Research Article Comparison of Neuroprotective...

Research ArticleComparison of Neuroprotective Effect of Bevacizumab andSildenafil following Induction of Stroke in a Mouse Model

Ivan Novitzky1 Neelan J Marianayagam12 Shirel Weiss23 Orkun Muhsinoglu4

Moran Fridman23 Tamar Azrad Leibovitch23

Nitza Goldenberg-Cohen234 and Shalom Michowiz13

1Department of Neurosurgery Rabin Medical Center-Beilinson Hospital 49100 Petach Tikva Israel2Krieger Eye Research Laboratory Felsenstein Medical Research Center 49202 Petach Tikva Israel3Sackler Faculty of Medicine Tel Aviv University 6997801 Tel Aviv Israel4Pediatric Ophthalmology Unit Schneider Childrenrsquos Medical Center of Israel 49100 Petach Tikva Israel

Correspondence should be addressed to Nitza Goldenberg-Cohen ncohen1gmailcom

Received 9 January 2016 Revised 11 April 2016 Accepted 21 April 2016

Academic Editor Monica Fedele

Copyright copy 2016 Ivan Novitzky et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

To evaluate the effect of bevacizumab and sildenafil on stroke parameters in a mouse model middle cerebral artery occlusionwas induced in male C57Bl6 mice using an intra-arterial filament method The filament was removed after 60 minutes and themice were immediately given a single intraperitoneal injection of saline bevacizumab or sildenafil An additional group of mice(119899 = 7) received bevacizumab 6 h after MCAO induction The mice were euthanized 24 hours later and evaluated for infarctarea and brain edema using triphenyltetrazolium chloride staining and ImageJ In the saline-treated mice (119899 = 16) total strokevolume was 1920 plusmn 638mm3 mean penumbra area was 45 plusmn 203mm3 and hemispheric asymmetry was 1065 Correspondingvalues in the bevacizumab group (119899 = 19) were 1779 plusmn 580mm3 73 plusmn 35mm3 and 1086 in the delayed (6 h) bevacizumabinjected mice (119899 = 7) they were 980 plusmn 800mm3 24 plusmn 20mm3 and 982 and in the sildenafil group (119899 = 16) they were1842 plusmn 541mm3 57 plusmn 202mm3 and 1099 The bevacizumab group had a significantly larger mean penumbra area whengiven immediately and smaller total stroke area in both groups than the saline- (119901 = 003) and sildenafil-treated (119901 = 0003)groups Only delayed bevacizumab group had reduced edema Bevacizumab injected immediately or delayed after injury exertsa neuroprotectivesalvage effect whereas immediate treatment with sildenafil does not Inflammation may play a role in theneuroprotective effect

1 Introduction

Bevacizumab is an inhibitor of vascular endothelial growthfactor (VEGF) that targets VEGF-A an isoform of VEGFthat stimulates endothelial cell proliferation and migrationBy specifically binding the VEGF-A protein it reduces theVEGF protein levels and restricts the growth of abnormalblood vessels thus reducing their leakage In addition it hasan anti-inflammatory role that reduces macrophage activityand inhibits infiltration of inflammatory materials causingneuronal damage [1] Bevacizumabhas been found to exert anantiangiogenic effect in tumors and a neuroprotective effectin the brain by reducing leakage and edema [1] It is used in

the treatment of colon cancer [2] and gliomas (low and highgrade) [3] and age-related macular degeneration [4] It wasalso recently found to be effective in reducingmacular edemain the sensory retina of patients with diabetes [5] Howeversome studies suggest that bevacizumab may play a role incausing cerebral ischemia [6 7]

Sildenafil citrate is a potent vasodilator which inhibits theaction of phosphodiesterase 5 (PDE 5) Sildenafil competeswith cyclic guanosine monophosphate (cGMP) responsiblefor smooth muscle relaxation for the substrate-binding siteon PDE 5 thereby increasing the concentration of cGMPin the tissue [8] Sildenafil is used mainly in the treatmentof erectile dysfunction It is beneficial for the treatment of

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 3938523 8 pageshttpdxdoiorg10115520163938523

2 BioMed Research International

pulmonary hypertension as well as PDE 5 is also distributedin the smoothmuscle arterial walls of the lungs [9] It was alsofound to induce retinal artery and vein dilatation in healthysubjects [10] Recent studies have shown that sildenafil mayexert a protective effect against ischemic injury in variousorgan systems such as the heart and kidney [11ndash13] In cardiactissue it acts as amimetic for super oxide dismutase prevent-ing the generation of hydrogen peroxide (H

