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. Author manuscript; available in PMC: 2015 Apr 1.
Published in final edited form as: Stroke. 2014 Feb 18;45(4):1108–1114. doi: 10.1161/STROKEAHA.113.004399

Combination treatment with AcSDKP and tissue plasminogen activator provides potent neuroprotection in rats after stroke

Li Zhang 1, Michael Chopp 1,3, Hua Teng 1, Guangliang Ding 1, Quan Jiang 1, Xiao Ping Yang 2, Nour Eddine Rhaleb 2, Zheng Gang Zhang 1
PMCID: PMC3966939  NIHMSID: NIHMS561226  PMID: 24549864

Abstract

Background and Purpose

N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP), an endogenously produced circulating peptide in humans and rodents, exerts anti-inflammatory and cardioprotective activities in various cardiovascular diseases.

Methods

The present study evaluated the neuroprotective effect of AcSDKP alone and in combination with thrombolytic therapy in a rat model of embolic focal cerebral ischemia.

Results

We found that treatment with AcSDKP alone at 1h or the combination treatment with AcSDKP and tissue plasminogen activator (tPA) at 4h after stroke onset substantially increased AcSDKP levels in plasma and cerebrospinal fluid (CSF) and robustly reduced infarct volume and neurological deficits, without increasing the incidence of brain hemorrhage compared with ischemic rats treated with saline, AcSDKP alone at 4h, and tPA alone at 4h. Moreover, the combination treatment considerably reduced the density of nuclear transcription factor-κB (NF-κB), transforming growth factorβ (TGFβ), and plasminogen activator inhibitor-1 (PAI-1) positive cerebral blood vessels in the ischemic brain, all of which were associated with reduced microvascular fibrin extravasation and platelet accumulation compared to tPA monotherapy. In vitro, AcSDKP blocked fibrin-elevated TGFβ1, PAI-1, and NF-κB proteins in primary human brain microvascular endothelial cells.

Conclusions

Our data indicate that AcSDKP passes the blood brain barrier (BBB) and that treatment of acute stroke with AcSDKP either alone at 1h or in combination with tPA at 4h of the onset of stroke is effective to reduce ischemic cell damage in a rat model of embolic stroke. Inactivation of TGFβ and NF-κB signaling by AcSDKP in the neurovascular unit may underlie the neuroprotective effect of AcSDKP.

Keywords: stroke, ischemia, vascular permeability, neuroprotection

Introduction

Stroke is a leading cause of death and disability worldwide. However, tissue plasminogen activator (tPA), the only FDA approved treatment for acute stroke is constrained by its narrow therapeutic window and potential adverse side effects of brain hemorrhage1. It becomes increasingly recognized that the perturbation of the neurovascular unit, a structure and functional interdependent microvascular and parenchyma network, following stroke leads to the activation of cascades of pro-inflammatory and pro-thrombotic and events, which hamper the thrombolytic effects of tPA and potentiates neurovascular disruption2, 3. Thus, successful treatment strategies for acute stroke will require novel therapies that maintain cerebral vascular integrity and patency and reduce ischemic neuronal damage2.

N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) is a naturally occurring peptide present in plasma and circulating mononuclear cells of humans and rodents, which is generated from its precursor thymosin-β4 (Tβ4) that contains AcSDKP in N-terminus4. In the circulation, AcSDKP has a 4.5-minute half-life and is hydrolyzed mainly by the N-terminal site of angiotensin-converting enzyme (ACE) at the Asp-Lys peptide bond, and eliminated via glomerular filtration5. ACE inhibitors prevent degradation of endogenous AcSDKP and raise its circulating concentrations approximately 5-fold in healthy subjects5, 6. An increase in AcSDKP level underlies the cardiovascular protective actions of ACE inhibitors without affecting blood pressure in experimental hypertension6, 7. In addition, administration of AcSDKP effectively reduces inflammatory responses associated extracellular matrix accumulation, and exerts anti-fibrotic effects after experimental myocardial infarction and renal injury6, 8. More importantly, clinical trials have shown that treatment with ACE inhibitors significantly reduced the incidence of stroke in individuals who were at high risk for cardiovascular events without apparent reduction of blood pressure9. Experimentally, administration of ACE inhibitors 2h prior to induction of stroke reduced infarct volume in the ischemic rat10. These data imply that an elevation of plasma AcSDKP levels may contribute to the neuroprotective effect of ACE inhibitors on stroke. However, the effect of AcSDKP on acute stroke has not been investigated. In the present study, using a rat model of embolic middle cerebral artery occlusion (MCAO), we examined the neuroprotective effect of AcSDKP on acute stroke. Our data showed that treatment of acute stroke with AcSDKP alone or in combination with tPA substantially reduced neurovascular damage and improved neurological outcome.

