49 , significantly reduced infarct volume, attenuated neurological deficits and increased survival rate in a rat model of embolic stroke (Faure et al. 2006).Despite IRAP being identified as the Ang IV binding site, Ang IV also binds to various other proteins including the angiotensin II AT1 and AT2 receptors and aminopeptidase N (Yamamoto et al. 2010).This promiscuity naturally gave rise to uncertainty as to whether IRAP was, in fact, the target responsible for the neuroprotection induced by exogenous administration of Ang IV.We now provide proof-ofprinciple that treatment with two different classes of small molecular weight selective IRAP inhibitors, HFI419 or SJM164, provided protection in a conscious model of stroke in the hypertensive rat.While the mechanism of IRAP inhibition remains to be fully elucidated, IRAP was upregulated in activated astrocytes and CD11b positive cells following stroke suggesting that IRAP plays an immunomodulatory role in the ischemic brain.Both HFI419 and SJM164 significantly reduced cortical infarct volume at 72 h after stroke, even when the therapeutic window was extended to 6 h.Impressively, when treatment was commenced at 2 h post-stroke, the neuroprotective effect of IRAP inhibition extended to the striatum, which is a region of the brain that is traditionally thought be refractory to therapeutic intervention 50 .Consistent with a significant reduction in infarct volume, treatment with HFI419 attenuated the physical symptoms of stroke, as demonstrated by improved performance on the tapered ledge-beam test and the rotarod as well as a reduction in the neurological deficit score.Impressively, these benefits were evident as early as 24 h post-stroke, following administration of just a single dose of either HFI419 or SJM164.This improvement in the physical symptoms of stroke, is most likely a reflection of the reduction of infarct volume and preservation of penumbral tissue.However, with early administration of treatment at 2 h post-ischemia, the IRAP inhibitor treated brains demonstrated increased neuronal survival within the infarcted cortical core.More importantly, we found that extending the interventional therapeutic window whereby the first dose of IRAP inhibitor was received at 6 h post-stroke, neuroprotection was still clearly evident by way of improvements in histological and behavioural assessments.From a clinical perspective, the therapeutic efficacy following delayed delivery of the first drug dose broadens potential patient candidacy, particularly as the symptom onset-to-hospital arrival times have remained largely unchanged over the last two decades with the majority of stroke patients presenting to hospital within 6 h from symptom onset 51,52 .
Immunologically, IRAP has been shown to be critical in major histocompatibility complex cross presentation 53 as well as T-cell receptor signalling 54 , and in this study we reveal for the first time that IRAP was upregulated in both activated astrocytes and CD11b positive CNS immune cells at 72 h post-stroke, which is suggestive of an inflammatory role for IRAP in the post ischaemic brain.Our finding is congruent with earlier studies that demonstrated an upregulation of IRAP in activated in peripherally activated macrophages following interferon gamma or lipopolysaccharide-induced inflammation (Nikolaou et al. 2014).In the current findings, treatment with 0.1 nmol HFI419 beginning at 2 h post-stroke increased recruitment of astrocytes and CD11b positive cells to the stroke-induced site of injury, both of which are critical for post-stroke neuronal repair and remodelling.Although cellular infiltration was not evident when treatment with an IRAP inhibitor commenced at 6 h poststroke, delayed treatment still reduced infarct volume and improved functional outcome suggesting that the benefits of IRAP inhibition are multifactorial and may vary depending on the temporality of the ischemic cascade.To further investigate the inflammatory role of IRAP, pro-and anti-inflammatory cytokine and chemokine gene expression were quantified at 24 and 72 h post-stroke where treatment with 0.1 nmol HFI419 began at 2 h post-stroke.At 24 h post-stroke, CCL2 and IL-6 were significantly upregulated following a single dose of HFI419.We first observed the benefits of IRAP inhibition at 24 h post-stroke following a single dose, with improved performance on the ledge-beam test and reduced neurological deficits.Taken together, these findings suggest that IL-6 and CCL2 may play a role in mediating early neurological protection following IRAP inhibition.
