Injectable VEGF Hydrogels Produce Near Complete ... - IngentaConnect

0963-6897/10 $90.00 + .00 DOI: 10.3727/096368910X498278 E-ISSN 1555-3892 www.cognizantcommunication.com

Cell Transplantation, Vol. 19, pp. 1063–1071, 2010 Printed in the USA. All rights reserved. Copyright  2010 Cognizant Comm. Corp.

Injectable VEGF Hydrogels Produce Near Complete Neurological and Anatomical Protection Following Cerebral Ischemia in Rats Dwaine F. Emerich,* Eduardo Silva,† Omar Ali,* David Mooney,‡ William Bell,* Seong Jin Yu,§ Yuji Kaneko,§ and Cesar Borlongan§ *InCytu, Inc., Lincoln, RI, USA †Wyss Institute for Biologically Inspired Engineering, Cambridge, MA, USA ‡School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, USA §Department of Neurosurgery and Brain Repair, University of South Florida, Tampa, FL, USA

Vascular endothelial growth factor (VEGF) is a potent proangiogenic peptide and its administration has been considered as a potential neuroprotective strategy following cerebral stroke. Because VEGF has a short halflife and limited access to the brain parenchyma following systemic administration, approaches are being developed to deliver it directly to the site of infarction. In the present study, VEGF was incorporated into a sustained release hydrogel delivery system to examine its potential benefits in a rat model of cerebral ischemia. The hydrogel loaded with VEGF (1 µg) was stereotaxically injected into the striatum of adult rats 15 min prior to a 1-h occlusion of the middle cerebral artery. Two days after surgery, animals were tested for motor function using the elevated bias swing test (EBST) and Bederson neurological battery. Control animals received either stroke alone, stroke plus injections of a blank gel, or a single bolus injection of VEGF (1 µg). Behavioral testing confirmed that the MCA occlusion resulted in significant deficits in the the EBST and Bederson tests. In contrast, the performance of animals receiving VEGF gels was significantly improved relative to controls, with only modest impairments observed. Cerebral infarction analyzed using 2,3,5-triphenyl-tetrazolium chloride staining confirmed that the VEGF gels significantly and potently reduced the lesion volume. No neurological or histological benefits were conferred by either blank gel or bolus VEGF injections. These data demonstrate that VEGF, delivered from a hydrogel directly to the brain, can induce significant functional and structural protection from ischemic damage in a rat model of stroke. Key words: Alginate; Stroke; Vascular endothelial growth factor (VEGF); Hydrogel; Neuroprotection; Transplantation

INTRODUCTION

creasingly clear that VEGF has potent effects on nonvascular, neuronal cells in the central nervous system (CNS) (18,25,26). VEGF and its receptors are located on neurons and astrocytes (18); VEGF induces neuritic growth ( 13) and is neuroprotective in models of numerous CNS diseases (16,19,20,28,29,31–33,38,40), including cerebral ischemia (5,14,27,37,43). Although VEGF appears promising in animal models of brain disease such as stroke, its use has been hampered by a short half-life and poor penetration across the blood–brain barrier when administered systemically (25). Direct intracerebral bolus injections of VEGF overcome these limitations but are impractical because repeated local injections into the ischemic region are needed to maintain elevated tissue levels for prolonged periods of time and this approach could cause further

Stroke is the third leading cause of death and a significant health care burden in developed countries (15). Although the incidence of stroke has declined over the past decades, there are no effective treatments for mitigating the neuronal loss after stroke (35). The ideal therapy would be one that significantly minimized or prevented the loss of neurons and associated neurological impairments (3,6,17,36). Delivery of neurotrophic factors such as vascular endothelial growth factor (VEGF) to the site of injury is one approach under investigation. VEGF is a secreted dimeric polypeptide that was traditionally believed to act as a specific mitogen for endothelial cells subserving angiogenesis and permeability in development and after injury (8). However, it is becoming in-

Received November 6, 2009; final acceptance March 26, 2010. Online prepub date: April 21, 2010. Address correspondence to Dwaine F. Emerich, Ph.D., InCytu, Inc., 701 George Washington Highway, Lincoln, RI 02865, USA. Tel: 401-4996662; E-mail: [email protected]