Materials and Methods

All experimental procedures were carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of Henry Ford Hospital. All outcome measurements were performed by observers blinded to the treatments.

Animal model

Male Wistar rats weighing 350–400 g (Charles River Laboratories) were subjected to embolic middle MCAO, as previously described (Please see Supplemental Methods for detail)11.

Experimental protocols

To examine the effect of AcSDKP on acute stroke, AcSDKP at a dose of 0.8 mg/kg/day was administered daily for 3 days starting 1, or 4 h after MCAO intra-arterially (IA) for 2h followed by a subcutaneous infusion with an osmotic pump (Alzet model 1003D) for 3 days (n=7/group). The dose of AcSDKP was selected based on our published studies showing that AcSDKP at a dose of 0.8mg/kg/day exerts potent cardiovascular protective actions in experimental hypertension in the rats6. In order to achieve rapid and broad distribution of AcSDKP in the brain, intra-arterial (IA) injection was initially chosen. Ischemic rats subjected to the same volume of saline infusion (n=12) with the identical protocol described above starting 1h after stroke onset were used as a control group. To examine whether combination of AcSDKP with tPA is effective for acute stroke, ischemic rats subjected to AcSDKP (n=13) or identical volume of saline treatment (n=12) with the same infusion method described above were co-treated with tPA (10mg/kg) intravenously (IV) starting 4h after MCAO. To examine whether blockage of AcSDKP with a neutralizing monoclonal antibody specifically against AcSDKP (mAb-Ac) abolishes the effects of the combination treatment with AcSDKP and tPA on stroke, mAb-Ac or a control antibody against rat IgG (0.4mg/kg, Sigma) were given intraperitoneally (I.P) 4 and 48h after MCAO in rats subjected to the combination treatment with AcSDKP and tPA (n=6/group).

AcSDKP enzyme immunoassay (EIA)

Plasma and cerebrospinal fluid (CSF) samples were obtained 24 after initiation of AcSDKP administration. Ac-SDKP levels in plasma and CSF were measured using a commercially available enzyme immunoassay kit accordingly to manufacture’s instruction (Cayman Chemicals).

Behavioral tests

Longa’s five point scale was used for acute assessment of neurological deficit 30 minutes after MCAO12. Rats with a score between 1 and 3 were randomized into treatment groups.

To detect sensorimotor deficits, a battery of behavioral tests including adhesive removal test11, foot-fault test13, and modified neurological severity score (mNSS)14 was performed 1 and 7 days after onset of MCAO by an observer blinded to the treatments (Please see Supplemental Methods for detail).

MRI measurements

MRI measurements were performed using a 7 Tesla Agilent MRI/MRS system (Santa Clara, CA) in rats subjected to tPA monotherapy and the combination treatment of AcSDKP and tPA initiated at 4h after MCAO (n=10/group). T2 weighted image (T2WI) and Diffusion-weighted images (DWI) were performed 1, 72 and 144 hr after MCAO. Areas of ischemic damage were calculated from MRI parameters of T2 map using threshold values of two standard deviations (SD) above the corresponding contralateral non-ischemic hemisphere15.

Histopathological analysis

Rats were sacrificed seven days after MCAO, and infarct volume was measured on hematoxylin and eosin (H&E) stained coronal sections using the microcomputer imaging device system (MCID, Imaging Research Inc), as previously described11. Gross hemorrhage, defined as blood evident to the unaided eye on the H&E stained coronal sections, was evaluated on seven H&E stained coronal sections for each animal. Data are presented as the percentage of gross hemorrhage in each experimental group.

