ARTICLE

Splenic blood-flow response following myocardial infarctionin ratSara A. Ruggiero, Jason S. Huber, Coral L. Murrant, Keith R. Brunt, and Jeremy A. Simpson

Abstract: During physiological stress (e.g., exercise, hypoxia), blood flow is shunted to specific anatomical regions to protectcritical organs; yet, splenic blood flow in these circumstances remains to be investigated. Despite being classically viewed as anon-critical organ, recent experimental and epidemiological evidence suggests the spleen plays a significant role in cardiovas-cular pathophysiology. We hypothesized that splenic blood flow is prioritized in the development of heart failure (i.e., chronicstate of reduced cardiac output). Five-week-old male Wistar rats were randomized for either myocardial infarction (MI; n = 58) orsham (n = 56) surgery. At 2, 5, and 9 weeks post-surgery, Doppler ultrasound measurements of the splenic, left renal, left commoncarotid, and left femoral arteries were performed. Cardiac function was assessed at all time points using echocardiography andat 9 weeks post-surgery using invasive hemodynamic analysis. Splenic and cerebral blood flow was preferentially maintained at9 weeks post-MI, whereas blood flow to the lower limb and kidney were reduced. Spleen size increased by 5 weeks post-MI andremained elevated. Splenic blood flow was maintained in conditions of decreased cardiac output, when other tissues showeddecreased blood flow. The maintenance of blood flow in the face of decreased cardiac output indicates that splenic function isbeing prioritized during heart failure.

Key words: heart failure, vascular, hypertension, monocytes, portal vein.

Résumé : En situation de stress physiologique (p. ex. exercice physique, hypoxie), la circulation sanguine est dérivée vers desrégions anatomiques spécifiques pour la protection d’organes cruciaux, mais la circulation splénique demeure à étudier dans cescirconstances. En dépit d’être typiquement perçue comme un organe non crucial, des données probantes expérimentales etépidémiologiques récentes laissent entendre que la rate jouerait un rôle important dans la physiopathologie cardiovasculaire.Nous avons posé que la circulation splénique est prioritaire dans l’évolution de l’insuffisance cardiaque (c.-à-d., l’état chroniquede diminution du débit cardiaque). Nous avons réparti aléatoirement des rats Wistar de 5 semaines dans des groupes d’infarctusdu myocarde (IM; n = 58) ou d’intervention chirurgicale factice (« sham »; n = 56). À l’aide de l’échographie-Doppler, nous avonsmesuré le débit de l’artère splénique, ainsi que des artères rénale, carotide commune et fémorale gauches. Nous avons évalué lafonction cardiaque à tous les jalons temporels à l’aide de l’échocardiographie, et à l’aide d’une étude hémodynamique effractive9 semaines après l’intervention chirurgicale. Neuf semaines après l’IM, la circulation se maintenait de façon préférentielle dansla rate et le cerveau, alors qu’elle était diminuée dans le membre inférieur et le rein. Cinq semaines après l’IM, le volume de larate était augmenté de manière persistante. La circulation splénique se maintenait en situation de diminution du débit cardi-aque, alors que l’on observait une diminution du débit sanguin dans d’autres tissus. Le maintien du débit sanguin en situationde diminution du débit cardiaque montre que la fonction splénique et prioritaire pendant l’insuffisance cardiaque. [Traduit parla Rédaction]

Mots-clés : insuffisance cardiaque, vasculaire, hypertension, monocytes, veine porte.

IntroductionThe spleen is classically known as the largest lymphoid organ,

responsible for clearing senescent erythrocytes, acting as a bloodreserve, and housing a significant portion of the adaptive immunesystem. Despite this, the spleen is also perceived as a non-criticalorgan. This is due in large part from the general surgical perspec-tive, as splenectomy is associated with excellent outcomes in thenear term, with �22 000 splenectomies being performed eachyear (DeFrances et al. 2007). Yet, the spleen has recently garnerednew attention for its role in cardiovascular homeostasis. Someepidemiological studies have concluded that splenectomy, or

damage to the spleen generally, elevates the risk of developingcardiovascular disease in the long term (Rørholt et al. 2017; Tsaiet al. 2015). Studies targeted at examining the contribution of thespleen to the cardiovascular system under stress show that thespleen has an important cellular housing function from whichmonocytes are mobilized after cardiac injury, and by which im-mune responses in animal models of disease are regulated(Ben-Mordechai et al. 2013; Kondo et al. 2016; Swirski et al. 2009;van der Laan et al. 2014). However, there are conflicting view-points as to whether the role of the spleen is adaptive, compensa-tory, or determinant to the pathophysiology of heart failure(Ismahil et al. 2014; Kondo et al. 2016; Tian et al. 2016).

Received 12 March 2018. Accepted 5 August 2018.