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mechanical injury. Other approaches under investigation, such as direct gene transfer or implantation of VEGF-secreting cells, are promising ways to maintain VEGF levels for long periods of time (perhaps permanently) but do not allow control over the duration of exposure or permit dose adjustments over time (21,43). The use of injectable hydrogels to deliver VEGF, and other potentially therapeutic molecules to the brain, might satisfy many of the characteristics of an ideal delivery system, including sustained delivery for days to weeks, controllable spatial distribution and dosage, and the ability to deliver VEGF directly to the site of damage. We previously reported on the benefits of VEGF delivered via a sustained release hydrogel delivery system following excitotoxic striatal lesions in rats (7). The VEGF-containing hydrogel was stereotaxically injected into the striatum and significantly ameliorated the neuronal loss and behavioral deficits that normally occur in that model. Here we used the same injectable hydrogel (alginate)-based sustained release delivery system to evaluate the effects of intrastriatal VEGF on the anatomical and functional consequences of transient occlusion of the middle cerebral artery (MCA) in rats. VEGF prevented both the neurodegeneration and behavioral impairments in this model of cerebral ischemia, providing the first demonstration that an injectable hydrogel-based system can be developed to deliver VEGF in therapeutic quantities sufficient to prevent the degeneration of neurons following cerebral ischemia. MATERIALS AND METHODS Animals All procedures adhered to NIH and Society for Neuroscience guidelines for use of animals in research. Ninety-nine adult male Sprague-Dawley rats (Harlen, Inc.) approximately 2–3 months of age and weighing 250–300 g were used. Animals were housed in a temperature (22 ± 1°C) and humidity (50 ± 5%)-controlled environment with free access to food and water, except for 24 h prior to surgery. Surgical procedures were conducted under aseptic conditions and all efforts were made to minimize animal suffering and to reduce the number of animals used. VEGF Gel Delivery System Gels capable of localized and sustained VEGF delivery were prepared as previously described (22). In brief, partially oxidized low (50 kDa) and high (250 kDa) molecular weight (MW) ultrapure MVG alginates (Pronova, Norway) were reconstituted in EBM-2 (Cambrex) to obtain a 2% w/v solution (75% LMW; 25% HMW used in all experiments) prior to gelation. Alginates solutions were first mixed with lyophilized recombinant

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human VEGF165 protein (R&D Systems, USA) by using two syringes coupled by a syringe connector, and the calcium slurry (25:1 ratio of alginate solution to saturated calcium sulfate solution) (Sigma, USA) was then mixed with the resulting alginate/VEGF solution. This process resulted in an alginate scaffold that provided 1 µg of VEGF when injected into the striatum in a 5 µl volume. Non-VEGF-containing gels were formulated directly as 2% w/v solutions. Hydrogels were allowed to cure at room temperature for at least 30 min and then was maintained at 4°C prior to animal injections. These gels continuously release bioactive VEGF for over 2 weeks (10,22). Surgery Rats were anesthetized with isoflurane (3–4%) and positioned in a stereotaxic instrument (Kopf, Tujunga, CA). A midline incision was made in the scalp and a hole drilled for injection of the VEGF-containing hydrogel into the striatum. The stereotaxic coordinates for implantation were: 0.5 mm anterior to bregma, 2.5 mm lateral to the sagittal suture, and 5.5 mm below the cortical surface. Five microliters of VEGF alone (in PBS) or VEGF-loaded alginate (total VEGF dose of 1 µg in each case) was injected into the striatum using a 10-µl Hamilton syringe mounted to a stereotaxic frame. Control animals received either no injections or identical injections of alginate that were not previously loaded with VEGF. Following implantation, the skin was sutured closed. Within 15 min, each animal received 60 min of transient unilateral focal ischemia produced using a well-established MCA occlusion model (1,2). For the occlusion, a midline neck incision was made and the right common carotid artery (CA) was exposed. This artery and the right external CA were ligated, and the right MCA was occluded by the insertion of a 4-0 nylon thread (17 mm long) with silicon coating through the common CA. The surgical incision was then closed and the animals were allowed to recover at an ambient temperature. After 60 min of occlusion, the thread was removed and regional cerebral blood flow (CBF) was restored. Based on our extensive experience with the MCA occlusion model, physiological parameters including blood gases of animals undergoing such surgical procedure remain within normal limits. These procedures resulted in the formation of four experimental groups: 1) stroke only (N = 8), 2) stroke + blank gel (N = 8), 3) stroke + VEGF gel (N = 9), and 4) stroke + VEGF bolus (N = 10). As previously shown in our studies (1,2), regional CBF as revealed by laser Doppler and examination of routine physiological variables (PaO2, PaCO2, and plasma pH) revealed that all animals subjected to the present MCA occlusion surgery exhibited about an 80% reduction in CBF and did not show any significant dif-