Immunohistochemistry analysis

Additional rats subjected to tPA monotherapy (n=4), the combination treatment with AcSDKP and tPA (n=6), and saline (n=4) at 4h after stroke onset were sacrificed 24 hours after initiation of treatment. The brains were removed and consecutive frozen coronal sections (8 μm) at bregma −0.4 to −1.4 mm were obtained for immunohistochamical analysis (Please see Supplemental Methods for detail).

Primary human cerebral endothelial cell culture

Primary human brain microvascular endothelial cells (HBECs) obtained from Applied Cell Biology Research Institute (Kirkland, WA) were cultured in endothelial cell growth media according to the manufacturer’s protocols. To directly examine whether AcSDKP blocks fibrin induced activated NF-κB (p65)16, plasminogen activator inhibitor 1 (PAI-1), and transforming growth factor β 1 (TGFβ1) expression in cerebral endothelial cells, AcSDKP (1nM) and fibrin (1.5μg/ml; Sigma) were simultaneously added into the cultured HBECs culture for 6h. The HBECs treated with fibrin in the absence of AcSDKP was used as a control. The HBECs were collected at 6h after treatment and the protein level of p65, PAI-1, and TGFβ1 were determined by Western blot analysis.

Statistic analysis

Data are presented as the mean ± SE. One-way ANOVA was used to compare multiple-group values (i.e., measurements of lesion volume, immunohistological data, MRI measurements, and AcSDKP levels). If the main effect of treatment group was significant at p<0.05, then all pair-wise comparisons between treatment groups were tested. Adjustments for multiple comparisons were made using Hochberg’s method. Fisher Exact test was used to test the gross hemorrhagic rates among the groups.

Results

Neuroprotective effect of AcSDKP

Prior to treatment, all ischemic rats exhibited neurological deficits measured by the Longa five point scale (1.9±0.1 for AcSDKP at 1h, 2.0±0.0 for AcSDKP at 4h, 1.9±0.1 for saline; p>0.05). However, ischemic rats treated with AcSDKP starting at 1h, but not 4h exhibited significant (p<0.05) improvement of neurological function measured by the adhesive removal test at days 1 and 7, and by foot-fault and mNSS tests at day 7 compared with ischemic rats treated with saline (Fig. 1). Histopathological analysis revealed that treatment of ischemia with AcSDKP initiated at 1h, but not 4h, significantly (p<0.05) reduced infarct volume compared to the saline treatment 7 days after MCAO (Fig. 1). There was no statistically significant difference in the incidence of gross hemorrhage between saline (1 out 8, 25%) and AcSDKP at 1h (1 out of 7, 28%) groups. These data indicate that AcSDKP treatment initiated at 1h after onset of stroke exerts a neuroprotective effect.

Figure 1. Infarct volume and neurological functional outcome.

Figure 1

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Panel A shows the effects of AcSDKP alone and in combination with tPA on infarct volume assessed 7 days after MCAO. Panel B, C, and D show the neurological functional outcome measured by neurological severity score, foot-fault test, and adhesive removal test at 1 (black bar) and 7 (open bar) days after stroke onset, respectively.

Using this model, we previously demonstrated that tPA administered 4h after MCAO did not have the neuroprotective effect11. We thus examined whether AcSDKP in combination with tPA can reduce ischemic neuronal damage. Consistent with our previous findings, monotherapy of tPA at 4h did not significantly improve and reduce neurological outcome and infarct volume, respectively, 7 days after MCAO. However, the combination therapy considerably improved neurological outcome and substantially reduced infarct volume compared to monotherapy of AcSDKP and tPA initiated at 4h and saline groups (Fig. 1). The combination therapy did not significantly increase the incidence of gross hemorrhage (1 out of 7, 14%) compared to AcSDKP alone (1 out of 7, 14%) and tPA alone (3 out 8, 38%).

To examine the specificity of AcSDKP therapeutic effect, a neutralizing antibody against AcSDKP was administered along with AcSDKP and tPA 4h after MCAO. The behavioral benefits of the combination treatment of AcSDKP and tPA were completely abolished by this neutralizing antibody compared to ischemic rats treated with a control antibody against rat IgG (Fig. 1). These data suggest that AcSDKP likely contributes to the observed therapeutic effect of the combination treatment.

