Role of botulinum neurotoxin–A in cerebral revascularization graft vasospasm prevention: current state of knowledge

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Graft stenosis and occlusion remain formidable complications in cerebral revascularization procedures, which can lead to significant morbidity and mortality. Graft vasospasm can result in early postoperative graft stenosis and occlusion and is believed to be at least partially mediated through adrenergic pathways. Despite various published treatment protocols, there is no single effective spasmolytic agent. Multiple factors, including anatomical and physiological variability in revascularization conduits, patient age, and comorbidities, have been associated with graft vasospasm pathogenesis and response to spasmolytics. The ideal spasmolytic agent thus likely needs to target multiple pathways to exert a generalizable therapeutic effect. Botulinum toxin (BTX)–A is a powerful neurotoxin widely used in clinical practice for the treatment of a variety of spastic conditions. Although its commonly described paradigm of cholinergic neural transmission blockade has been widely accepted, evidence for other mechanisms of action including inhibition of adrenergic transmission have been described in animal studies. Recently, the first pilot study demonstrating clinical use of BTX-A for cerebral revascularization graft spasm prevention has been reported. In this review, the mechanistic basis and potential future clinical role of BTX-A in graft vasospasm prevention is discussed.

ABBREVIATIONS BTX = botulinum toxin; CGRP = calcitonin gene-related peptide; eNOS = endothelial nitric oxide synthase; KCl = potassium chloride; MLC = myosin light chain; MLCP = MLC phosphatase; MYPT1 = myosin phosphatase targeting subunit 1; NE = norepinephrine; NO = nitric oxide; RhoA = ras homolog gene family member A; ROCK = rho-associated protein kinase; SNAP = synaptosomal-associated protein; VEGF = vascular endothelial growth factor; VSMC = vascular smooth-muscle cell.

Abstract

Graft stenosis and occlusion remain formidable complications in cerebral revascularization procedures, which can lead to significant morbidity and mortality. Graft vasospasm can result in early postoperative graft stenosis and occlusion and is believed to be at least partially mediated through adrenergic pathways. Despite various published treatment protocols, there is no single effective spasmolytic agent. Multiple factors, including anatomical and physiological variability in revascularization conduits, patient age, and comorbidities, have been associated with graft vasospasm pathogenesis and response to spasmolytics. The ideal spasmolytic agent thus likely needs to target multiple pathways to exert a generalizable therapeutic effect. Botulinum toxin (BTX)–A is a powerful neurotoxin widely used in clinical practice for the treatment of a variety of spastic conditions. Although its commonly described paradigm of cholinergic neural transmission blockade has been widely accepted, evidence for other mechanisms of action including inhibition of adrenergic transmission have been described in animal studies. Recently, the first pilot study demonstrating clinical use of BTX-A for cerebral revascularization graft spasm prevention has been reported. In this review, the mechanistic basis and potential future clinical role of BTX-A in graft vasospasm prevention is discussed.

Early graft occlusion after cerebral revascularization occurs in approximately 5%–10% of cases despite advancements in surgical technique and pharmacological treatments.41,42,53 Although rare, graft vasospasm remains a formidable and potentially lethal complication that affects multiple surgical specialties,28 including plastic and reconstructive surgery, cardiac surgery, and neurosurgery. Patency and spasm rates vary by graft type, with arterial grafts currently preferred for revascularization procedures over venous grafts due to their lower risk of developing intimal hyperplasia and atherosclerosis, and greater long-term patency rates.11 Nevertheless, long-term failure of arterial grafts still occurs in 2%–18% of cases, and arterial conduits have a significantly greater risk of vasospasm. Whether from occlusions or flow-limiting vasospasm, perfusion deficits in downstream territories from graft compromise can have devastating clinical consequences.17,25,38

Despite extensive study to date, the exact cause of graft vasospasm remains unclear, and there is no single effective spasmolytic agent for preventing graft vasospasm.27,55 While complex pharmacological antispastic protocols have been proposed in coronary bypass grafting (often involving systemic vasodilators), their use in cerebrovascular bypass is generally limited by the need to maintain cerebral perfusion in these patients.31

Botulinum toxin (BTX) is a potent neurotoxin widely used for the treatment of a variety of spastic and hyperactive autonomic conditions.12,33 The potent spasmolytic properties of BTX have sparked interest in its possible application for the prevention of graft spasm. In this review, we examine the current state of knowledge regarding BTX as it relates to graft vasospasm prevention, with a focus on revascularization conduit anatomy, physiology, and vasomotor mechanistic pathways.

Background

Graft Vasospasm and Current Treatment Options

Multiple factors affect vessel wall contractility and must be considered during graft selection and evaluation/optimization of potential spasmolytic agents. In addition to physical and chemical stimuli, vessel contractility is determined by anatomical features such as wall structure and the density and composition of receptors (Fig. 1), both of which can be affected by patient age and comorbidities such as atherosclerosis, hypertension, and diabetes.25,28,30,35,58 Although it is known that dynamic changes in blood vessel diameter are mediated by vascular smooth-muscle cells (VSMCs) and vessel elasticity is determined by the amount of elastic laminae in the vessel wall,24,25 less is known about vasomotor receptor composition, which is only described in select arteries and may vary within different segments of the same artery.24,25,29,55 Despite their higher contractility, the common utilization of limb arteries in cerebrovascular bypass due to their ease of access and harvest makes the development of effective antispastic treatments critical.24,52

FIG. 1.
FIG. 1.

Factors involved in vasoreactivity. Vasoreactivity is determined not only by external/systemic factors (presence of physical stimuli and vasoactive substances), but also by intrinsic anatomical and physiological contractile properties of blood vessels as dictated by the density and composition of receptors and vessel wall structure.

The extensive research on coronary artery bypass graft spasm possesses mixed relevance for cerebral revascularization.24,27,29,55 Given the multifaceted nature of vasospasm, variability of revascularization conduits, and pharmacological agents each targeting different pathways, a common conclusion of this work has been that there is no single ideal spasmolytic agent. Hence, complex spasmolytic protocols with combinations of different vasodilators applied pre-, intra-, and postoperatively, including both systemic and topical applications, have been developed. Besides emphasizing the importance of atraumatic graft harvest, these protocols typically contain papaverine or calcium channel antagonists (e.g., verapamil or nicardipine) and a nitrate (e.g., nitroglycerin).24,27 The utility of these protocols, which emphasize systemic vasodilator therapy, is limited in cerebrovascular bypass patients given the need for blood pressure/cerebral perfusion maintenance in the postoperative period.31,60 Additionally, the effects of intraoperative preventive and postoperative salvage vasodilators such as verapamil or papaverine are typically short-lived, and may require repeat interventions with increasing complication risks.7,19,37 Addressing these limitations, the ideal therapeutic agent would be safe, long-lasting, and quickly and easily applied intraoperatively for spasm prevention rather than treatment.