VEGF GELS EXERT NEUROPROTECTION AGAINST STROKE

ferences in the physiological parameters examined. A heating pad and a rectal thermometer allowed maintenance of body temperature at normal limits. Mean body weights of stroke animals did not significantly differ across treatment groups over the poststroke period. Behavioral Testing: Motor Asymmetry Two days after surgery, the elevated body swing test (EBST) was used to evaluate the functional consequences of the MCA occlusion and to quantify improvements in motor function produced by VEGF. The EBST reliably detects stable motor asymmetry at this early time point (1,2). For the EBST, each rat was elevated by its tail and the direction of swings made by the animal was recorded. After a single test, the animal was lowered and allowed to move freely for 30 s prior to retesting. These steps were repeated 20 times for each animal. Healthy intact rats exhibit a 50% swing bias; that is, the same number of swings to the left and right are observed. In ischemic animals a greater than 75% biased swing activity is observed. Behavioral Testing: Neurological Evaluation Animals were tested for neurological function using the Bederson test battery immediately following the EBST. Using previously described methods, a neurologic score for each rat was obtained using three tests: 1) contralateral hindlimb retraction measuring replacement of the hindlimb after it was displaced laterally by 2–3 cm, graded from 0 (immediate replacement) to 3 (replacement after minutes or no replacement); 2) beam walking ability graded 0 for a rat that readily traversed a 2.5-cm-wide, 80-cm-long beam to 3 for a rat unable to stay on the beam for 10 s; and 3) bilateral forepaw grasp measuring the ability to hold onto a 2-cm diameter wooden rod, graded 0 for normal forepaw grasping behavior to 3 for a rat unable to grasp with the forepaws. The three tests were conducted over approximately 15 min and the scores were combined to give an average neurologic deficit score.

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performed using ice baths. The tissue sample and 1.0 ml of T-PER were transferred to a Dounce homogenizer. PBS (1.0 ml; pH 7.0) was added to the vial then transferred to the homogenizer to recover any residual sample remaining in the vial. The sample was homogenized on ice without foaming and the sample was transferred to a 2.0-ml cryovial and maintained on ice. The VEGF in the samples was quantified using a commercially available ELISA (R&D Systems, Minneapolis, MN). Samples were diluted in the buffer supplied with the ELISA according to the manufacturer’s instructions. The contralateral striatum was processed identically and used as a control tissue. Additional controls consisted of spiking 1 µg of a VEGF bolus or VEGF gel into a striatal homogenate prepared as described above (Ns = 4). The final data were expressed relative to controls in which VEGF (1 µg in a gel or as a bolus) was spiked into a striatal homogenate processed identically to tissues removed from treated brains. Histology After completion of behavioral testing, animals were anesthetized using CO2 and transcardially perfused with 100 ml of ice-cold saline. The brain tissue was removed, immersed in cold saline for 5 min, and sliced into sections 2 mm thick. These slices were incubated in 2% 2,3,5-triphenyl-tetrazolium chloride (TTC; Nacalai Tesque, Inc., Kyoto, Japan), immersed in saline for 30 min at 37°C, and transferred to a 10% formalin solution for fixation. The TTC-stained brain slices were photographed using a digital camera, and the volume of any infarct was calculated as described previously (1,2). The volume of infarction in each animal was calculated from the product of the average slice thickness and the sum of infarction areas in all brain slices that were examined. To minimize artifacts produced by postischemic edema in the infarcted area, the infarct volume was calculated using a routine technique with the infarction area in the ipsilateral hemisphere indirectly measured by subtracting the noninfarcted area in the ipsilateral hemisphere from the total intact area of the contralateral hemisphere.