MRI measurements were performed to non-invasively assess evolution of the of ischemic lesion in rats treated with the combination therapy of AcSDKP and tPA or monotherapy of tPA, starting at 4h of MCAO. MRI analysis revealed that at 1h of MCAO, all rats exhibited ischemic lesion within the territory supplied by the MCA as measured by DWI (Fig. 2). Ischemic lesion in rat treated with tPA alone increased over 144h (Fig. 2). In contrast, ischemic rats treated with the combination of AcSDKP and tPA yielded a significantly reduced lesion over the same period compared to tPA alone (Fig. 2). Together, these data indicate that the combination therapy has the neuroprotective effect on acute stroke.

Figure 2. MRI measurement.

Figure 2

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Panels A and B show T2 weighted MRI at 1, 72and 144 h after MCAO of a representative rat treated with tPA (A) and the combination of AcSDKP and tPA (B) 4 h after stroke. Panels C and D are H&E stained sections obtained from the same representative rats 7 days after MCAO, respectively. Bar graph shows quantitative data of T2 values.

To examine whether AcSDKP passes the BBB, we measured AcSDKP levels in plasma and CSF by means of EIA. Normal or ischemic rats treated with AcSDKP alone, or the combination of AcSDKP and tPA at 4h after MCAO exhibited significant elevation of AcSDKP levels in plasma and CSF 24h after the initiated treatment (Fig. 3). The neutralizing antibody against AcSDKP completely suppressed augmentation of AcSDKP levels in rats subjected to the combination treatment (Fig. 3). These data indicate that AcSDKP can pass the BBB.

Figure 3. Plasma and CSF AcSDKP level.

Figure 3

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Bar graph shows plasma and CSF AcSDKP level measured by EIA in normal and ischemic rats 24h after initiation of treatment.

Effects of treatments on thrombosis and vascular permeability

In addition to its thrombolytic effect, tPA induces an impairment of the BBB, especially when tPA is given beyond a therapeutic window11, 17. We thus, examined the effect of the adjuvant treatment with AcSDKP and tPA on BBB integrity and cerebral microvascular patency. Monotherapy of tPA 4h after MCAO significantly increased thrombosis in cerebral microvessels and BBB leakage measured by microvascular platelet accumulation and parenchymal fibrin deposition, respectively, compared to the saline treatment (Fig. 4). However, the adjuvant treatment with AcSDKP completely blocked microvascular thrombosis and BBB leakage induced by tPA and stroke (Fig. 4). These data suggest that improvement of microvascular patency and integrity by AcSDKP likely results in reduction of infarct volume and consequently leads to a decrease of neurological deficits.

Figure 4. Fibrin and platelet accumulation.

Figure 4

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Panel A shows immunoreactivity of fibrin (red) and EBA (green) in representative rats treated with tPA monotherapy and the combination of AcSDKP and tPA. Bar graph B shows the quantitative data of extravascular fibrin leakage in the ischemic brain. Panel C show thrombocyte immunoreactivity (green) in EBA immunoreactive vessels (red) in the representative rats treated with tPA monotherapy and the combination of AcSDKP and tPA. Panel D shows the quantitative data of thrombocyte immunoreactive vessels in the ischemic brain. Bars=100 μm.

Effects of treatments on TGF-β1, NF-κB, and PAI-1 expression

We previously demonstrated that AcSDKP inactivated the TGF-β1/pSmad 2 signaling in vascular fibrosis, observed in the experimental hypertension and myocardial infarction7, 18. Our immunohistochemistry data showed that stroke induced TGF-β and p-Smad2/3 immunoreactive vessels 24h after MCAO (Fig. 5). Compared to the saline treatment, the combination of AcSDKP and tPA significantly reduced the densities of TGF-β1 and p-Smads2/3 immunoreactive vessels, whereas tPA monotherapy failed to reduce the densities of TGF-β1 and p-Smads2/3 immunoreactive vessels (Fig. 5). In addition, the combination treatment resulted in a significant decrease in TGF-β1 immunoreactive vessels, and a trend towards reduction in p-Smads2/3 immunoreactive vessels compared with the tPA monotherapy (Fig. 5).

Figure 5. Cerebrovascular TGFβ1, p65, and PAI-1 expression.