BTX-A as a Spasmolytic Agent: Classic Concepts

BTXs are potent biological toxins produced by anaerobic, gram-positive spore-forming bacteria from the Clostridium genus. There are 7 well-known, serotypically different BTXs (A–G) produced by 6 distinct groups of clostridia.56 The primary BTX mechanism of action is cholinergic neuromuscular transmission blockade through inhibition of the release of acetylcholine from nerve terminals.33,51,56 Specifically, BTX binds to a polysialoganglioside receptor on the presynaptic membrane and enters the nerve terminal via receptor-mediated endocytosis. The light chain of the BTX is a metalloproteinase that then cleaves one or more proteins from the soluble N-methylmaleimide sensitive factor attachment protein receptor complex that mediates synaptic vesicle exocytosis. BTX-B, -D, -F, and -G cleave vesicle-associated membrane protein, while BTX-A and -E cleave synaptosomal-associated protein–25 (SNAP-25), and BTX-C cleaves SNAP-25 and syntaxin.33,56 Although this BTX-mediated cholinergic neural transmission blockade is irreversible, the duration of BTX paralytic activity varies significantly depending on toxin type, dose, species, and type of cholinergic nerve terminals targeted (autonomic or skeletal muscle).51 BTX-A, one of the most commonly used subtypes, induces skeletal muscle paralysis that can be detected 2–3 days after injection and typically reaches its maximum at 1–2 weeks. This effect is gradually lost over time as nerve terminals are remodeled, with complete restoration of neural transmission to baseline levels in 3–4 months.51

Currently, BTX is FDA approved for a very limited number of spastic conditions, with the majority of the more than 50 reported therapeutic applications being unlabeled, including hemifacial spasm, hyperhidrosis, headache, urinary incontinence, and Raynaud’s phenomenon.12

BTX-A Utility in Vasospasm Prevention: Relevant Animal Studies

To date, BTX use has been reported in 6 animal studies evaluating its effects on microvascular anastomosis patency, vessel diameter, and response to vasospastic challenge on in situ blood vessels or grafts (Table 1).5,14,18,34,45,48 In these studies, rat (n = 5/6) and rabbit (n = 1/6) models were used to evaluate various BTX types (n = 3 BTX-A, n = 2 BTX-B, and n = 1 BTX-C), doses (1–1500 IU), application methods (injection, direct application to the vessels in situ or in a tissue bath), and target vessels (femoral, aortic, and posterior auricular) with variable follow-up (30 minutes to 28 days). BTX efficacy was reported in all of these studies, demonstrating its utility for vasospasm prevention.14,18,34,48 Specifically, BTX improved microvascular anastomosis patency rates18,48 and counteracted vasospastic challenges from phenylephrine,14,34 potassium chloride (KCl),45 norepinephrine (NE),45 and cold,14,18,34 with these effects detectable as early as 30 minutes45 after treatment. The spasmolytic potential of BTX in these studies was shown to be dependent on both dose and application time.5,45

TABLE 1.

Animal studies investigating BTX effects on in situ blood vessels and grafts

BTX
Authors & YearSpeciesTypeDoseApplicationBlood VesselsTx TimeVasospastic ChallengeTime PointsResearch MethodsVasomotor Factors EvaluatedOutcome
Clemens et al., 2009RatA10 IU/0.5 mlInject in perivascular spaceFemoral5 days preopPhenylephrine & LE cold exposureVessel anast 5 days post-Tx, challenge after anast, anast eval 0 & 1 hr after challengeVisual insp & vessel diam measurementsNonePatency maintained in 100% of vessels pretreated w/ BTX-A & only 44% pretreated w/ saline, sig shorter anast time & ease in BTX-A group vs saline
Murakami et al., 2009RatC1, 2.5, 5, 10, 20 IU/mlTissue bathAortic rings30 minsKCl & NEIsometric contractile responses measured post-TxIsometric contractile response measurement using myograph, hist exam using Elastica van Gieson stainNoneComplete inhibition of KCl-induced graft spasm w/ 5 IU/ml BTX-C Tx; dose-dependent NE-induced graft spasm prevention by BTX-C w/ max effect at 5 IU/ml; BTX-C effects on NE exposure longer lasting than w/ papaverine
Arnold et al., 20095RatA5 IUDirect application to vesselsFemoral3 mins before skin closureNonePOD 1, 7, 14, & 28Visual insp & vessel diam measurementsNoneMax arterial diam after BTX Tx on POD 14, BTX-treated artery sig larger than cntrl on POD 14, BTX-treated vein sig larger than cntrl on POD 28
Fathi et al., 2010RabbitA20 IU/mlSubcut inject around vesselsPst auricular arteries & marginal veins7 days before vessel dissectionCold exposureVessel measurement immediately after dissection, vessels then divided & re-anastomosed, exposed to cold challenge & vessel patency evaluated 1 hr laterVisual insp & vessel diam measurementsNoneArtery & vein diams sig larger & patency rate higher in BTX-A pre-Tx group
Janz et al., 2011RatB100 IUAnast tissue bath & direct inject in perivascular region 2 cm proximal to anastFemoralImmediately after anastPhenylephrine & LE cold exposureChallenge at 12, 24, 48, 72, & 120 hrs after adventitia stripping, anast & Tx w/ subsequent patency evalVisual insp & vessel diam measurementsNoneVessel thrombosis rate sig lower in BTX-B group at all time points except 120 hrs, when there were no thrombotic events; BTX-B produced sig greater ↑ in vessel diam vs cntrl
Park et al., 2014RatB1500 IU/0.3 mlPerivascular injectFemoral3 days before vessel dissectionNoneVessel diams & blood flow measured 3 days after inject on dissection & after re-anast of vesselsVessel diam & blood flow measurements using laser Doppler flowmetryNoneVein & artery diam & peak blood flow velocity sig ↑ in BTX-B vs cntrls

Anast = anastomosis; cntrl = control; diam = diameter; eval = evaluation; hist = histological; inject = injection; insp = inspection; LE = lower extremity; POD = postoperative day; pst = posterior; sig = significant(ly); subcut = subcutaneous; Tx = treatment; ↑ = increase(d).

Multiple reports on the effects of BTX in animal models of free and pedicled cutaneous, myocutaneous, and muscular flaps also support a potentially multifaceted mechanism of action. We identified 15 animal studies to date (Table 2) where the effects of BTX-A (n = 14) and BTX-B (n = 1) on flap viability, vasodilation, blood flow, angiogenesis, and inflammation were evaluated.1,6–8,10,22,32,36,39,40,49,50,54,57,62 In these studies, a rat model was most commonly used (n = 14/15) and there was significant variability in BTX dose (0.1–20 IU), application method (subdermal, intradermal, subcutaneous, intramuscular or perivascular injection, direct application to the vessels, or tissue bath), treated flaps (random cutaneous, abdominal or dorsal cutaneous, transverse rectus abdominis myocutaneous, cremaster, spinotrapezius, or gastrocnemius muscular), and evaluation time points (5 minutes to 21 days). Better flap survival rates,10,22,32,36,39,50,54,57 increased angiogenesis and angiogenic marker expression,36,39,40,49,50,54 improved blood flow,54,57 vasodilation,8,39,50,62 and reduced inflammation and inflammatory marker expression1,6 were observed in the BTX treatment groups as compared to controls. Interestingly, BTX-A also improved random cutaneous flap survival in rats after short- and long-term tobacco exposure, demonstrating its potential efficacy for the prevention of reconstructive and revascularization surgery complications in smokers.10,36

TABLE 2.