2O2) [11] This

reduces the formation of oxygen-free radicals the drivingforce behind ischemia reperfusion injury and consequentmitochondrial dysfunction and cardiacmyocyte injury Silde-nafil also protects the heart by activating the ATP-sensitivepotassium channels on the mitochondrial walls [12] therebyhelping to maintain the mitochondrial membrane potentialduring ischemia reperfusion injury Furthermore sildenafilhas at least a partial anti-inflammatory effect through iNOSinhibition [14] It was found to reduce the expression ofcytokines COX-2 and GFAP (glial fibrillary acidic protein)in a demyelinating model induced in wild type mice andin iNOSminusminus mice sildenafil reduced Iba-1 IFN-120574 and IL-1120573 levels following cuprizone induced myelin damage andincreased glutathione S-transferase pi improving the myelinstructureultrastructure [14] Prompted by findings that silde-nafil citrate is protective against ischemia reperfusion injuryin neonatal rats researchers postulated that it may be helpfulin the treatment of neonatal cerebral ischemia [15] At thesame time several reports have described sildenafil as a strokeinducer in the presence of preexisting conditions or a historyof stroke [16]

The aim of the present study was to determine if beva-cizumab and sildenafil have a protective influence on cerebralischemic events using a mouse model of stroke

2 Methods

21 Animals All protocols were conducted in accordancewith the ARVO Statement for the Use of Animals inOphthalmic and Visual Research and were approved andmonitored by the Animal Care Committee of Rabin MedicalCenter (Permit Number 030813 022-b6640-2) Adult maleC57Bl6 mice (wild type) were purchased from HarlanLaboratories (Jerusalem Israel) Animals were maintained inour animal colony at 22∘C on a 12-hour light12-hour darkcycle All procedures and analyses were performed by thesame researcher (Ivan Novitzky) to reduce variability

22 Induction of Middle Cerebral Artery Occlusion Ourmiddle cerebral artery occlusion (MCAO) model is basedon previously published protocols [17 18] In brief micewere anesthetized with ketaminexylazine (80mgkg and8mgkg resp) and the neck was incised to expose the leftcommon carotid artery (LCCA) Two separate LCCA liga-tures were made using 60 surgical sutures The left internalcarotid artery (LICA) and the left pterygopalatine artery wereclipped The LCCA was perforated with a 27-gauge needleand a sterile 50-nylon monofilament with cautery-enlargedtip was inserted through the hole and pushed up to the clipin the LICA Both microvascular clips were then removed

The filament was advanced and inserted about 10mm intothe middle cerebral artery After 60 minutes of occlusion athird ligature was placed around the LCCA the filament wasremoved and the LCCA was permanently ligated

23 Treatment Immediately after removal of the filamentandwound closure with nylon suture themice were allocatedto one of three groups 03mL (300 120583L) saline treatment(09 sterile NaCl solution sham injection) single peri-toneal injection of bevacizumab (Avastin GenentechRoche75 120583g300 120583L) or single peritoneal injection of sildenafil(Revatio 08mgmL solution for injection Pfizer LimitedSandwich United Kingdom 24 120583g300 120583L) An additionalgroup of 7 mice received a single bevacizumab injection6 h after induction All mice were given fluid replenishment(05mL sterile saline) postoperatively and topical lidocainegel was placed on the incision site for pain relief

24 Triphenyltetrazolium Chloride Staining At 24 hours afterMCAO induction the animals were euthanized with carbondioxide The brains were removed incubated in 05 235-triphenyltetrazolium chloride (TTC) solution in 09 salinefor 10 minutes at 37∘C and cut coronally

25 Fluorescent Gelatin Perfusion for Brain Vascular ImagingVascular imaging was performed by transcardial perfusionof gelatin containing fluorescently labeled bovine serumalbumin (BSA) as previously reported [18 19] Briefly themice were terminally anesthetized with pentobarbital over-dose and a series of solutions was perfused transcardiallyas follows 20mL heparinized 09 saline 10mL 2 type Agelatin in saline with fluorescein-conjugated BSA (approxi-mately 001mgmL) and 2mL 4 type A gelatin in salinewith cyanin-conjugated BSAThereafter the ascending aortawas clamped with a hemostat while under pressure andthe animal was immersed in an ice-water bath to congealthe gelatin Perfusion of both hemispheres (unaffected andinjured) was compared

26 Immunostaining Cryosections of untreated (saline)MCAO induced brain sections were taken at 24 h washedwith phosphate buffer saline times1 blocked with 2 BSA inphosphate buffer saline with 05 Triton X-100 for 15minand incubated at 4∘C overnight with the primary antibodyrat anti-CD45 (1 100Millipore Temecula CA)The sectionswere washed with 02 PBS with 05 Triton X-100 andincubated at room temperature for 1 h with the secondaryantibody goat anti-rat IgGAlexa Fluor 488 (1 200) (Molecu-lar Probes Invitrogen Corporation Carlsbad CA USA)Thebrain sections underwent nuclear counterstaining with DAPI(Invitrogen) Images were generated using a conventional flu-orescence microscope (Fluoview X Olympus Tokyo Japan)Excitation wavelengths were 405 nm for DAPI and 488 nmfor Alexa Fluor

27 Calculation of Lesion Volume and Hemispheric Asymme-try Digital images of the TTC-stained coronal sections wereanalyzed for lesion volume and edema using Fiji imaging