Quantification of VEGF Levels in Brain Separate groups of normal (nonstroke) gender and age-matched animals were used to determine the levels of VEGF in the striatum following injections of either a VEGF bolus or the VEGF-loaded gel as described above. The injected striatum was removed at 5 min, 1, 2, 4, or 8 h after a bolus injection and 5 min, 1, 8, 24 h, and 3, 7, 14, and 21 days following injection of the VEGF hydrogel (Ns = 4/time point). Excised striata were placed in 2-ml cryovials containing 1.0 ml of Tissue Protein Extraction Reagent (T-PER, Thermo Scientific, Rockford, IL). The vials were frozen immediately and stored at −80°C until analysis. All processing was

RESULTS Behavioral Testing: Injections of VEGF-Releasing Hydrogels Reduce Stroke-Induced Motor Deficits Statistical analysis using a one-way ANOVA revealed a significant benefit of VEGF on the EBST (p < 0.0001). Whereas the performance of animals receiving intrastriatal injections of blank gels was indistinguishable from the stroke-only controls, Bonferroni post hoc t-tests confirmed that VEGF gels significantly ameliorated (p < 0.0001) motor asymmetry (Fig. 1) relative to animals receiving stroke alone (45%) or injections of a blank alginate gel (42%). The resulting average motor

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Figure 1. A VEGF hydrogel delivery system reduces motor asymmetry as assessed using the elevated bias swing test. Animals were tested 2 days following surgery. Data are presented as the mean ± SD number of ipsilateral swings during 20 trials. *p < 0.0001 versus animals receiving stroke only and injections of blank gel. Note that no benefits were observed following a single bolus injection of VEGF.

asymmetries in animals treated with the VEGF gel were less than the conventionally accepted 75% (15/20 biased swings) criterion for stroke rats to be considered significantly impaired. Those animals receiving bolus injections of VEGF were not significantly improved on this task, as these animals exhibited a robust bias in swing activity that was comparable to that seen in controls. Performance on the Bederson neurological battery paralleled that seen on the EBST with controls exhibiting pronounced deficits in performance (Fig. 2). Consistent with the EBST, prior treatment with the VEGF gel significantly normalized performance (p < 0.0001). Relative to animals receiving stroke alone or stroke plus blank gel injections, performance of the VEGF geltreated animals was improved more than fourfold. Again, treatment with a bolus injection of VEGF did not impact performance (ps > 0.45) as these animals were indistinguishable from controls (Fig. 2). Of note, neurological ratings of ipsilateral forepaw grasping and hindlimb retraction were 80% vs. controls). In contrast, no statistically significant effect on lesion size was observed in animals subjected to either stroke alone or stroke plus blank gels. Similar to the motor and neurological test results, a bolus injection of VEGF did not impact the extent of the lesion relative to controls. Quantification of VEGF Levels in Brain Consistent with previous reports (10,22), the alginate gels provided a prolonged delivery of VEGF to the rat striatum. VEGF delivered as a bolus injection exhibited a half-life of approximately 90 min and was undetectable within 4–8 h. In contrast, VEGF levels remained high for 1–3 days and were readily detectable at 7–14 days after injection (Fig. 5). VEGF was never detectable in nontreated brain tissue. DISCUSSION This study provides the first demonstration that implants of a sustained release hydrogel system delivering VEGF can prevent anatomical and behavioral consequences of transient cerebral ischemia. Based on quantification of lesion volume in TTC-stained brain sections, the size of the lesion in VEGF gel-treated animals was reduced >80% relative to controls. Behavioral analysis using the EBST and Bederson neurological battery confirmed the potent beneficial effects of sustained delivery of VEGF. The 1-h MCA occlusion produced marked