Animal studies investigating BTX effects on free and pedicled flaps

BTX
Authors & YearSpeciesTypeDoseApplicationFlapTx TimeVasospastic ChallengeTime PointsResearch MethodsVasomotor Factors EvaluatedOutcome
Kim et al., 2009RatA1.5 IU/0.05 mlSingle intradermal inject in central portion of proximal 3rd of flap after its elevationRandom cutaneous flapOn flap elevationNoneFlap evaluated on POD 1, 3, 5, & 7Gross & laser-induced fluorescein fluoroscopy optical flap eval; surviving flap area measurements; hist eval using H&E; detection of CD31, VEGF, & iNOS mRNA expression using RT-PCRVEGF, iNOSSig better flap survival rate in BTX A–treated group vs cntrl; hist eval showed more prominent vasodilation & endothelial proliferation in BTX-A Tx group vs cntrl; higher CD31, VEGF, & iNOS gene mRNA expression rates in BTX-A group vs cntrl
Arnold et al., 20097RatANRDirect Tx to vessels for several mins before closureVentral pedicled island cutaneous flap, inferior epigastric vesselsOn flap elevationNoneOn POD 4 flap evaluated for necrosis & ischemiaVisual insp & percentage area of flap necrosis calculationsNoneNo sig differences of necrosis area btwn Tx groups & adventitia intact vs disrupted groups; papaverine Tx group showed least ischemic changes & least amount of abnormal perfusion vs cntrl & BTX-A Tx groups
Stone et al., 2012RatA4, 6, & 10 IUTissue bathCremaster muscle microvasculatureAfter muscle dissection & placement in tissue bathNone; sympathetic contribution evaluated by adding α adrenergic blockers prazosin & rauwolscine; phenylephrine used to evaluate successful adrenergic blockadeVessel diams measured immediately after BTX-A Tx & every 5 mins up to 20 mins post-Tx; sympathetic contribution evaluated after adding pharmacologic agents & evaluating diam every 5 mins up to 20 minsMicrovascular measurements using live video microscopy, direct systemic arterial pressure measurements using femoral catheterNoneBTX-A Tx caused sig more prominent arteriolar vasodilation vs cntrls w/ sig dose & time effects; max vasodilation at 10 mins post-Tx w/ 6 & 10 IU doses being more effective than 4 IU; BTX-A vasodilatory effects likely mediated by sympathetic adrenergic blockade
Schweizer et al., 2013MouseA & B0.1 IU & 0.5 IUSubcut inject near vascular pedicleAxially perfused dorsal skin flapPreconditioning group, 24 hrs before op; Tx group, intraoperativelyNonePOD 1, 3, & 5 blood flow & tissue oxygenation measurements; microdialysis performed on POD 1 & development of tissue necrosis evaluated on POD 5Blood flow measurements using laser Doppler flowmetry; tissue partial O2 tension measured using microprobes; microdialysis measurements of tissue metabolism (glucose, lactate/pyruvate ratio, glycerol); tissue necrosis eval using laser Doppler camera; immunofluorescent IHC expression of TUNEL, eNOS, & RhoAeNOS, RhoABTX Tx groups had sig ↑ blood flow vs cntrl; sig improved tissue oxygenation, higher glucose & lower lactate levels were observed in BTX Tx groups vs cntrl; flap viability was sig ↑ in BTX groups as were levels of eNOS & RhoA immunoexpression; no difference btwn BTX types & application time
Akcal et al., 2013RatA3.5 IUIM inject in midportion of flap, perivascular inject in connective tissue around vesselsGastrocnemius muscle flapPre-Tx 7 days before flap elevation & global flap ischemia induction then 7-day reperfusion periodNone7 days post-reperfusion, 14 days post-TxMacro- & microscopic eval of angiogenesis, amount of lymphocyte infiltration, edema, & myocyte damage; IHC eval of CGRP, FLT-4, VEGF, & substance P expressionVEGF, substance PDegree of muscle flap injury not sig different among Tx groups; sig ↑ no. of fibroblasts in BTX-A Tx groups; no sig general differences in CGRP, VEGF, & substance P immunoexpression among the groups; CGRPR1 expression marked in thick-walled vessels of BTX-A groups; VEGF & substance P expression marked in thin-walled vessels of BTX-A groups; perivascular BTX-A application showed less necrosis, inflammation, & edema than IM application group
Arnold et al., 2014RatA2 IU/ml, 1 mlInject in soft tissue around base of pediclePedicled ab flap elevated on superficial inferior epigastric vesselsOn flap elevationNonePOD 1, 2, & 7mRNA expression of TNFα, IL-1, & VEGF using RT-PCRVEGF↓ IL-1 mRNA expression on POD 2, ↓ TNFα mRNA expression on POD 2 & 7
Kucukkaya et al., 2014RatA0.5 IUInject in open wound & graft areaDorsal cutaneous flapOn flap elevationNoneGraft area evaluated on POD 5, 15, 30, & 60Macro- & microscopic exam using simple stainsNoneSig ↑ in neovascularization & fibroblast density, ↓ in no. of hair follicles, sweat, & sebaceous glands in BTX-A group vs cntrls; nonsig ↑ in mononuclear cell infiltration in BTX-A group vs cntrls; lesser degree of wound contraction observed in BTX-A vs cntrls
Karayel et al., 2015RatA2 IUAdministration in center of proximal 3rd of flapRandom cutaneous flap1 wk before flap elevationCigarette smokeFlap evaluated for viability daily for 14 days; hist eval on POD 7Clinical necrotic area eval & measurements, hist exam using H&E stainSig ↓ necrotic area in BTX-A Tx group undergoing tobacco exposure along w/ ↑ angiogenesis & reduced epithelial damage
Park et al., 2015RatA20 IUSubdermal injects distributed evenly through flapTransverse rectus abdominis myocutaneous flap5 days after initial midline incision, 5 days before flap elevationNoneFlap survival evaluated on POD 0, 1, 3, & 5Gross eval of flap survival, hist eval using H&E, IHC of CD31, CD31, & VEGF mRNA expression level detection using RT-PCRVEGFBTX-A group showed sig improved flap survival, larger artery & vein lumen area, higher CD31 immunoexpression in certain flap areas, lower CD31 & higher VEGF mRNA expression
Camargo et al., 2016RatA20 IUMultiple intradermal injects to cover flap areaRandom dorsal cutaneous flap7 days before flap elevationCigarette smoke2 & 4 mos tobacco exposure; eval on POD 7Necrotic flap area measurements, carboxyhemoglobin measurement in blood samplesNoneBTX-A ↑ random flap viability in tobacco-exposed rats at 2 & 4 mos
Ghanbarzadeh et al., 2016RatA24 IU/kgIntradermal injects distributed throughout flapRandom dorsal cutaneous flap2 wks before flap elevationNoneNecrosis evaluated on POD 7Necrotic flap area measurementsNoneBTX-A Tx sig reduced distal flap necrosis vs cntrl & topical nitroglycerine application groups
Park et al., 2016RatA10 IUSubdermal injectTransverse rectus abdominis myocutaneous flap5 days before flap elevationNoneFlap survival area evaluated on POD 5, gene expression analysis evaluated on POD 1, 3, & 5Surviving flap area measurements, RhoA, Rac1, & Cdc42 mRNA expression quantification using RT-PCRRhoARhoA, Rac1, & Cdc42 (angiogenesis regulators) mRNA expression sig higher in BTX-A group vs cntrls in all zones of flap
Human dermal fibroblasts in vitroA1, 5, & 10 IUAdded to cell cultureNA1 day before gene expression analysisNAGene expression analysis 1 day after BTX-A TxRhoA, Rac1, & Cdc42 mRNA expression quantification using RT-PCRRhoARhoA mRNA expression sig higher in 1 IU BTX-A group while all 3 gene mRNA expression sig upregulated in 5 & 10 IU groups vs cntrls w/ most prominent effects observed in 10 IU group
Aru et al., 2017RatA10 IU/ml, 0.2 mlNRSpinotrapezius muscle flap arteriole2 wks before flap elevationNone2 wksMicrocirculation assessment using transillumination through microscopeNoneSig greater mean baseline arteriolar diam in BTX-A pre-Tx group
Roh et al., 2017RatA29 IU/kgInject in center of flapRandom dorsal cutaneous flap3 days before flap elevationNoneFlap survival & blood flow eval, HPLC analysis of amount of NE, WB expression of eNOS & NPY & calorimetric assay of NO performed on POD 3 & 7; on POD 7, IHC eval of VEGF & PECAM/CD31 immunoexpressionSurviving flap area measurements, in vivo microcirculation assessment using laser Doppler flowmetry, VEGF & CD31 IHC expression, NE level measurements using HPLC, NPY & eNOS protein expression analysis using WB, colorimetric assay of NO end productsVEGF, NE, eNOS, NOBTX-A Tx sig improved flap survival & ↑ blood flow in all flap areas on POD 3 & in distal flap area on POD 7 vs cntrls; sig higher CD31 immunoexpression was found in BTX-A groups vs cntrls; expression of NE in BTX-A group was sig lower immediately after flap elevation & on POD 3 vs cntrls; eNOS protein level was sig higher in BTX-A Tx group immediately after flap elevation & on POD 3; expression of NPY & levels of NO were not affected by BTX-A
Huang, 2018RatA5 IUSubcut inject near vascular pedicleAb skin flap raised on epigastric artery12 hrs before flap elevation & ischemia-reperfusion inductionNone5 days after 2 ischemia-reperfusion challenges & areas of ischemia & necrosis were evaluatedMeasurement of necrotic & ischemic flap areasNoneNo sig differences on necrosis btwn Tx & cntrl groups; extent of arterial & venous ischemia was sig smaller in BTX-A group vs cntrls