BioMed Research International 3

Table 1 List of primers

Gene Forward primer sequence (51015840-31015840) Reverse primer sequence (51015840-31015840)Mouse ACTB TAGGCACCAGGGTGTGATGGT CATGTCGTCCCAGTTGGTAACAMouse SOD-1 GCCCGGCGGATGAAGA CGTCCTTTCCAGCAGTCACAMouse HO-1 CAGGTGTCCAGAGAAGCTTT TCTTCCAGGGCCGTGTAGATMouse CD45 GAACATGCTGCCAATGG TGTCCCACATGACTCCTTMouse BAX CTGAGCTGACCTTGGA GACTCCAGCCACAAAGMouse BCL-2 CCTGTGGATGACTGAG GAGCAGGGTCTTCAGAMouse TNF-120572 TCT CAA AAT TCG AGT GAC AAG C ACT CCA GCT GCT CCT CCA CMouse GFAP CCAGCTTCGAGCCAAGG GAAGCTCCGCCTGGTAGMouse MIP-2 CAGGGTGTGATGGTGGGAAT TGCTCTGGGCCTCGTCA

processing software (ImageJ National Institutes of HealthUSA) Stroke area and penumbra and hemispheric volumewere calculated The stroke lesions were identified as whiteareas completely devoid of TTC staining and the penumbrawas segmented by any color noticeably lighter than healthytissue which appears bright red [20] Total stroke area wasdefined as the sum of the stroke and penumbra region Brainedema was defined by hemispheric asymmetry calculatedas the ratio of infarcted hemisphere to normal healthyhemisphere

28 Molecular Analysis

281 RNA Extraction Brain tissues from 11 mice afterMCAO induction 6 treated 6 hours after induction bybevacizumab were dissected and frozen in RNAlater (LifeScience Division Sigma-Aldrich St Louis MO USA) Themiddle third of each hemisphere was used The mRNAsamples were analyzed in pairs per mice (injured anduninjured hemisphere) and also pooled (normal unaffectedhemispheres versus stroke hemispheres for treated anduntreated groups) Total RNA was isolated using TRIzolreagent (Life Technologies Invitrogen) according to themanufacturerrsquos protocol and then reverse-transcribed intocDNA using random hexamers (Amersham BiosciencesBuckinghamshire UK) and Moloney murine leukemia virusreverse transcriptase (Promega Madison WI USA)

282 Real-Time Quantitative PCR Real-time quantitativePCR (RT-qPCR) was applied to study gene expression ofapoptosis-related BAX and BCL-2 genes inflammatory-related CD45 and MIP-2 (IL-8) and stress-related HO-1genes in the brain using gene-specific primers (Table 1) Thereaction efficiency was tested using a standard curve Geneexpression was normalized tomouse beta actin a housekeep-ing gene StepOne Software v222 (Applied BiosystemsFoster City CA USA) was used for RT-qPCR Reactionswere performed in a 10 120583L volume containing 1 120583L cDNA05 120583M of each of the forward and reverse primers andbuffer included in the Master Mix (SYBR Green I AppliedBiosystems)

Cycling conditions consisted of an initial denaturationstep of 95∘C for 10min followed by 50 cycles of 1min at 95∘C

and 1min of annealing and extension at 60∘C Duplicate RT-qPCR reactions were performed for each gene to minimizeindividual tube variability and an average was taken foreach time point Threshold cycle efficiency corrections werecalculated andmelting curves were obtained using cDNA foreach individual-gene PCR assay The results were quantifiedby the comparative threshold cycle (Ct) method whereΔΔCt = ΔCt (sample) minus ΔCt (reference gene) (Data AssistSoftware v222 Applied Biosystems)

29 Statistical Analysis Experimental data are expressed asmean plusmn standard error Studentrsquos 119905-test was used to determinethe significance of differences between groups 119901 lt 005 wasconsidered significant

3 Results

Mean values of the stroke and edema parameters afterMCAO induction in the mice treated with saline beva-cizumab or sildenafil are shown in Table 2 and Figure 1The early bevacizumab-treated animals had a significantlygreater mean penumbra area (salvageable tissue surroundingthe infarct) than both the control mice (119901 = 003) andthe sildenafil-treated mice (119901 = 0003) and both groupsof bevacizumab had smaller total stroke area than theother groups The percentage of injured brain hemisphererelative to total healthy hemispheric tissue was lower inthe bevacizumab-treated mice than in the sildenafil-treatedmice (119901 = 034) The saline-treated animals had the lowesthemispheric asymmetry There was no decrease in brainedema after ischemic injury in either the bevacizumab- orsildenafil-immediately treated group

Figure 2 shows the lack of perfusion in the injuredhemisphere as compared to the unaffected hemisphere ofuntreated mice (control) Immunostaining for inflammation(CD45) of the same brains (Figure 3) shows the extravascularinfiltration of inflammatory cells into the damaged areawhere the vascular perfusion was interrupted Figure 4 showsinfiltration of CD45 into the stroke area in the delayedbevacizumab-treated brain

Table 3 shows the MCAO-related morbidity and mortal-ity in the three groups Mortality was categorized as eitherperioperative (traumatic bleeding during the procedure oranesthesia related) or postoperative (death after surgery)