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Figure 2. A VEGF hydrogel delivery system reduces motor asymmetry as assessed using the Bederson neurological test battery. Animals were tested 2 days following surgery for paw grapsing, forelimb akinesia, and beam walking (see text for details). Data are presented as the mean ± SD of the three tests combined. *p < 0.0001 versus animals receiving stroke only and injections of blank gel. Note that no benefits were observed following a single bolus injection of VEGF.

deficits that were comparable in magnitude between animals receiving a stroke only and a stroke plus a blank hydrogel implant. In contrast, those animals receiving VEGF gels were significantly improved on all tests with performance approaching that of normal animals. Inter-

estingly, the benefits of VEGF were observed only using the sustained release gel as a single high-dose bolus injection of VEGF was ineffective at reducing lesion size or behavioral deficits. The importance of the method of delivering VEGF has been pointed out previously (25)

Figure 3. Histogram demonstrating the neuroprotective effects of a VEGF hydogel implanted into the striatum prior to MCA occlusion. Data are presented as the mean ± SD of infarct volume determined using 2,3,5-triphenyl-tetrazolium chloride staining. *p < 0.0001 versus animals receiving stroke only and injections of blank gel. As seen with the behavioral analyses, no benefits were observed following a single bolus injection of VEGF.

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as high-dose intravenous infusion of VEGF immediately after the onset of focal cerebral embolic ischemia induces leakage of the blood–brain barrier with hemorrhagic transformation of the ischemic lesions (42). However, both intraventricular infusion and topical application of VEGF on the cerebral cortex reduce edema formation and infarct volume (11,12). The lack of benefit afforded by bolus VEGF in the present study might be related to the very high dose used because previous reports have highlighted the critical role that proper dosage plays in protecting neurons from trauma (14,41). The difficulties in maintaining proper dosage following bolus injections is further complicated by the short half-life of VEGF. Both issues alone or combined can be addressed to a significant degree with the hydrogel delivery system used in the present studies. The potent reduction in the lesion normally produced by MCA occlusion supports the possibility that VEGF might be of benefit in diseases such as cerebral ischemia. Although VEGF has long been suggested as a therapeutic for stroke, its potential is limited by its short

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half-life and poor penetration into the desired brain region following systemic administration (25). Intuitively, an ideal VEGF delivery system would provide elevated, dose-controlled delivery to the site of infarction for a relatively short time frame following a single administration. While other approaches such as repeated direct bolus injections, gene transfer, cell transplantation, etc., address some of these issues, each also suffers from one or more potential drawbacks. On the other hand, the use of injectable hydrogels might satisfy many of the characteristics of an ideal delivery system including sustained, dose-controlled delivery for day to weeks, controllable drug distribution, controllable dosage delivered, and ability to deliver the VEGF directly to the site of injury (5,7,10,22). In addition, hydrogels such as the ones used in these studies have known and controllable degradation profiles and excellent biocompatibility (4, 30). While we did not confirm the in vivo degradation profile of the alginate gel in the current studies, previous studies have directly addressed this issue. In a rat model of Huntington’s disease we implanted this exact same

Figure 4. Photomicrograph of 2,3,5-triphenyl-tetrazolium chloride-stained sections illustrating the protective effects of prior injection of a VEGF-loaded alginate gel.


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Figure 5. Time course of the levels of VEGF in the striatum following injection (1 µg total) of either a sustained release alginate gel (solid line) or a single bolus injection of VEGF (1 dashed line). At various times postinjection the injected striatum was removed and levels of VEGF were quantified by ELISA. Data are expressed relative to controls in which VEGF (1 µg in a gel or as a bolus) was spiked into a striatal homogenate treated identically to tissues removed from treated brains. Note that while the VEGF levels rapidly dissipate following a bolus injection, VEGFloaded gel provides a sustained and elevated level of VEGF for several days. Data are expressed as mean ± SD.