Ab = abdominal; CD31 = cluster of differentiation 31; Cdc42 = cell division control protein 42; CGRPR1 = CGRP receptor 1; FLT-4 = Fms-related tyrosine kinase 4; HPLC = high-performance liquid chromatography; IHC = immunohistochemistry; IL-1 = interleukin-1; IM = intramuscular; iNOS = inducible nitric oxide synthase; mRNA = matrix RNA; NA = not applicable; NPY = neuropeptide Y; NR = not reported; PECAM = platelet endothelial cell adhesion molecule; Rac1 = Rac family small GTPase 1; RT-PCR = real-time polymerase chain reaction; TNFα = tumor necrosis factor–alpha; TUNEL = terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling; WB = Western blot; ↑ = increase(d); ↓ = decrease(d).

Although these animal studies provide valuable insights into the potential benefit of BTX in revascularization surgery, marked differences in innervation and response to spasmogens have been demonstrated between human and animal blood vessels.26,28,29 The first translational application of BTX-A for cerebrovascular bypass graft spasm prevention in humans was only recently published.63

Potential Mechanisms of BTX A–Mediated Vasospasm Prevention

BTX and Adrenergic Vascular Innervation

Understanding the spasmolytic effects of BTX requires a mechanistic understanding of vasoconstriction. Postganglionic sympathetic axons form a plexus at the adventitia-media border in most arteries, arterioles, and veins across animal species, including humans.68,70 Norepinephrine (NE), produced in sympathetic nerves, is released from perivascular axon varicosities after sympathetic stimulation and binds to VSMC postsynaptic α and β adrenoreceptors. NE-induced VSMC contraction in most major arteries is thought to be primarily mediated by postsynaptic α1 adrenoreceptor activation, with variable contribution from α2 receptors (Fig. 2),23 although the relative contributions of α1 and α2 adrenoreceptors to vasoconstriction depends on the anatomical location and segment of the vessel. In fact, in limb arteries the α2 adrenoreceptor role in vasoconstriction increases when moving proximal to distal,23 a finding that, when combined with the predisposition of the distal end of grafts to vasospasm, suggests a potentially important role of α2 receptors in this process.55

FIG. 2.
FIG. 2.

RhoA/ROCK and adrenergic pathways mediating VSMC contraction. The ROCK pathway is primarily activated by vasoconstrictive substance binding to a G protein–coupled receptor and activating guanine nucleotide exchange factor (GEF). It can also be activated by reactive oxygen species (ROS) produced by the VSMC mitochondria upon cold exposure or via mechanical stimulus. In the case of cold exposure, activated ROCK promotes α2c receptor recruitment from the endoplasmic reticulum to the cell membrane. Phosphorylation of the MLC by MLC kinase (MLCK) activated by Ca2+-calmodulin is the main molecular event mediating VSMC contraction. VSMC contraction can also be promoted by MLCP (which mediates MLC dephosphorylation and subsequent inhibition) inactivation by the ROCK downstream effector MYPT1. Adrenergic VSMC contraction occurs via two pathways depending on which receptor is activated. NE binding to the α1 receptor activates phospholipase C β (PLCβ), which increases intracellular Ca2+ levels mediating MLCK activation. NE binding to the α2 receptor inhibits adenylyl cyclase (AC), subsequently preventing inactivation of MLCK. BTX A–mediated VSMC relaxation could be mediated via inhibition of the ROCK pathway (1–3), or by blocking release of NE from perivascular adrenergic nerves (4). 5-HT = 5-hydroxytryptamine; cAMP = cyclic adenosine monophosphate; GDP = guanosine diphosphate; GTP = guanosine triphosphate; IP3 = inositol triphosphate.