Table 2 Penumbra stroke and brain edema in sildenafil- bevacizumab- (immediate and 6 h) and saline-treated MCAO induced mice

Treatment 119873

Meanpenumbra area

(mm3)

Mean strokearea (mm3)

Total strokearea (mm3)

Total injuredhemisphere(mm3)

of injuredhemisphere

Normalhemisphere

Hemisphericasymmetry(ratio)

Sildenafil 16 57 plusmn 202 1272 plusmn 676 1842 plusmn 541 3263 plusmn 160lowast 5610 plusmn 1531 2968 plusmn 313 1099

Bevacizumab 19 73 plusmn 35 1050 plusmn 553 1779 plusmn 580 3467 plusmn 204lowast 510 plusmn 1552 3189 plusmn 325 10867 24 plusmn 20 740 plusmn 542 980 plusmn 800 3290 plusmn 293 2900 plusmn 223 3351 plusmn 223 0982

Saline 16 45 plusmn 203 1470 plusmn 675 1920 plusmn 638 3272 plusmn 295 5809 plusmn 167 3070 plusmn 320 1065lowast119901 = 003 versus saline and 119901 = 0003 versus sildenafil

Figure 1 TTC (235-triphenyltetrazolium chloride) staining of MCAO infarcted brain tissue from mice treated with saline (control firstcolumn) sildenafil (2nd column) and bevacizumab (3rd and 4th columns) Infarcted tissue is pale while healthy tissue is pink It is evidentthat the mice treated with bevacizumab (immediate and late) have the largest amount of healthy tissue indicating a neuroprotective effectEach line represents different animal

(a) (b)

Figure 2 Perfusion studies of injured hemisphere versus control Two brain sections following fluorescent perfusion of MCAO inducedbrains without treatment (saline group) Note the good vascularization on the healthy side with infarct and lack of perfusion in the infarctedarea (Low magnification times5 panoramic view)


(a) (b) (c)

(d) (e) (f)

Figure 3 CD45 staining (red) of brains section of mice following MCAO induction without treatment (saline injected controls) andperfusion with fluorescent gelatin (green) The nuclei are stained with DAPI (blue) (a) CD45 immunostaining (red) showing infiltrationof the inflammatory cells to the injured hemisphere and increased cellularity (DAPI blue) (times5) while the unaffected hemisphere is wellperfused (green) (b) Higher magnification of the injured versus unaffected brain (times20) (c) Incomplete perfusion of the vessels in the infarctarea with extravasation of inflammatory cells to the tissue as compared to the unaffected brain (green) without inflammatory cells (dndashf)Incomplete vascularization with intra- and extracellular inflammatory cells (times40)

Morbidity was defined as visible neurological deficit aftersurgery andor treatment The highest mortality (75) wasdetected in the late bevacizumab injection followed by 60in the saline group probably due to three experiments wherenone of the mice survived the anesthesia Following theMCAO induction the bevacizumab-treated mice had lowermorbidity and mortality rates than controls The sildenafil-treated group had lower mortality than controls but highermorbidity

The molecular analysis of the untreated group versusthe bevacizumab 6 h treated group showed significantlyincreased BAX levels and BAXBCL-2 ratio (507-fold versus090-fold for BAX and BAXBcl-2 ratio 384 versus 051) andincreased CD45 levels in the bevacizumab 6 h treated group(204-fold) versus untreated group (084-fold) SOD1 andGFAP levels remained at baseline in the untreated group andmildly reduced (082 foldNS) in the 6 h bevacizumab-treatedgroup MIP-2 remained at baseline in both groups

4 Discussion

This study shows that bevacizumab a VEGF inhibitor has aneuroprotective effect on mice after MCAO induction Thebevacizumab-treated mice had a lower volume of injuredbrain and a larger penumbra meaning more salvageable tis-sue than the untreated mice Furthermore injection 6 h afterMCAO induction still showed a neuroprotective effect withreduced brain edema However there was no between-group

difference in brain edema in the early treated groups Wedid not provide any evidence to support the assumption thatbevacizumab decreases the leakage and therefore protects thebrain However the neuroprotective effect might be exertedthrough the reduced inflammatory cell infiltration into theinjured area Inhibition of the inflammatory reaction plays arole in the neuroprotective effect We hypothesize that thisneuroprotective effect of bevacizumab is directly related tothe anti-inflammatory properties of the drug It is of notethat the mortality was highest in the group treated only 6hours after MCAO induction (Table 2)This warrants furtherinvestigation

Molecular analysis showed a neuroprotective effect inreducing apoptosis reaction While CD45 was elevated inthe 6 h bevacizumab-treated group the untreated groupshowed a trend to receding inflammatory reaction which isnot significant to discuss protection or destruction MIP-2and GFAP levels were similar near baseline in both groupsPrevious studies from our laboratory have demonstratedprotective effect in reducing acute inflammation in strokemodel in TLR4 knockout mice in brain [20] and optic nerve[21]