Certainly, peak IL-6 serum levels post-stroke correlated with the extent of infarct size and neurological deficit 55 however whether or not this is causative is equivocal 56 particularly given that upregulation of IL-6 is protective against NMDA-mediated excitotoxicity 57 .Experimentally, IL-6 is neuroprotective in in vivo stroke models whereby intracerebroventricular injection of recombinant IL-6 reduced infarct volume and improved neurological deficits 58 and blockade of IL-6 signalling with intraperitoneal injections of an IL-6 receptor antibody exacerbated ischaemic brain injury 59 .Additionally, IL-6 is essential for promoting post stroke angiogenesis 60 as well as neurogenesis and long-term functional recovery 61 .In the current study, HFI419 treatment resulted in IL-6 upregulation in both the cortical and striatal core regions of damage consistent with a neuroprotective role for IL-6 as infarct volume was significantly reduced in both of these regions.Despite these findings, there is still ambivalence surrounding the role IL-6 following stroke as global IL-6 gene deletion demonstrate comparable infarct volume to controls with reductions in body temperature 62 .However, if body temperature is controlled for by external warming, global IL-6 deletion resulted in a larger infarct volume and worse neurological outcome favouring an overall neuroprotective role of IL-6 independent of its pyrogenic properties 63 .www.nature.com/scientificreports/ The monocyte chemoattractant protein-1 (MCP1/CCL2) was upregulated in the penumbra with IRAP inhibitor treatment following stroke.CCL2 is a potent monocyte chemoattractant that mediates its effects through its receptor CCR2 64 .Acutely following stroke, CCR2 KO mice exhibited larger infarct volume, cerebral oedema, worse neurological function as well as increased blood brain barrier permeability 65,66 .However, long-term poststroke follow-up studies in these mice demonstrated increased mortality rates and abolished long-term neurological and functional recovery 67,68 .Moreover, aberrant CCL2/CCR2 signalling resulted in a larger infarct volume 69 , a higher incidence of haemorrhagic transformation 70 and reduced angiogenesis 71 .The benefits of CCL2 and CCR2 are likely related to the recruitment of Ly6C hi peripheral monocytes and their ability to differentiate into the antiinflammatory/reparative M2 phenotype 69,71,72 .In our study, HFI419 treatment upregulated CCL2 expression in the cortical penumbra and unsurprisingly this was associated with a regionally matched increase in CD11b cell counts.Despite an elevated CCL2 with drug treatment, IRAP inhibition with a low dose of HFI419 reduced stroke related increased forebrain volume suggestive of reduced cerebral oedema.The reduction of forebrain volume likely represents a dose response effect of HFI419 given that the higher dose of HFI419 did not reduce oedema despite reducing infarct volume.Indeed, IRAP is expressed in peripheral macrophages with greater expression in M1 macrophages, however inhibition of the catalytic domain with Ang IV did not reduce pro-inflammatory cytokines 14 , a finding consistent with the current study.Nevertheless, acutely following stroke, recruited microglia are predominantly polarised to the M2 phenotype 73 .Although further studies are required to confirm, it is plausible that IRAP inhibition post-stroke increases monocyte recruitment through elevated CCL2 and therefore promotes M2 polarisation, or that direct IRAP inhibition preferentially differentiates monocyte/microglia M2 phenotype via an alternative mechanism.Lastly, in addition to monocyte recruitment, CCL2 and its receptor CCR2 play an integral role in endogenous neuroblast differentiation and migration to injured tissue 74,75 as well as the homing of peripherally delivered stem cell therapy following stroke 74,76,77 .Interestingly, IRAP is highly expressed in the subventricular zone 78 and postulated to play a role in neuronal development 49,79 and although this mechanism requires further investigation, it may hold significant implications for long-term stroke recovery.
In addition to immunomodulation, augmentation of collateral blood flow may be another potential contributor to the therapeutic benefits of post-stroke IRAP inhibition.Inhibition of IRAP, in both the IRAP KO mice or rats treated with the IRAP inhibitor Ang IV, have been shown to enhance collateral blood flow post-stroke 11,80 .Consistent with this, IRAP inhibitors delivered locally to the pulmonary 81 , renal 82 and cerebral vasculature 83,84 have been demonstrated to promote dilatation and increased blood flow.In these studies, the dilatory effects are inhibited in the presence of L-NAME and therefore likely mediated via a nitric oxide dependent pathway possibly through the increased availability of IRAP substrates such as the NO-dependent vasodilator bradykinin 85 .We were not able to directly measure cerebral blood flow following MCA occlusion in the current study, a limitation of the conscious stroke model, however HFI419 did not elicit any vasoactive responses in the isolated healthy basilar artery, which suggests that the protection we have demonstrated is unlikely to have a vascular component.Furthermore, we did not detect increased endothelial NOS expression following treatment with HFI419 at either 24 or 72 h post-stroke nor was there a difference in cerebral microvascular density at 72 h post-stroke.Nevertheless, our findings do not exclude the possibility of an acute dilatory effect in an injured vessel following IRAP inhibition, as improved collateral blood flow in previous stroke studies was demonstrated either during the transient occlusion period 11 , or within minutes after stroke and drug administration 80 in the dysfunctional vessel 86 .Additionally, considering long term IRAP inhibition reverses chronic vessel dysfunction 87 and that IRAP inhibition improves learning and memory 21,22,24,28,29 as well as reducing seizure susceptibility 30,88 , there is strong rationale to further explore the effects of chronic IRAP inhibition following stroke in the context of preventing complicating vascular and neurological sequelae.