S.A. Ruggiero and C.L. Murrant. Department of Human Health and Nutritional Science, College of Biological Sciences, University of Guelph, Guelph,ON N1G 2W1, Canada.J.S. Huber and J.A. Simpson. Department of Human Health and Nutritional Science, College of Biological Sciences, University of Guelph, Guelph,ON N1G 2W1, Canada; IMPART team Canada Investigator Network.K.R. Brunt. IMPART team Canada Investigator Network; Department of Pharmacology, Dalhousie Medicine New Brunswick, Dalhousie University,Saint John, NB E2K 5E2, Canada.Corresponding author: Jeremy A. Simpson (email: jeremys@uoguelph.ca).Copyright remains with the author(s) or their institution(s). Permission for reuse (free in most cases) can be obtained from RightsLink.

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Can. J. Physiol. Pharmacol. 96: 1060–1068 (2018) dx.doi.org/10.1139/cjpp-2018-0134 Published at www.nrcresearchpress.com/cjpp on 13 August 2018.

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Cardiac output is the driving force behind maintaining suffi-cient nutrient supply to meet the metabolic demands of the body.In times of stress (e.g., cold or hypoxia) or demand (e.g., exercise),cardiac output shifts to support the requirements for homeo-stasis. This sophisticated preferential maintenance of blood flowenables compensation and protects critical organ function tomaintain life in times of stress. This regulation is undermined inconditions that lead to heart failure, thus blood flow is oftenshunted and prioritized to essential organs (e.g., brain) and awayfrom nonessential areas (e.g., epidermis). Yet, in heart failurespecifically, it has not been determined how cardiac output isprioritized through the transition from insult to cardiac de-compensation. This could provide essential information as tothe risk of end-organ damage after cardiac injuries, such asacute myocardial infarction (MI). As well, it is important infor-mation for the development and application of new and existingtherapies. This is especially critical in maintaining blood volumeand perfusion of the kidney after MI, given the important role oftargeting the renin–angiotensin–aldosterone systems (RAAS) incardiovascular disease. As such, post-MI prioritization of bloodflow significantly contributes to the pharmacokinetics (i.e., liver) orpharmacodynamics (i.e., kidney) of RAAS-targeted pharmacology.

Interestingly, the spleen has been linked to the regulation ofboth blood pressure and blood volume during periods of hemo-dynamic stress (Andrew et al. 2000; Kaufman 1992), although themechanisms remain to be revealed. Muller et al. reported renal,gut, and limb muscle blood flow is reduced in heart failure, withno correlation to cardiac output (Muller et al. 1992). Splenic bloodflow is prioritized in hemodynamic challenges such as endotox-emia (Andrew et al. 2000), hypervolemia (Kaufman and Deng1993), and portal hypertension (Kaufman and Levasseur 2003). Incontrast, splenic blood flow is reduced in hemorrhagic trauma(Wang et al. 1992). The spleen is generally understudied andwhether MI alters the form or function (blood flow) of the spleenremains unknown. Thus, we hypothesized that splenic blood flowis preserved during development of MI-induced heart failure (achronic state of reduced cardiac output).

Here we investigated the temporal profiles of hemodynamiccardiac function, blood flow, and organ morphometry followingthe progression of MI in rats. Our findings show that following MI,splenomegaly progressively develops and splenic blood flow ispreserved despite declining cardiac output while blood flows tothe kidney and lower limbs are reduced. These data suggest thatthe spleen is a critical organ, specifically in heart failure.

Methods and materials

AnimalsMale Wistar rats (140–180 g; n = 114) were bred and housed in

cages of 3–4 rats on a 12 h light– 12 h dark cycle. Food and waterwere provided ad libitum. Housing and experimental procedureswere approved by the University of Guelph Animal Care Commit-tee and in conformity with the Guide to the Care and Use of Experi-mental Animals from the Canadian Council on Animal Care.

MIAt 5 weeks of age, rats were randomly assigned for either MI

(n = 58) or sham (n = 56) surgery. Animals were anesthetized (2.5%isoflurane in 100% O2), intubated, connected to a ventilator (Hal-lowell EMC, Pittsfield, Massachusetts, USA), and ventilated at 100–125 breaths/min; 6–8 mL/breath. The left lateral rib cage wasshaved and the incision area was cleaned using a soap and watersolution followed by 70% ethanol and 5% iodine. A left-sided tho-racotomy was performed at the 3rd intercostal space and thepericardium was opened. A cotton ball was used to temporarilydisplace the left lung and access the heart. The left anterior de-scending artery was identified under the left atrial appendagetravelling along the anterior left ventricle towards the apex and

was ligated using surgiproII polypropylene suture (Covidien, Min-neapolis, Minnesota, USA). Ribs and skin were sutured using 3-0vicryl (Ethicon, USA) and Sofsilk (Covidien, Minneapolis, Minne-sota, USA) suture. Sham surgeries followed the same protocol butdid not involve ligation. A subcutaneous injection of carprofenwas administered as a general analgesic; an injection of a lido-cane/bupivacaine was administered directly to the incision area asa local anesthetic intra/post-operatively. Buprenorphine was ad-ministered during the animal’s recovery and thereafter as neededfor pain management.