We have demonstrated here the increased infiltration ofinflammatory cells to the damage area in the brain (usingimmunohistochemistry) in untreated mice but showedincreased molecular gene expression levels of CD45 butnot MIP-2 in the 6 h treated brain We assume that theCD45 represent inflammatory reaction that can be protective


(a) (b)

(c) (d) (e)

Figure 4 Immunohistochemistry staining for inflammatory cells using CD45marker demonstrating Inflammatory infiltration to the infarctarea (a) Inflammatory cells in the injured hemisphere but not in the other side (times20) (b) The location of the area immunostained in (a) isdemonstrated in TTC staining (times5) (c) Highermagnification of (a) (times40) (d)The injured hemisphere with positive staining for inflammatorycells (e) The unaffected hemisphere negative for CD45 staining

Table 3 Mortality and morbidity in treated rats after MCAO induction

Saline Bevacizumab SildenafilImm Imm 6 h Imm

Total number of mice at study onset 58 32 35 27Mortality

After surgery 15 5 14 1After anesthesia 20 4 9 1Total 35 (60) 9 (28) 23 (68) 2 (074)

MorbidityAfter surgery 3 (13) 0 0 6 (24)Survived procedure 20 23 12 19Included in study 16 19 12lowast 16No stroke 4 (20) 3 (13) 0 (0) 1 (63)Extreme damage 0 1 (43) 0 (0) 2 (105)

Imm immediately injected after MCAO induction lowast7 of 11 for histology (TTC) and immunohistochemistry and 5 for molecular analysis

or destructive depending on its expression levels Furtheranalysis of IL-8MIP-2 and GFAP did not show differencesbetween the groups

It has been postulated that there might be a connectionbetween hypoxia and inflammations [22] This relationship

could be explained by the fact that the hypoxic endothe-lium expresses inflammatory chemokine receptors such asCXCR4 and CXCR7 as well as the angiogenic chemokines(such as CXCL8 and CXCL1) [22] These are all regulated bythe transcription factor hypoxia inducible factor and serve


as potential future drug targets for anti-inflammatory drugsWe measured the inflammatory-related gene expression andconfirmed the increased inflammatory effect after strokeinduction Immunostaining for CD45 and molecular expres-sion levels confirmed its effect

Unlike bevacizumab sildenafil did not exert a signifi-cant generalized neuroprotective effect Although sildenafilwas found to reduce the inflammatory reaction in modelsof demyelinating brain injury [14] it was not effective inthe MCAO model There was no significant difference inpoststroke brain edema between sildenafil- and saline-treatedmice

These findings contrast with earlier clinical studies Beva-cizumab has been shown to induce cerebral ischemic eventsin patients being treated for glioma and to reduce brainedema in patients with brain tumor [3 7] We attributeits protective effect to the restriction of proinflammatoryactivities Sildenafil has been found to protect against cerebraledema [15 16] and to serve as an appropriate therapy forpatients with stroke [23] Accordingly in an experimen-tal study of the poststroke effects of bevacizumab [24]researchers treated Sprague-Dawley rats with bevacizumabor saline following induction of MCAO and analyzed thefindings with positron emission tomography in conjunctionwith behavioral analysis Their results suggested that beva-cizumab has no effect on functional recovery after stroke[24] However positron emission tomography showed adecreased metabolic demand for glucose and inhibition ofvascular formation which could explain our finding of aneuroprotective effect In an analogous study of sildenafilrats in the study group had consistently better behavioralscores than controls and a reduced cerebral infarction volume[25] Transmission electron microscopy showed a significantdecrease in the number of degenerated neurons in thesildenafil group and molecular analysis showed a significantdecrease in the extent of synapse damage via the cGMP-dependent Nogo-R pathway

The difference between similar studies in the brain [2425] and our study has several explanations These experi-mental studies used rat models of stroke instead of a mousemodel which we utilized Amuch smaller number of animals(119899 = 8ndash12) were used in these studies compared to ourwork (bevacizumab 1951 and sildenafil 1651) with muchhigher doses of the study drugs (up to 32mgkg times3) [2425] The time course of our experiments was also differentOur animals were investigated only 24 hours after MCAOinduction whereas other studies used varying time points[24 25] Also the neuroprotective effect of bevacizumab wasobserved in the context of solid tumors where it could be dueto a decrease in glucose demand in neural tissue as shown onpositron emission tomography [24]

Sildenafil was also found to have a protective effect in var-ious organ systems such as heart and kidney [13] probablyvia a reduction in oxidative stress in ischemia reperfusioninjury Specifically sildenafil induced mitochondrial biogen-esis in the injured cells in the renal cortex Mitochondrialdysfunctiondisruption of mitochondrial biogenesis is one ofthe mechanisms underlying ischemiareperfusion injury inthe kidney and apparently plays a role in acute kidney injury

and renal tubular dysfunction [13] It has also been implicatedin ischemia reperfusion injury in the brain Sildenafil wasfound to induce mitochondrial biogenesis in the mouse renalcortex [12] and a postmortem analysis of brain tissue frompatients after ischemic events showed that mitochondrialoxidative stress was responsible for neuronal cell death [23]Therefore sildenafil is considered a promising therapeuticagent for patients with acute kidney injury or stroke