VEGF-loaded gel into the striatum (7). When the tissue was examined histologically 17 days later no residual gel was observed within the implant site. These data are also similar to those obtained when implanting this gel system intramyocardially along the border of the myocardial infarction in rats (10). In those studies, the gels exhibited a rapid degradation in vitro; within 7 days, approximately 60% of the initial gel mass was lost and only small gel fragments could be occasionally observed in tissue sections of the myocardium 2–4 weeks after injection. The clearance of the gels from multiple tissue types including the brain is consistent with its anticipated hydrolytic degradation rate. The angiogenic effects of VEGF have been well documented, but its role in neuroprotection is also becoming increasingly clear. Exogeneous administration of VEGF provides anatomical and behavioral protection in animal models of ALS, PD, stroke, and acute spinal cord injury. While these benefits are becoming more and more widely reported, the mechanism(s) underlying the neuroprotective effects of VEGF in the present and other studies is unclear and underinvestigated. In vitro studies have suggested that VEGF is capable of producing direct protective effects on cultured cerebellar neurons (34), serum-deprived endothelial cells (9), peripheral nerve grafts (24), and neurons exposed to hypoxic con-

ditions (13). These effects are likely mediated by interactions with VEGF receptors including flt-1 that have been previously suggested to underlie the direct neuroprotective effects of VEGF (9,39). Alternatively, many of the neuroprotective effects of VEGF may result from indirect mechanisms. VEGF has marked proliferative effects on astrocytes and glial cells. Silverman and colleagues (23) reported that VEGF promoted the growth/survival of neurons in a manner that was largely dependent on the presence of astroglial cells. VEGF has also been reported to act as a neuronal maturation factor in vitro (23) and might locally induce a proliferation of local glial cells that release trophic factors such as GDNF, BDNF, and FGF2 to further augment the effects of VEGF itself (39). Understanding whether these factors alone, or in combination, account for the effects observed in present studies and across other animal models is a fertile area of future research. Future studies should also carefully consider the complex interplay between the dose of VEGF, timing of delivery, and its spatial presentation. For instance, this gel is capable of inducing significant angiogenesis, alleviating ischemia, and preventing necrosis in a hindlimb ischemia model in mice in a manner dependent on the release and distribution of VEGF (22). Mice were submitted to ischemic hindlimb surgery, and immedi-

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ately injected with either a bolus of VEGF or the VEGF hydrogels used currently. During the first 24 h after injection, higher concentrations of VEGF in the injection region were found when VEGF was delivered from the alginate, compared with a bolus injection. At 12 h after injection, 95% of the VEGF dose in the gels was still localized in the region closest to the injection site, whereas VEGF delivered via bolus injection was much more widely dispersed. Bolus VEGF injection led to small amounts of bioactive VEGF at the injection site at 24 h, and complete VEGF loss occurred with 3 days. Physiologic levels of VEGF were still present in the ischemic hindlimbs 15 days after delivery with the alginate. Confirmation of this pattern of distribution in the CNS would be worthwhile as would studies using sustained release and/or bolus injections of VEGF to more carefully tease out the essential timing and dosage of VEGF needed poststroke for optimal effects. In conclusion, we report here that implants of alginate hydrogels providing sustained delivery of VEGF prevents the infarction and neurological deficits that typically occur following MCA occlusion. Based on these data, further studies are warranted to evaluate the use of sustained release VEGF systems across a range of CNS diseases during and after neuronal degeneration. Regarding cerebral ischemia, future studies should rigorously evaluate the magnitude and persistence of its benefits across both motor and cognitive domains as well as develop an understanding of the therapeutic window of opportunity after cerebral ischemia. CNS implants of VEGF, together with a refined understanding of its underlying mechanism of protection, might be of benefit because of its potent neuroprotective and symptomatic benefits. A final fruitful avenue of research might be to use the present hydrogel-based system to induce focal angiogenesis in ischemic brain tissue. Because controlling VEGF expression and maintaining a certain level of VEGF in the ischemic region appear to be crucial for focal angiogenesis, the current system might be uniquely suited for such efforts. CONCLUSIONS We have demonstrated that intracerebral implantation of an alginate provides therapeutic sustained delivery of VEGF in a rodent model of stroke. The VEGF-secreting gels provided nearly complete anatomical and neurological protection, suggesting that this unique approach might have significant clinical potential for stroke and other acute CNS diseases. REFERENCES 1. Borlongan, C. V.; Hadman, M.; Sanberg, C. D.; Sanberg, P. R. Central nervous system entry of peripherally injected

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