EchocardiographyAnimals were anaesthetized using 2% isoflurane in 100% O2 and

body temperature was maintained at 37 °C. All images were ob-tained using the 2D Vevo 2100 system (Visual Sonics Inc., Toronto,Ontario, Canada) and MS250S transducer (21 MHz). 2D brightness-mode (B-mode) and motion-mode (M-mode) parasternal long-axisimages of the left ventricle were obtained at all 3 time points aspreviously described (Platt et al. 2017). Left ventricle dimensionsand heart rate were measured and standard cardiac parameterswere calculated.

Doppler blood flowDoppler flow was obtained from colour Doppler images of the

splenic, left renal, left femoral, and left common carotid arteriesas an index of blood flow to the spleen, kidney, lower limb, andbrain at all time points. The flow velocity was measured between0° and 55° from a vertical reference in the Y-plane, perpendicularto the arterial diameter (DA). Blood flow was calculated from themeasured velocity time integral (VTI), time of acquisition (T), andDA (Flow = VTI/T × 2�(0.5DA)2); values were averaged over3–4 consecutive heartbeats. Measurements were excluded fromanalysis if the Doppler reading could not detect a velocity, thevelocity showed dual-directional flow, or the angle of the arterywas >55° from the vertical reference. In these cases, measure-ments were re-collected 24–48 h after the initial collection.

Ultrasound cross-sectional areaFollowing Doppler flow measurements, B-mode images of the

spleen and left kidney were acquired. Transverse splenic cross-sectional area (CSA) images displayed a triangular shape and weretaken from the mid-splenic region. Longitudinal renal CSA imageswere taken from the largest mid-renal region with the renal veinfully visible. The images were traced with the ultrasound cursor toestimate the organ CSA.

Invasive hemodynamics and morphometricsAt 9 weeks post-surgery, rats were anesthetized (2.5% isoflurane

in 100% O2) and body temperature was maintained at 37 °C. Anincision was performed on the left anterior surface of the neckand the left common carotid artery was isolated. A 1.9F pressurecatheter (Scisense Inc., London, Ontario, Canada) was insertedinto the left common carotid artery and advanced into the leftventricle. Signals were collected using iWorx software (Labscribe3)and digitized at a sampling rate of 2 kHz. Following data collec-tion, animals were sacrificed via cardiac excision. The right kid-ney, right soleus muscle, right extensor digitorum longus muscleand spleen were rapidly excised and the kidney and spleen wereweighed. Tibial length was measured following manual cleaningof the proximal and distal ends of the tibia.

Statistical methodsAll statistical analyses were performed using GraphPad (Prism 6,

GraphPad Software, Inc.). Data are presented as mean ± SE. Nor-mality was tested using the Shapiro–Wilk test. A one-way ANOVAwas performed to identify significant differences between groupsand a protected Fisher’s least squared difference (LSD) test wasperformed to specify those differences. A Student’s two-tailedt test was used for single time-point comparisons. Linear regres-

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sion analysis was performed to determine the correlation coeffi-cients between blood flow and organ size. Significance wasdefined as P < 0.05.

Results

MI causes significant systolic and diastolic dysfunction withdeclining cardiac output

Echocardiographic parameters including cardiac output, ejec-tion fraction, stroke volume, and end-diastolic dimensions deter-mine cardiac function post-MI. Accordingly, cardiac function wasassessed in our model by echocardiography at 2, 5, and 9 weekspost-MI in comparison with age-matched sham animals. MIcaused a reduction in cardiac output at all time points andreached approximately 50% of sham values by 9 weeks post-surgery (Fig. 1A). There was a reduction in stroke volume, due tolarger end-systolic dimensions and a decrease in heart rate inage-matched animals (Fig. 1A, Table 1). Ejection fraction was alsoreduced at all time points (Fig. 1A). The decreases in systolic func-tion coincided with increases in left ventricular end-diastolicdimensions at all time points, indicative of left ventricle en-largement due to dilation post-MI (Table 1).