Figure 5

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Panel A shows immunoreactivity of TGFβ1 in a representative normal, ischemic rats treated with tPA monotherapy, and ischemic rats treated with the combination of AcSDKP and tPA. Panel B shows colocalization of pSmad2/3 (red) with TGFβ1 immunoreactive vessels (green) in a representative ischemic rat treated with saline. Panel C shows double immunofluorescent staining of PAI-1 (green) with EBA (red) from a representative normal rat, and in ischemic rats treated with saline, tPA, or AcSDKP+tPA. Panel D shows p65 immunoreactivities from a representative normal rat, and in ischemic rats treated with saline, tPA, or AcSDKP+tPA. Bar graphs show the quantitative data of TGFβ1, pSmad2/3, PAI-1, and p65 immunoreactive vessels in the ischemic brain, respectively. Bars=50 μm.

Activation of TGF-β1 upregulates PAI-119. Double immunostaining showed that stroke considerably induced PAI-1 immunoreactive vessels, and monotherapy with tPA further increased the PAI-1 immunoreactive vessel density compared with saline treated rats 24h after MCAO (Fig. 5). However, adjuvant treatment with AcSDKP completely restrained the densities of PAI-1 immunoreactive vessels evoked by stroke and tPA (Fig. 5).

NF-κB modulates cerebral vascular patency and integrity20. We found that stroke substantially induced the active form of NF-κB, p65 immunoreactivity in vessels within the ipsilateral hemisphere, and monotherapy of tPA augmented p65 immunoreactive vessels 24h after MCAO, which was completely suppressed by the combination therapy of AcSDKP and tPA (Fig. 5). Collectively, these in vivo data suggest that AcSDKP suppresses TGF-β1/pSmad2/3 and NF-κB signals in cerebral vessels triggered by stroke and tPA.

To examine whether AcSDKP acts directly on cerebral endothelial cells, HBECs were treated with fibrin in the presence or absence of AcSDKP. Western blot analysis showed that incubation of cerebral endothelial cells with fibrin for 6h considerably increased levels of TGF-β1, PAI-1, and p65, while AcSDKP substantially blocked fibrin-increased TGF-β1, PAI-1, and p65 (Fig. 6). These data suggest that AcSDKP can suppress fibrin-evoked expression of TGF-β1, PAI-1, and p65 in cerebral endothelial cells, which is consistent with our in vivo findings.

Figure 6. Effects of AcSDKP on cerebral endothelial cells.

Figure 6

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Panel A shows the representative Western blots of TGFβ1, PAI-1, and p65 in HBECs cultured with fibrin in the presence or absence of AcSDKP. Bar graphs show the quantitative data of TGFβ1(B), PAI-1(C), and p65(E) protein levels on HBECs, respectively. *p<0.05 vs Control. +p<0.05 vs Fibrin.

Discussion

The present study, for the first time, demonstrates that administration of AcSDKP initiated 1h after MCAO or combination of AcSDKP and tPA given 4h after stroke onset substantially reduced infarct volume and neurological deficits. Improved cerebral microvascular patency and integrity by AcSDKP likely contribute to the observed neuroprotective effect.

Treatment of acute stroke requires rapid restitution of cerebral blood flow (CBF) in the ischemic cerebral microvascular bed, preserving BBB integrity, and minimizing ischemic cell death1. Thrombolysis with tPA restores blood flow through its desirable fibrinolytic action. However, delayed tPA treatment may adversely exacerbate stroke induced neurovascular dysfunction via aggravating the prothrombotic cascade, inflammatory response, and BBB disruption2, 3. The present study demonstrated that AcSDKP potently blocked secondary thrombus formation and BBB leakage, which leads to neuroprotective effects when treatment is initiated acutely (1h) after stroke onset. More importantly, we found that adjuvant treatment with AcSDKP completely blocked tPA aggravated secondary thrombus formation and BBB leakage, and concomitantly blocked the lesion expansion in the ischemic rats. Elevation of plasma AcSDKP level has been observed in patients treated with ACE inhibitors, which is well known to maintain the hemostatic balance of fibrinolytic and procoagulant factors, and to attenuate inflammatory and fibrotic responses in patients with cardiovascular disease and stroke21, 22. Collectively, these data suggest that AcSDKP has therapeutic potential for treatment of patients with acute stroke.