Despite numerous clinical candidates being effectively trialled in preclinical stroke models, translation to human clinical trials has been largely unsuccessful in part due to limitations in study design.The current study design attempts to address some of these shortcomings by the choice of the model, the ET-1 model of stroke in a conscious animal which (i) eliminates the confounding variable of anaesthesia 89 , (ii) blood flow pattern that closely mimic what is seen in humans and (iii) includes hypertension as a co-morbidity.Moreover, administration of the neuroprotective agent was delayed to 6 h after the induction of stroke, which is a clinically feasible timeline for treatment delivery.One limitation of the current study is the central route of drug administration.An intracerebroventricular mode of delivery was chosen to ensure adequate central access of the IRAP inhibitors in this proof of principle study.As such, the impact of the BBB on drug delivery was not considered.Nevertheless, approximately 20% of intraperitoneal injected HFI419 reaches the brain 5 min post-injection in the healthy Sprague Dawley 90 .Moreover, the BBB is known to be compromised following ischemic stroke 91 and therefore the central nervous system bioavailability of peripherally delivered IRAP inhibitor following stroke is likely greater in the injured brain.The compounds used in the present study, HFI419 and SJM164, are small molecular weight, specific inhibitors of IRAP.In contrast to Ang IV or other peptide inhibitors, they demonstrate superior selectivity for IRAP and a reduced susceptibility to systemic degradation 28 which is favourable for a peripheral and clinically relevant route of administration.
Currently, the only pharmacological intervention following ischemic stroke is tPA, however this therapy is only accessible to a limited number of patients and offers no additional therapeutic benefit beyond the restoration of cerebral blood flow.This underscores the urgent need for novel therapeutics with a wider therapeutic window.We have shown, for the first time, that pharmacological inhibition of IRAP via central administration of two distinct classes of small molecular weight IRAP inhibitors significantly improved morphological and functional outcomes following stroke in the SHR, even when treatment was delayed to 6 h post stroke.Although further research is required to elucidate the cellular mechanism responsible for the neuroprotection demonstrated, our current understanding suggests that there are likely various components to the therapeutic benefit of IRAP inhibition, including modulation of the innate immune response and preservation of neuronal survival ultimately leading to improved motor and neurological function.The findings of this study provide compelling evidence https://doi.org/10.1038/s41598-023-46072-5

Figure 2 .Figure 3 .
Figure 2. Inhibition of IRAP beginning at 2 h post-stroke attenuated cortical neuronal cell death and increased microglia and astrocyte cell counts within the ischaemic core.Immunohistochemistry (IHC) using a neuronal marker NeuN (A-C) to visualise neuronal bodies within the striatal and cortical core as well as the cortical penumbra at 72 h post-stroke revealed that treatment with 0.1 or 1 nmol HFI419 beginning at 2 h, and then at 24, 48 and 70 h post-stroke, attenuated neuronal cell death within the cortical core.IHC using the astrocyte specific marker GFAP (D-E), revealed an increased GFAP positive cell count within the cortical core following treatment with 0.1 nmol HFI419.Lastly, IHC using the microglial marker CD11b (F-I) revealed an increased CD11b cell count within the striatal and cortical core following treatment with 0.1 or 1 nmol HFI419.The data is represented as mean ± SEM and analysed using a two-way ANOVA followed by a Dunnett's test where *P < 0.05 and ***P < 0.001 (n = 4-9).

Figure 4 .
Figure 4. IRAP inhibition beginning at 2 h post-stroke upregulated IL-6 and CCL2 24 h.Following treatment with 0.1 nmol HFI419 2 h post-stroke, the gene expression of inflammatory mediators was determined in the ipsilateral and contralateral striatum, cortical core, and cortical penumbra at 24 h post-stroke.Following treatment with an IRAP inhibitor at 2, 24, 48 and 70 h post-stroke, (J & K) IL-6 was upregulated in the striatal and cortical core compared to vehicle control.In the cortical penumbra, treatment with an IRAP inhibitor upregulated (M-O) CCL2 compared to vehicle treatment.The data is represented as mean ± SEM and analysed using a two-way ANOVA followed by a Dunnett's test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; n = 5-6).

Figure 6 .
Figure 6.Neuroprotection mediated by IRAP inhibition was independent of changes in blood pressure, vascular reactivity and vascular density.(A) Systolic blood pressure determined pre stroke and at 70 h post stroke was not different between sham operated, vehicle and HFI419 (1 nmol) treated animals.(B) Myograph preparations of the SHR basilar artery indicated no change in vascular reactivity in response to increasing concentrations of HFI419 (n = 3-5; data represented by mean ± SEM).Comparison of GLUT1 immunopositively stained cell area within the striatal core (C), cortical core (D) and cortical penumbra (E) revealed no differences with either stroke or treatment with an IRAP inhibitor (n = 4-9, data represented by mean ± SEM).
Vol.:(0123456789) Scientific Reports | (2023) 13:19722 | https://doi.org/10.1038/s41598-023-46072-5www.nature.com/scientificreports/Statistical analysis Data are expressed as the mean ± SEM unless stated otherwise with statistical analysis completed using GraphPad Prism 9. Infarct volume was analysed with a one-way ANOVA followed by a Dunnett's test for drug treatment beginning at 2 h post stroke or with an unpaired t-test for drug treatment beginning at 6 h post stroke.The neurological score, ledge-beam test, immunohistochemical cell counts, RT-qPCR, hemispheric brain volume and wire myograph were analysed with a two-way repeated measures ANOVA followed by a Tukey's test.The sample size and P values are provided with each figure with a P < 0.05 considered significant.Vol:.(1234567890)Scientific Reports | (2023) 13:19722 | https://doi.org/10.1038/s41598-023-46072-5www.nature.com/scientificreports/