The affinity of BTX for cholinergic transmission blockade has been found to be related to the abundance of high-affinity acceptor sites on cholinergic neurons.9 Although noncholinergic nerve terminals have a relatively low number of these acceptor sites, BTX can still be taken up via low-affinity sites if its concentration is high enough.9,44 Moreover, while BTX-A cleavable SNAP-25 has been found in sympathetic axons innervating guinea pig blood vessels, NE-mediated vascular constriction was shown to be reduced after BTX-A administration with only partial cleavage of SNAP-25.43,44 In light of these findings, it was hypothesized that BTX-A might also be inhibiting adrenergic neurotransmission via a mechanism distinct from SNAP-25 cleavage.44

A recent study utilized α1 and α2 receptor blockers to indirectly examine the mechanism of BTX A–mediated vasodilation in rat cremaster muscle microvasculature.62 Sympathetic blockade seemed to be the likely mechanism, as BTX-A and adrenergic antagonist administration caused similar levels of vasodilation when applied separately, while BTX application after adrenergic antagonist administration caused no additional vasodilation. Another, more recent study evaluated the effects of BTX-A injection on the survival of random dorsal cutaneous flaps in a rat model,54 and reported significantly improved flap survival, increased blood flow, and elevated expression of angiogenesis markers and endothelial nitric oxide synthase (eNOS) in the BTX A–treated as compared to the control group. Interestingly, flap tissue NE levels, as measured by high-performance liquid chromatography assay, were found to be significantly reduced immediately and 3 days after flap elevation in the BTX group (3 and 6 days after BTX-A treatment, respectively), suggesting that BTX A–mediated blockade of NE release might be contributing to its vasodilatory effects (Fig. 2). Although the reduction of NE found 3 days after BTX-A treatment in this work is likely mediated in part via SNAP-25 cleavage, earlier time points after BTX-A treatment were not investigated, leaving open questions regarding the mechanisms of early BTX-A effects seen when applied to cerebral revascularization and Raynaud’s phenomenon (discussed below). Although these studies provide valuable insights into the possible mechanisms of BTX-A vascular spasmolytic activity, they may not be directly translatable to humans given the arterial graft variability and differences in small animal versus human vascular innervation.

Insights From BTX-A Utility in the Treatment of Raynaud’s Phenomenon

Raynaud’s phenomenon is characterized by an exaggerated vasospastic response to cold or emotional stress stimuli in cutaneous arterioles.20,46,71 The first report of BTX-A in patients with Raynaud’s phenomenon was published in 2004.64 Since then, multiple case series have demonstrated that direct perivascular BTX-A injections can provide rapid and long-lasting digit vasospasm relief.46,47,69 Immediate pain relief was reported in more than 80% of patients, while improved digit perfusion (as evaluated by laser Doppler scans) was achieved within 30 minutes of injection in about 70% of patients, with a striking 300% flow improvement as early as 15 minutes after injection.47 Notably, the effect of BTX-A in this setting appears to be prolonged, ranging from 13 to 59 months after a single injection.47 Direct vasodilatory effects of BTX have also been reported in a rat model as early as 5 minutes after treatment, with a maximum effect seen at 15 minutes.62 This differs from classic BTX-A characteristic effects, which have a known delay of several days and a duration of approximately 3–4 months.46,47,51 Insights from the current literature on BTX-A indicate that the mechanism of vasospasm prevention is likely different from the classic paradigm.

BTX-A and RhoA/ROCK Pathway

Growing evidence also demonstrates ras homolog gene family member A (RhoA)/rho-associated protein kinase (ROCK) pathway involvement in not only hypertension pathogenesis via increased inflammation, VSMC contractility, and endothelial dysfunction from negative regulation of the nitric oxide (NO) pathway,16,59,61,72 but also in graft vasospasm via similar mechanisms.28 Takagi et al. investigated the effects of the ROCK inhibitor fasudil on radial artery graft vasospasm prevention.65 Their findings showed an increase of in situ free blood flow with a corresponding decrease of myosin phosphatase targeting subunit 1 (MYPT1) and myosin light chain (MLC) phosphorylation that strongly counteracted the effects of major vasoconstrictors (NE and 5-hydroxytryptamine). Enhanced ROCK activity was also identified in skeletonized, spastic radial arteries as compared to nonmanipulated, nonspastic arteries, indicating that mechanical stimulation–induced vasospasm is likely mediated via the ROCK pathway as well. Moreover, ROCK pathway inhibition via abolished MYPT1 and MLC phosphorylation yielded a greater increase of in situ free blood flow as compared to that produced by the widely used antispastic agent verapamil, which showed no effect on ROCK.65 Currently, however, the clinical use of fasudil is approved only in Japan and China.

The two main mechanisms of ROCK-mediated upregulation of VSMC contractility are calcium-dependent translocation of α2c adrenoreceptors from the Golgi apparatus to the plasma membrane, and inhibition of myosin light chain phosphatase (MLCP; Fig. 2).61 Findings from Smith et al.61 also demonstrate that ROCK activity (specifically, phosphorylation of MYPT1) and ROCK expression itself can change independently, as MYPT1 phosphorylation activity can increase while ROCK expression remains unchanged in hypertensive as compared to normotensive subjects. This underlines the distinction between ROCK expression and the functionality of its downstream effectors, and provides a potential explanation for the recent controversial findings of BTX A–mediated Rho kinase upregulation in murine myocutaneous and skin flaps.49,57 In these and numerous other animal studies, improved flap survival, increased tissue perfusion, vasodilation, and angiogenesis after BTX-A pretreatment has been demonstrated.6,8,39,49,57,66 Hence, the reported upregulation of Rho kinase expression in response to BTX-A seems to be contradictory to its well-reported vasodilatory effects in animal models and Raynaud’s phenomenon treatments in humans.20,46,64 Accordingly, Schweizer et al. hypothesized that increased RhoA immunoexpression after BTX-A administration could result from a compensatory overexpression in response to functional inhibition,57 however, no targeted investigations of BTX A–specific effects on RhoA downstream effectors in the ROCK pathway have been reported.

BTX-A and Other Vasoactive Substance Release

Evidence supporting other mechanistic pathways for BTX-mediated spasmolytic effects has also been reported. Calcitonin gene-related peptide (CGRP) is a known microvascular dilator found in sensory and motor nerve endings,20,67 and CGRP upregulation in rat cholinergic nerve terminals and neurons after BTX-mediated muscular paralysis has been found.67 Substance P is a neuropeptide involved in neurogenic inflammation and endothelium-dependent NO-mediated vasodilation.20 Although no significant CGRP and substance P overall immunoexpression differences in BTX A–treated muscular flaps as compared to controls has been demonstrated, prominent CGRP expression was specifically found in thick-walled vessels, while substance P expression was limited to the thin-walled vessels of BTX A–treated tissues.1 Expression of vascular endothelial growth factor (VEGF), a factor in NO-mediated angiogenesis and vasodilation, has also been shown to be increased following BTX administration in multiple animal studies.1,39,50 Although more detailed investigation is necessary, these findings indicate that the role of BTX in vasodilation is likely multifactorial.