However it must be mentioned that the fundamentalphysiology of neural tissue is different to other tissuesmaking it less forgiving to ischemic injury It has been shownthat mitochondrial dysfunction due to oxidative stress afterischemic injury is most likely the mechanism of neuronaldamage in stroke [23] It is likely however that sildenafil has aless protective effect in neural tissue compared to other tissuesin the body due to the less forgiving nature of the centralnervous system to ischemic injury

5 Conclusions

This study demonstrated that bevacizumab has a slight neu-roprotective effect after ischemic stroke whereas sildenafildoes not This is in contradiction to previous studies [2425] However we have utilized a larger sample size Cautionshould be used in attempting to identify neuroprotectivedrugs Neural tissue is complex and a large amount ofanimalssamples are needed before meaningful results can beobtained

Disclosure

The authors alone are responsible for the content andwriting of the paper This work was presented in part atthe International Symposium on Ocular Pharmacology andTherapeutics (ISOPT) Berlin Germany in July 2015

Competing Interests

The authors declare no competing financial interests

Authorsrsquo Contributions

Ivan Novitzky conducted the study analyzed the resultsand edited the figures Neelan J Marianayagam analyzedthe results and wrote the paper draft Shirel Weiss analyzedthe results Orkun Muhsinoglu performed the study MoranFridman performed the immunohistochemistry studies andmicroscopy Tamar Azrad Leibovitch performed the molec-ular analysis and analyzed the results Nitza Goldenberg-Cohen designed analyzed and wrote the paper ShalomMichowiz designed performed analyzed the results andcritically revised the paper All authors discussed the resultsand implications and commented on the paper at all stagesIvan Novitzky and Neelan J Marianayagam contributedequally to this paper


Acknowledgments

This study was partially supported by the Zanvyl and IsabelleKrieger Fund Baltimore Maryland USA Israel ScientificFoundation (Grant no 118912) and Rabin Young Investiga-tor Award (Ivan Novitzky)

References

[1] Y Sun K Jin L Xie et al ldquoVEGF-induced neuroprotectionneurogenesis and angiogenesis after focal cerebral ischemiardquoThe Journal of Clinical Investigation vol 111 no 12 pp 1843ndash1851 2003

[2] J H Strickler andH I Hurwitz ldquoBevacizumab-based therapiesin the first-line treatment of metastatic colorectal cancerrdquo TheOncologist vol 17 no 4 pp 513ndash524 2012

[3] S J Peak and V A Levin ldquoRole of bevacizumab therapy inthe management of glioblastomardquo Cancer Management andResearch vol 2 no 1 pp 97ndash104 2010

[4] J M Pitlick K F Vecera K N Barnes J W Reski and A BForinash ldquoBevacizumab for the treatment of neovascular age-related macular degenerationrdquo Annals of Pharmacotherapy vol46 no 2 pp 290ndash296 2012

[5] J A Wells A R Glassman A R Ayala et al ldquoAfliberceptbevacizumab or ranibizumab for diabetic macular edemardquoTheNew England Journal of Medicine vol 372 no 13 pp 1193ndash12032015

[6] A A Grivas D T Trafalis and A E Athanassiou ldquoImplicationof bevacizumab in fatal arterial thromboembolic incidentsrdquoJournal of BUON vol 14 no 1 pp 115ndash117 2009

[7] T J Fraum T N Kreisl J Sul H A Fine and F MIwamoto ldquoIschemic stroke and intracranial hemorrhage inglioma patients on antiangiogenic therapyrdquo Journal of Neuro-Oncology vol 105 no 2 pp 281ndash289 2011

[8] J D Corbin ldquoMechanisms of action of PDE5 inhibition in erec-tile dysfunctionrdquo International Journal of Impotence Researchvol 16 supplement 1 pp S4ndashS7 2004

[9] C F Barnett and R F Machado ldquoSildenafil in the treatmentof pulmonary hypertensionrdquoVascular Health and RiskManage-ment vol 2 no 4 pp 411ndash422 2006

[10] M Pache PMeyer C Prunte SOrgul I Nuttli and J FlammerldquoSildenafil induces retinal vasodilatation in healthy subjectsrdquoThe British Journal of Ophthalmology vol 86 no 2 pp 156ndash1582002

[11] M A S Fernandes R J F Marques J A F Vicente etal ldquoSildenafil citrate concentrations not affecting oxidativephosphorylation depress H

2O2generation by rat heart mito-

chondriardquoMolecular and Cellular Biochemistry vol 309 no 1-2pp 77ndash85 2008

[12] XWang PW Fisher L Xi and R C Kukreja ldquoEssential role ofmitochondrial Ca2+-activated andATP-sensitiveK+ channels insildenafil-induced late cardioprotectionrdquo Journal of Molecularand Cellular Cardiology vol 44 no 1 pp 105ndash113 2008

[13] RMWhitaker L PWills L J Stallons andRG SchnellmannldquoCGMP-selective phosphodiesterase inhibitors stimulate mito-chondrial biogenesis and promote recovery from acute kidneyinjuryrdquo The Journal of Pharmacology and Experimental Thera-peutics vol 347 no 3 pp 626ndash634 2013