Following ischemic insult, the cerebral endothelial cells rapidly converted into a prothrombotic and proinflammatory state, which leads to a neurovascular dysfunction that aggravates the progression of ischemic brain damage3. In the present study, we found that stroke induced neurovascular disruption was associated with upregulation of cerebrovascular TGFβ1, PAI-1, and NF-κB expression, all of which play important roles in regulating thrombosis and inflammatory responses after stroke. TGFβ1 is a multipotent cytokine involved in cell growth, matrix protein synthesis, and inflammation. The activation of TGFβ1 induces the expression of PAI-1, a major inhibitor of fibrinolysis derived from endothelial cells, and modulates vascular barrier function via promoting matrix metalloprotease production19. In addition, the activation of NF-κB signaling evokes the transcription of proinflammatory genes, which has a direct effect on modulating neurovascular homeostasis after stroke20. Thus, the activation of cerebrovascular TGFβ1 and NF-κB signaling by stroke likely trigger secondary thrombosis, inflammation, and BBB disruption. In the present study, we found that administration of AcSDKP substantially reduced cerebrovascular TGFβ1 and NF-κB signals elicited by stroke and tPA, which were closely associated with decreases of thrombosis and BBB leakage. Moreover, our in vitro data indicate that AcSDKP suppresses TGFβ1 and NF-κB signals in cerebral endothelial cells, which are consistent with published studies showing that AcSDKP treatment protects against renal and cardiac damage via inhibiting TGFβ1 and NF-κB mediated inflammatory responses and fibrosis23. Therefore, suppression of cerebrovascular TGFβ1 and NF-κB signals by AcSDKP may be one of the mechanisms underlying the neuroprotective effects of AcSDKP on acute stroke.

Alternatively, AcSDKP may confer the neuroprotection by directly acting on parenchymal neural cells. In the present study, we found that the CSF and plasma AcSDKP remain at basal levels 24h after stroke onset, suggesting that stroke does not affect endogenous AcSDKP level. However, systemic administration of AcSDKP was accompanied with the elevation of CSF AcSDKP level in the ischemic rats, indicating that AcSDKP can readily cross the BBB. Although the biological role of endogenous and exogenous AcSDKP in the CNS is not clear, neuroprotective and neurorestorative activities of Tβ4, the putative precursor of AcSDKP, have been previously reported24, 25. In vitro, the application of Tβ4 protects against excitotoxicity-induced neuronal death24. In experimental stroke and traumatic brain injury (TBI), Tβ4 treatment leads to neuroprotection, neurorestoration, and improvement of neurological functional recovery25. These findings raise a possibility that AcSDKP acts on multiple cellular targets within the neurovascular unit, and subsequently exerts beneficial effects in the treatment of experimental stroke. Additional studies are warranted to investigate the effects of exogenous AcSDKP on neuronal damage and the biological functions of endogenous AcSDKP in the brain.

Several characteristics of AcSDKP indicate that this tetrapeptide is a promising neurovascular protective agent for ischemic stroke, and therefore merits further preclinical development and evaluation. Firstly, as a naturally occurring peptide, the pharmacokinetics and metabolism of Ac-SDKP have been well established and there is no apparent toxicity in rodents. Secondly, while the BBB poses significant challenges to the permeation of neuroprotective agents, AcSDKP can readily cross the BBB in the intact and ischemic brain. More importantly, with the identification of multiple molecular mechanisms and mediators on the pathogenesis of stroke, the multi-targeted effects of AcSDKP on the neurovascular unit could achieve optimized therapeutic efficacy. Furthermore, with increased utilization of thrombolytic therapy in patients with acute stroke, the safety and efficacy of the combination treatment observed in the present study should encourage further efforts to develop clinical trials of AcSDKP in combination with tPA for the treatment of acute stroke.

Supplementary Material

Supplemental material

Acknowledgments

Funding Sources: This work was supported by the National Institutes of Health grants, RO1 NS 079612 (ZGZ), RO1 AG037506 (MC), and RO1 NS62832 (LZ).

The authors wish to thank Cynthia Roberts, Yisheng Cui, Min Wei, Qing-e Lu, Sutapa Santra, and Gulser Gurocak for technical assistance.

Footnotes

Disclosures: None.

The content is solely the responsibility of the authors and does not necessarily represent the official view of the National Institutes of Health.

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NIHMS561226-supplement-Supplemental_material.pdf (14.2KB, pdf)

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