BTX-A Clinical Use and Safety

BTX is currently FDA approved for therapeutic and cosmetic indications and has demonstrated an excellent safety profile in labeled as well as unlabeled applications.3,15 Serious adverse events such as death, generalized muscle weakness, dysphagia, respiratory insufficiency, and anaphylaxis after BTX injections have been rarely reported and are typically associated with excessively high toxin doses and serious predisposing comorbidities in affected patients.15 Generalized side effects have been associated with retrograde axonal migration of BTX from the site of injection.2–4 The proposed ex-vivo perivascular toxin application by soaking the graft in a reconstituted BTX-A solution intraoperatively during cerebral bypass does not involve injections.63 Moreover, after approximately 30 minutes of soaking in BTX-A solution, the graft is thoroughly flushed and washed to remove any residual toxin, thus minimizing risks of local and systemic spread. While more extensive studies determining optimal dosing and exposure times for BTX-A use are certainly necessary, the concentration of the toxin proposed in cerebral revascularization grafts is only 10 IU/ml.63 As a comparison, recommended doses of BTX-A used in injection form for spastic disorders reaches up to 100 IU/ml, with total dosages of 360 IU per treatment session.3

Similar to numerous currently practiced unlabeled BTX-A and other off-label medication uses,12,13 BTX-A use in graft vasospasm is unlabeled and will likely remain so given the costly and time-consuming nature of obtaining FDA approval for new indications of currently approved drugs.21,73 Unlabeled drug use, defined as prescribing currently available medications for an indication that does not have FDA approval (including unapproved patient populations, doses, or administration forms),73 is nonetheless at the treating physician’s discretion for indications that they believe are in the patient’s best interests based on credible evidence.21,73

Conclusions

Animal studies suggest that BTX effects on the vasculature likely reach beyond its classic cholinergic paradigm and involve multiple pathways. The optimal application conditions and therapeutic outcomes for BTX use in revascularization patients remain ill defined. Elucidating the role of BTX-A in graft spasm prevention will aid in determining whether and how the findings from animal studies are translated to clinical practice. This knowledge will aid in optimizing patients, conduit selection, and application conditions for BTX treatment. Based on experimental data and initial clinical reports of BTX-A for spasmolysis, the applications for BTX-A will likely extend to cardiac revascularization as well as reconstructive surgery.

Disclosures

The authors report no conflict of interest concerning the materials or methods used in this study or the findings specified in this paper.

Author Contributions

Conception and design: all authors. Acquisition of data: Russin, Ravina, Carey. Analysis and interpretation of data: all authors. Drafting the article: Russin, Ravina, Rennert. Critically revising the article: Russin, Ravina, Strickland. Reviewed submitted version of manuscript: all authors. Administrative/technical/material support: Russin, Carey. Study supervision: Russin.

References

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    Akcal ASevim KZYesilada AKiyak VSucu DOTatlidede HS: Comparison of perivascular and intramuscular applied botulinum toxin a pretreatment on muscle flap ischemia-reperfusion injury and chemical delay. J Craniofac Surg 24:2782832013

  • 2

    Alimohammadi MPunga AR: Neurophysiological measures of efficacy and safety for botulinum toxin injection in facial and bulbar muscles: special considerations. Toxins (Basel) 9:E3522017

  • 3

    Alter KEWilson NA: Botulinum Neurotoxin Injection Manual. New York: Demos Medical Publishing2015

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    Antonucci FRossi CGianfranceschi LRossetto OCaleo M: Long-distance retrograde effects of botulinum neurotoxin A. J Neurosci 28:368936962008

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    Arnold PBCampbell CARodeheaver GMerritt WMorgan RFDrake DB: Modification of blood vessel diameter following perivascular application of botulinum toxin-A. Hand (N Y) 4:3023072009

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    Arnold PBFang TSongcharoen SJZiakas GZhang F: Inflammatory response and survival of pedicled abdominal flaps in a rat model after perivascular application of botulinum toxin type A. Plast Reconstr Surg 133:491e498e2014

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    Arnold PBMerritt WRodeheaver GTCampbell CAMorgan RFDrake DB: Effects of perivascular botulinum toxin-A application on vascular smooth muscle and flap viability in the rat. Ann Plast Surg 62:4634672009

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    Aru RGSongcharoen SJSeals SRArnold PBHester RL: Microcirculatory effects of botulinum toxin A in the rat: acute and chronic vasodilation. Ann Plast Surg 79:82852017

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    Black JDDolly JO: Selective location of acceptors for botulinum neurotoxin A in the central and peripheral nervous systems. Neuroscience 23:7677791987

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    Camargo CPFernandes FALee MHSilva LCBesteiro JMGemperli R: The positive effect of Botulinum toxin type A on the viability of random flap in tobacco exposed in rats. Acta Cir Bras 31:7207232016

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    Carrel TWinkler B: Current trends in selection of conduits for coronary artery bypass grafting. Gen Thorac Cardiovasc Surg 65:5495562017

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    Cheng CMChen JSPatel RP: Unlabeled uses of botulinum toxins: a review, part 1. Am J Health Syst Pharm 63:1451522006

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    Cheng CMChen JSPatel RP: Unlabeled uses of botulinum toxins: a review, part 2. Am J Health Syst Pharm 63:2252322006

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    Clemens MWHiggins JPWilgis EF: Prevention of anastomotic thrombosis by botulinum toxin a in an animal model. Plast Reconstr Surg 123:64702009

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    Fabbrocini MFattouch KCamporini GDeMicheli GBertucci CCioffi P: The descending branch of lateral femoral circumflex artery in arterial CABG: early and midterm results. Ann Thorac Surg 75:183618412003

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    Fathi MFathi HMazloumi MKhalilzadeh OAmanpour SMeysamie A: Preventive effect of botulinum toxin A in microanastomotic thrombosis: a rabbit model. J Plast Reconstr Aesthet Surg 63:e720e7242010

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    Feng LFitzsimmons BFYoung WLBerman MFLin EAagaard BD: Intraarterially administered verapamil as adjunct therapy for cerebral vasospasm: safety and 2-year experience. AJNR Am J Neuroradiol 23:128412902002

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    Fonseca CAbraham DPonticos M: Neuronal regulators and vascular dysfunction in Raynaud’s phenomenon and systemic sclerosis. Curr Vasc Pharmacol 7:34392009

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    Ghanbarzadeh KTabatabaie ORSalehifar EAmanlou MKhorasani G: Effect of botulinum toxin A and nitroglycerin on random skin flap survival in rats. Plast Surg (Oakv) 24:991022016

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    Guimarães SMoura D: Vascular adrenoceptors: an update. Pharmacol Rev 53:3193562001

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    He GWTaggart DP: Antispastic management in arterial grafts in coronary artery bypass grafting surgery. Ann Thorac Surg 102:6596682016

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    He GWTaggart DP: Spasm in arterial grafts in coronary artery bypass grafting surgery. Ann Thorac Surg 101:122212292016

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    He GWYang CQ: Radial artery has higher receptor-mediated contractility but similar endothelial function compared with mammary artery. Ann Thorac Surg 63:134613521997

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    Heiferman DMSouter JRReynolds MRAnderson DESerrone JC: Extracranial-to-intracranial bypass for pressor dependent cerebrovascular insufficiency: modified classification and representative case. Curr Neurovasc Res 15:2562612018

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    Huang L: Beneficial effect of botulinum toxin A on secondary ischaemic injury of skin flaps in rats. Br J Oral Maxillofac Surg 56:1441472018

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    Jankovic J: Botulinum toxin: state of the art. Mov Disord 32:113111382017

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    Janz BAThomas PRFanua SPDunn REWilgis EFMeans KR Jr: Prevention of anastomotic thrombosis by botulinum toxin B after acute injury in a rat model. J Hand Surg Am 36:158515912011