[14] C Raposo A K D S Nunes R L D A Luna S M D RAraujo M A da Cruz-Hofling and C A Peixoto ldquoSildenafil(Viagra) protective effects on neuroinflammation the role of

iNOSNO system in an inflammatory demyelination modelrdquoMediators of Inflammation vol 2013 Article ID 321460 11 pages2013

[15] C Charriaut-Marlangue T Nguyen P Bonnin et al ldquoSildenafilmediates blood-flow redistribution and neuroprotection afterneonatal hypoxia-ischemiardquo Stroke vol 45 no 3 pp 850ndash8562014

[16] M Lorberboym I Mena J Wainstein M Boaz and Y LamplldquoThe effect of sildenafil citrate (Viagra) on cerebral blood flowin patients with cerebrovascular risk factorsrdquo Acta NeurologicaScandinavica vol 121 no 6 pp 370ndash376 2010

[17] O Engel S Kolodziej U Dirnagl and V Prinz ldquoModelingstroke in micemdashmiddle cerebral artery occlusion with thefilament modelrdquo Journal of Visualized Experiments no 47article 2423 2011

[18] D Rappoport D Morzaev S Weiss et al ldquoEffect of intravitrealinjection of bevacizumab on optic nerve head leakage andretinal ganglion cell survival in a mouse model of optic nervecrushrdquo InvestigativeOphthalmologyampVisual Science vol 54 no13 pp 8160ndash8171 2013

[19] J D Nicholson A C Puche Y Guo D Weinreich B J Slaterand S L Bernstein ldquoPGJ

2provides prolonged CNS stroke

protection by reducing white matter edemardquo PLoS ONE vol 7no 12 Article ID e50021 2012

[20] D Pushkov J D Nicholson S Michowiz et al ldquoRelativeneuroprotective effects hyperbaric oxygen treatment and TLR4knockout in a mouse model of temporary middle cerebralartery occlusionrdquo International Journal of Neuroscience vol 126no 2 pp 174ndash181 2016

[21] D Morzaev J D Nicholson T Caspi S Weiss E Hochhauserand N Goldenberg-Cohen ldquoToll-like receptor-4 knockoutmice are more resistant to optic nerve crush damage than wild-type micerdquo Clinical and Experimental Ophthalmology vol 43no 7 pp 655ndash665 2015

[22] C R Mackay ldquoMoving targets cell migration inhibitors as newanti-inflammatory therapiesrdquo Nature Immunology vol 9 no 9pp 988ndash998 2008

[23] L T Watts R Lloyd R J Garling and T Duong ldquoStrokeneuroprotection targetingmitochondriardquoBrain Sciences vol 3no 2 pp 540ndash560 2013

[24] Y Dong F Song J Ma et al ldquoSmall-animal PET demonstratesbrain metabolic change after using bevacizumab in a rat modelof cerebral ischemic injuryrdquoNeuroscience Bulletin vol 30 no 5pp 838ndash844 2014

[25] X-M Chen N-N Wang T-Y Zhang F Wang C-F Wu andJ-Y Yang ldquoNeuroprotection by sildenafil neuronal networkspotentiation in acute experimental strokerdquo CNS NeuroscienceandTherapeutics vol 20 no 1 pp 40ndash49 2014

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Evidence-Based Complementary and Alternative Medicine

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pulmonary hypertension as well as PDE 5 is also distributedin the smoothmuscle arterial walls of the lungs [9] It was alsofound to induce retinal artery and vein dilatation in healthysubjects [10] Recent studies have shown that sildenafil mayexert a protective effect against ischemic injury in variousorgan systems such as the heart and kidney [11ndash13] In cardiactissue it acts as amimetic for super oxide dismutase prevent-ing the generation of hydrogen peroxide (H

2O2) [11] This

reduces the formation of oxygen-free radicals the drivingforce behind ischemia reperfusion injury and consequentmitochondrial dysfunction and cardiacmyocyte injury Silde-nafil also protects the heart by activating the ATP-sensitivepotassium channels on the mitochondrial walls [12] therebyhelping to maintain the mitochondrial membrane potentialduring ischemia reperfusion injury Furthermore sildenafilhas at least a partial anti-inflammatory effect through iNOSinhibition [14] It was found to reduce the expression ofcytokines COX-2 and GFAP (glial fibrillary acidic protein)in a demyelinating model induced in wild type mice andin iNOSminusminus mice sildenafil reduced Iba-1 IFN-120574 and IL-1120573 levels following cuprizone induced myelin damage andincreased glutathione S-transferase pi improving the myelinstructureultrastructure [14] Prompted by findings that silde-nafil citrate is protective against ischemia reperfusion injuryin neonatal rats researchers postulated that it may be helpfulin the treatment of neonatal cerebral ischemia [15] At thesame time several reports have described sildenafil as a strokeinducer in the presence of preexisting conditions or a historyof stroke [16]