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    Joyner MJDietz NM: Sympathetic vasodilation in human muscle. Acta Physiol Scand 177:3293362003

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    Keyrouz SGDiringer MN: Clinical review: Prevention and therapy of vasospasm in subarachnoid hemorrhage. Crit Care 11:2202007

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    Khot UNFriedman DTPettersson GSmedira NGLi JEllis SG: Radial artery bypass grafts have an increased occurrence of angiographically severe stenosis and occlusion compared with left internal mammary arteries and saphenous vein grafts. Circulation 109:208620912004

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    Kim TKOh EJChung JYPark JWCho BCChung HY: The effects of botulinum toxin A on the survival of a random cutaneous flap. J Plast Reconstr Aesthet Surg 62:9069132009

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    Kucukkaya DIrkoren SOzkan SSivrioglu N: The effects of botulinum toxin A on the wound and skin graft contraction. J Craniofac Surg 25:190819112014

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    Lawton MTHamilton MGMorcos JJSpetzler RF: Revascularization and aneurysm surgery: current techniques, indications, and outcome. Neurosurgery 38:83941996

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    Liu JKKan PKarwande SVCouldwell WT: Conduits for cerebrovascular bypass and lessons learned from the cardiovascular experience. Neurosurg Focus 14(3):e32003

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    Morris JLJobling PGibbins IL: Botulinum neurotoxin A attenuates release of norepinephrine but not NPY from vasoconstrictor neurons. Am J Physiol Heart Circ Physiol 283:H2627H26352002

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Article Information

Correspondence Jonathan J. Russin: University of Southern California Neurorestoration Center, The Keck School of Medicine, Los Angeles, CA. jonathan.russin@med.usc.edu.

INCLUDE WHEN CITING DOI: 10.3171/2018.11.FOCUS18514.

Disclosures The authors report no conflict of interest concerning the materials or methods used in this study or the findings specified in this paper.

© AANS, except where prohibited by US copyright law.

Headings

Figures

  • View in gallery

    Factors involved in vasoreactivity. Vasoreactivity is determined not only by external/systemic factors (presence of physical stimuli and vasoactive substances), but also by intrinsic anatomical and physiological contractile properties of blood vessels as dictated by the density and composition of receptors and vessel wall structure.

  • View in gallery

    RhoA/ROCK and adrenergic pathways mediating VSMC contraction. The ROCK pathway is primarily activated by vasoconstrictive substance binding to a G protein–coupled receptor and activating guanine nucleotide exchange factor (GEF). It can also be activated by reactive oxygen species (ROS) produced by the VSMC mitochondria upon cold exposure or via mechanical stimulus. In the case of cold exposure, activated ROCK promotes α2c receptor recruitment from the endoplasmic reticulum to the cell membrane. Phosphorylation of the MLC by MLC kinase (MLCK) activated by Ca2+-calmodulin is the main molecular event mediating VSMC contraction. VSMC contraction can also be promoted by MLCP (which mediates MLC dephosphorylation and subsequent inhibition) inactivation by the ROCK downstream effector MYPT1. Adrenergic VSMC contraction occurs via two pathways depending on which receptor is activated. NE binding to the α1 receptor activates phospholipase C β (PLCβ), which increases intracellular Ca2+ levels mediating MLCK activation. NE binding to the α2 receptor inhibits adenylyl cyclase (AC), subsequently preventing inactivation of MLCK. BTX A–mediated VSMC relaxation could be mediated via inhibition of the ROCK pathway (1–3), or by blocking release of NE from perivascular adrenergic nerves (4). 5-HT = 5-hydroxytryptamine; cAMP = cyclic adenosine monophosphate; GDP = guanosine diphosphate; GTP = guanosine triphosphate; IP3 = inositol triphosphate.

References

1

Akcal ASevim KZYesilada AKiyak VSucu DOTatlidede HS: Comparison of perivascular and intramuscular applied botulinum toxin a pretreatment on muscle flap ischemia-reperfusion injury and chemical delay. J Craniofac Surg 24:2782832013

2

Alimohammadi MPunga AR: Neurophysiological measures of efficacy and safety for botulinum toxin injection in facial and bulbar muscles: special considerations. Toxins (Basel) 9:E3522017

3

Alter KEWilson NA: Botulinum Neurotoxin Injection Manual. New York: Demos Medical Publishing2015

4

Antonucci FRossi CGianfranceschi LRossetto OCaleo M: Long-distance retrograde effects of botulinum neurotoxin A. J Neurosci 28:368936962008

5

Arnold PBCampbell CARodeheaver GMerritt WMorgan RFDrake DB: Modification of blood vessel diameter following perivascular application of botulinum toxin-A. Hand (N Y) 4:3023072009

6

Arnold PBFang TSongcharoen SJZiakas GZhang F: Inflammatory response and survival of pedicled abdominal flaps in a rat model after perivascular application of botulinum toxin type A. Plast Reconstr Surg 133:491e498e2014

7

Arnold PBMerritt WRodeheaver GTCampbell CAMorgan RFDrake DB: Effects of perivascular botulinum toxin-A application on vascular smooth muscle and flap viability in the rat. Ann Plast Surg 62:4634672009

8

Aru RGSongcharoen SJSeals SRArnold PBHester RL: Microcirculatory effects of botulinum toxin A in the rat: acute and chronic vasodilation. Ann Plast Surg 79:82852017

9

Black JDDolly JO: Selective location of acceptors for botulinum neurotoxin A in the central and peripheral nervous systems. Neuroscience 23:7677791987

10

Camargo CPFernandes FALee MHSilva LCBesteiro JMGemperli R: The positive effect of Botulinum toxin type A on the viability of random flap in tobacco exposed in rats. Acta Cir Bras 31:7207232016

11

Carrel TWinkler B: Current trends in selection of conduits for coronary artery bypass grafting. Gen Thorac Cardiovasc Surg 65:5495562017

12

Cheng CMChen JSPatel RP: Unlabeled uses of botulinum toxins: a review, part 1. Am J Health Syst Pharm 63:1451522006

13

Cheng CMChen JSPatel RP: Unlabeled uses of botulinum toxins: a review, part 2. Am J Health Syst Pharm 63:2252322006

14

Clemens MWHiggins JPWilgis EF: Prevention of anastomotic thrombosis by botulinum toxin a in an animal model. Plast Reconstr Surg 123:64702009

15

Coté TRMohan AKPolder JAWalton MKBraun MM: Botulinum toxin type A injections: adverse events reported to the US Food and Drug Administration in therapeutic and cosmetic cases. J Am Acad Dermatol 53:4074152005

16

Dong MYan BPLiao JKLam YYYip GWYu CM: Rho-kinase inhibition: a novel therapeutic target for the treatment of cardiovascular diseases. Drug Discov Today 15:6226292010

17

Fabbrocini MFattouch KCamporini GDeMicheli GBertucci CCioffi P: The descending branch of lateral femoral circumflex artery in arterial CABG: early and midterm results. Ann Thorac Surg 75:183618412003

18

Fathi MFathi HMazloumi MKhalilzadeh OAmanpour SMeysamie A: Preventive effect of botulinum toxin A in microanastomotic thrombosis: a rabbit model. J Plast Reconstr Aesthet Surg 63:e720e7242010

19

Feng LFitzsimmons BFYoung WLBerman MFLin EAagaard BD: Intraarterially administered verapamil as adjunct therapy for cerebral vasospasm: safety and 2-year experience. AJNR Am J Neuroradiol 23:128412902002

20

Fonseca CAbraham DPonticos M: Neuronal regulators and vascular dysfunction in Raynaud’s phenomenon and systemic sclerosis. Curr Vasc Pharmacol 7:34392009

21

França KLitewka S: Controversies in off-label prescriptions in dermatology: the perspective of the patient, the physician, and the pharmaceutical companies. Int J Dermatol [epub ahead of print] 2018

22

Ghanbarzadeh KTabatabaie ORSalehifar EAmanlou MKhorasani G: Effect of botulinum toxin A and nitroglycerin on random skin flap survival in rats. Plast Surg (Oakv) 24:991022016

23

Guimarães SMoura D: Vascular adrenoceptors: an update. Pharmacol Rev 53:3193562001

24

He GW: Arterial grafts: clinical classification and pharmacological management. Ann Cardiothorac Surg 2:5075182013

25

He GW: Arterial grafts for coronary artery bypass grafting: biological characteristics, functional classification, and clinical choice. Ann Thorac Surg 67:2772841999

26

He GWRosenfeldt FLBuxton BFAngus JA: Reactivity of human isolated internal mammary artery to constrictor and dilator agents. Implications for treatment of internal mammary artery spasm. Circulation 80:I141I1501989

27

He GWTaggart DP: Antispastic management in arterial grafts in coronary artery bypass grafting surgery. Ann Thorac Surg 102:6596682016

28

He GWTaggart DP: Spasm in arterial grafts in coronary artery bypass grafting surgery. Ann Thorac Surg 101:122212292016

29

He GWYang CQ: Characteristics of adrenoceptors in the human radial artery: clinical implications. J Thorac Cardiovasc Surg 115:113611411998

30

He GWYang CQ: Radial artery has higher receptor-mediated contractility but similar endothelial function compared with mammary artery. Ann Thorac Surg 63:134613521997

31

Heiferman DMSouter JRReynolds MRAnderson DESerrone JC: Extracranial-to-intracranial bypass for pressor dependent cerebrovascular insufficiency: modified classification and representative case. Curr Neurovasc Res 15:2562612018

32

Huang L: Beneficial effect of botulinum toxin A on secondary ischaemic injury of skin flaps in rats. Br J Oral Maxillofac Surg 56:1441472018

33

Jankovic J: Botulinum toxin: state of the art. Mov Disord 32:113111382017

34

Janz BAThomas PRFanua SPDunn REWilgis EFMeans KR Jr: Prevention of anastomotic thrombosis by botulinum toxin B after acute injury in a rat model. J Hand Surg Am 36:158515912011

35

Joyner MJDietz NM: Sympathetic vasodilation in human muscle. Acta Physiol Scand 177:3293362003

36

Karayel HKaya BCaydere MTerzioğlu AAslan G: Prevention of unfavourable effects of cigarette smoke on flap viability using botulinum toxin in random pattern flaps: an experimental study. Plast Surg (Oakv) 23:1771822015

37

Keyrouz SGDiringer MN: Clinical review: Prevention and therapy of vasospasm in subarachnoid hemorrhage. Crit Care 11:2202007

38

Khot UNFriedman DTPettersson GSmedira NGLi JEllis SG: Radial artery bypass grafts have an increased occurrence of angiographically severe stenosis and occlusion compared with left internal mammary arteries and saphenous vein grafts. Circulation 109:208620912004

39

Kim TKOh EJChung JYPark JWCho BCChung HY: The effects of botulinum toxin A on the survival of a random cutaneous flap. J Plast Reconstr Aesthet Surg 62:9069132009

40

Kucukkaya DIrkoren SOzkan SSivrioglu N: The effects of botulinum toxin A on the wound and skin graft contraction. J Craniofac Surg 25:190819112014

41

Lawton MTHamilton MGMorcos JJSpetzler RF: Revascularization and aneurysm surgery: current techniques, indications, and outcome. Neurosurgery 38:83941996

42

Liu JKKan PKarwande SVCouldwell WT: Conduits for cerebrovascular bypass and lessons learned from the cardiovascular experience. Neurosurg Focus 14(3):e32003

43

Morris JLJobling PGibbins IL: Botulinum neurotoxin A attenuates release of norepinephrine but not NPY from vasoconstrictor neurons. Am J Physiol Heart Circ Physiol 283:H2627H26352002

44

Morris JLJobling PGibbins IL: Differential inhibition by botulinum neurotoxin A of cotransmitters released from autonomic vasodilator neurons. Am J Physiol Heart Circ Physiol 281:H2124H21322001

45

Murakami EIwata HImaizumi MTakemura H: Prevention of arterial graft spasm by botulinum toxin: an in-vitro experiment. Interact Cardiovasc Thorac Surg 9:3953982009

46

Neumeister MW: The role of botulinum toxin in vasospastic disorders of the hand. Hand Clin 31:23372015

47

Neumeister MWChambers CBHerron MSWebb KWietfeldt JGillespie JN: Botox therapy for ischemic digits. Plast Reconstr Surg 124:1912012009

48

Park BYKim HKKim WSBae TH: The effect of botulinum toxin B pretreatment to the blood flow in the microvascular anastomosis. Ann Plast Surg 72:2142192014

49

Park THPark JHChang CHRah DK: Botulinum toxin A upregulates Rac1, Cdc42, and RhoA gene expression in a dose-dependent manner: in vivo and in vitro study. J Craniofac Surg 27:5165202016

50

Park THRah DKChong YKim JK: The effects of botulinum toxin A on survival of rat TRAM flap with vertical midline scar. Ann Plast Surg 74:1001062015

51

Pirazzini MRossetto OEleopra RMontecucco C: Botulinum neurotoxins: biology, pharmacology, and toxicology. Pharmacol Rev 69:2002352017

52

Ramanathan DStarnes BHatsukami TKim LJDi Maio SSekhar L: Tibial artery autografts: alternative conduits for high flow cerebral revascularizations. World Neurosurg 80:3223272013

53

Roh SWAhn JSSung HYJung YJKwun BDKim CJ: Extracranial-intracranial bypass surgery using a radial artery interposition graft for cerebrovascular diseases. J Korean Neurosurg Soc 50:1851902011

54

Roh TSJung BKYun ILew DHKim YS: Effect of botulinum toxin A on vasoconstriction and sympathetic neurotransmitters in a murine random pattern skin flap model. Wound Repair Regen 25:75852017

55

Rosenfeldt FLHe GWBuxton BFAngus JA: Pharmacology of coronary artery bypass grafts. Ann Thorac Surg 67:8788881999

56

Rossetto OPirazzini MMontecucco C: Botulinum neurotoxins: genetic, structural and mechanistic insights. Nat Rev Microbiol 12:5355492014

57

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