The aim of the present study was to determine if beva-cizumab and sildenafil have a protective influence on cerebralischemic events using a mouse model of stroke

2 Methods

21 Animals All protocols were conducted in accordancewith the ARVO Statement for the Use of Animals inOphthalmic and Visual Research and were approved andmonitored by the Animal Care Committee of Rabin MedicalCenter (Permit Number 030813 022-b6640-2) Adult maleC57Bl6 mice (wild type) were purchased from HarlanLaboratories (Jerusalem Israel) Animals were maintained inour animal colony at 22∘C on a 12-hour light12-hour darkcycle All procedures and analyses were performed by thesame researcher (Ivan Novitzky) to reduce variability

22 Induction of Middle Cerebral Artery Occlusion Ourmiddle cerebral artery occlusion (MCAO) model is basedon previously published protocols [17 18] In brief micewere anesthetized with ketaminexylazine (80mgkg and8mgkg resp) and the neck was incised to expose the leftcommon carotid artery (LCCA) Two separate LCCA liga-tures were made using 60 surgical sutures The left internalcarotid artery (LICA) and the left pterygopalatine artery wereclipped The LCCA was perforated with a 27-gauge needleand a sterile 50-nylon monofilament with cautery-enlargedtip was inserted through the hole and pushed up to the clipin the LICA Both microvascular clips were then removed

The filament was advanced and inserted about 10mm intothe middle cerebral artery After 60 minutes of occlusion athird ligature was placed around the LCCA the filament wasremoved and the LCCA was permanently ligated

23 Treatment Immediately after removal of the filamentandwound closure with nylon suture themice were allocatedto one of three groups 03mL (300 120583L) saline treatment(09 sterile NaCl solution sham injection) single peri-toneal injection of bevacizumab (Avastin GenentechRoche75 120583g300 120583L) or single peritoneal injection of sildenafil(Revatio 08mgmL solution for injection Pfizer LimitedSandwich United Kingdom 24 120583g300 120583L) An additionalgroup of 7 mice received a single bevacizumab injection6 h after induction All mice were given fluid replenishment(05mL sterile saline) postoperatively and topical lidocainegel was placed on the incision site for pain relief

24 Triphenyltetrazolium Chloride Staining At 24 hours afterMCAO induction the animals were euthanized with carbondioxide The brains were removed incubated in 05 235-triphenyltetrazolium chloride (TTC) solution in 09 salinefor 10 minutes at 37∘C and cut coronally

25 Fluorescent Gelatin Perfusion for Brain Vascular ImagingVascular imaging was performed by transcardial perfusionof gelatin containing fluorescently labeled bovine serumalbumin (BSA) as previously reported [18 19] Briefly themice were terminally anesthetized with pentobarbital over-dose and a series of solutions was perfused transcardiallyas follows 20mL heparinized 09 saline 10mL 2 type Agelatin in saline with fluorescein-conjugated BSA (approxi-mately 001mgmL) and 2mL 4 type A gelatin in salinewith cyanin-conjugated BSAThereafter the ascending aortawas clamped with a hemostat while under pressure andthe animal was immersed in an ice-water bath to congealthe gelatin Perfusion of both hemispheres (unaffected andinjured) was compared

26 Immunostaining Cryosections of untreated (saline)MCAO induced brain sections were taken at 24 h washedwith phosphate buffer saline times1 blocked with 2 BSA inphosphate buffer saline with 05 Triton X-100 for 15minand incubated at 4∘C overnight with the primary antibodyrat anti-CD45 (1 100Millipore Temecula CA)The sectionswere washed with 02 PBS with 05 Triton X-100 andincubated at room temperature for 1 h with the secondaryantibody goat anti-rat IgGAlexa Fluor 488 (1 200) (Molecu-lar Probes Invitrogen Corporation Carlsbad CA USA)Thebrain sections underwent nuclear counterstaining with DAPI(Invitrogen) Images were generated using a conventional flu-orescence microscope (Fluoview X Olympus Tokyo Japan)Excitation wavelengths were 405 nm for DAPI and 488 nmfor Alexa Fluor

27 Calculation of Lesion Volume and Hemispheric Asymme-try Digital images of the TTC-stained coronal sections wereanalyzed for lesion volume and edema using Fiji imaging











3 Results






Treatment 119873

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5 Conclusions


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3 Results






Treatment 119873

Meanpenumbra area

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(a) (b) (c)

(d) (e) (f)




4 Discussion






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5 Conclusions


Disclosure


Competing Interests





Acknowledgments


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of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Treatment 119873

Meanpenumbra area

(mm3)





Normalhemisphere






(a) (b)



(a) (b) (c)

(d) (e) (f)




4 Discussion






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(c) (d) (e)



















5 Conclusions


Disclosure


Competing Interests





Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b) (c)

(d) (e) (f)




4 Discussion






(a) (b)

(c) (d) (e)



















5 Conclusions


Disclosure


Competing Interests





Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

(c) (d) (e)



















5 Conclusions


Disclosure


Competing Interests





Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























5 Conclusions


Disclosure


Competing Interests





Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Comparison of Neuroprotective...

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