Invasive left ventricular hemodynamics was collected 9 weekspost-MI to further characterize cardiac function. The maximumfirst derivative of pressure, +dp/dtmax, is used as an index of con-tractility, whereas the minimum first derivative of pressure,−dp/dtmin, is an index of relaxation. Animals had systolic (i.e.,decreased left ventricle pressure, decreased +dp/dtmax and dia-stolic (i.e., elevated end-diastolic pressure, increased −dp/dtmin)dysfunction compared with sham animals 9 weeks post-MI(Fig. 1B). Both echocardiographic and invasive hemodynamic mea-surements reveal a reduced heart rate in the MI animals com-pared with sham at 9 weeks (Figs. 1A and 1B) consistent withexpected cardiac response to MI. Taken together, these data con-firm that animals displayed both systolic and diastolic cardiacdysfunction following a MI.

MI causes splenomegaly, reduces cardiac output and renalmass

To investigate whether spleen enlargement (i.e., splenomegaly),occurs in our model, we measured organ mass and splenic CSAwith ultrasound, as a noninvasive surrogate to estimate organ size(Figs. 2A–2C) progressively. Splenic CSA remained unchanged at

Fig. 1. Myocardial infarction (MI) caused progressive systolic and diastolic dysfunction over 9 weeks. (A) Echocardiographic parameters measured2, 5, and 9 weeks post-MI or sham surgery. (B) Invasive hemodynamic measurements 9 weeks post-MI (n = 18) or sham surgery (n = 9).Remaining n values are reported in Table A1. LVP, left ventricular pressure; EDP, end diastolic pressure; dp/dtmax, index of left ventriclecontractility; dp/dtmin, index of left ventricle relaxation; MAP, mean arterial pressure. *, Statistical significance from age-matched sham(P < 0.05). [Colour online.]

0

5000

10000

dp/d

tmax

(mm

Hg/

s)-10000

-5000

0

dp/d

tmin

(mm

Hg/

s )

0

10

20

EDP

(mm

Hg)

80

90

100

110

120

LVP

(mm

Hg)

300

350

400

Hea

rtR

ate

(bea

ts/m

in) SHAM

MI

0 2 4 6 8 100

50

100

150

Weeks Post Surgery

SHAMMI ***

0 2 4 6 8 100

30

60

90

Weeks Post Surgery

Ejec

tion

Frac

tion

(%)

0 2 4 6 8 10300

350

400

450

Weeks Post Surgery

Hea

rtR

ate

(bea

ts/m

in)

**

*

* * *

* *

A. B.

*

*

*

*

*

60

80

100

120

MA

P(m

mH

g)

*

Car

diac

Out

put (

ml/m

in)

Table 1. Echocardiography measurements 9 weeks post myocardial infarction (MI) or sham surgery.

Echocardiography

HR(beats/min)

BM(g)

EF(%)

FS(%)

ESD(mm)

EDD(mm)

PWTs(mm)

PWTd(mm)

SV(�L)

CO(mL/min)

CI(mL/min·kg)

Temperature(°C)

Sham2 weeks (n=37) 380±5 276±6 72.4±1.2 43.2±1.1 4.0±0.1 7.0±0.1 2.3±0.04 1.5±0.04 186.6±5.2 70.8±2.1 0.260±0.008 37.1±0.15 weeks (n=30) 376±6 372±7 72.2±1.5 43.4±1.3 4.4±0.1 7.7±0.1 2.5±0.05 1.7±0.04 226.8±5.3 85.3±2.4 0.232±0.008 37.1±0.19 weeks (n=22) 374±7 450±10 74.5±1.5 45.3±1.3 4.3±0.1 7.9±0.1 2.7±0.07 1.9±0.05 250.8±7.7 93.3±3.1 0.209±0.008 37.1±0.1

MI2 weeks (n=37) 369±5 251±7* 29.4±1.4* 14.7±0.8* 7.7±0.1* 9.0±0.1* 2.2±0.05 1.6±0.03 131.4±5.6* 48.1±2.0* 0.195±0.008* 37.1±0.15 weeks (n=48) 359±6* 368±6 27.5±1.1* 13.7±0.6* 8.5±0.1* 9.9±0.1* 2.1±0.05* 1.7±0.03 148.5±5.1* 53.2±2.0* 0.146±0.006* 37.0±0.19 weeks (n=20) 348±9* 461±7 23.5±1.5* 11.6±0.8* 9.1±0.2* 10.2±0.2* 2.1±0.10* 1.8±0.06 137.8±7.6* 48.2±3.2* 0.105±0.008* 37.0±0.1

Note: Data is expressed as mean ± SE. HR, heart rate; BM, body mass; EF, ejection fraction; FS, fractional shortening; ESD, end-systolic dimension; EDD, end-diastolicdimension; PWTs, systolic posterior wall thickness; PWTd, diastolic posterior wall thickness; SV, stroke volume; CO, cardiac output; CI, cardiac index.

*Significant difference from age-matched sham (P < 0.05).

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2 weeks, with a significant increase at 5 and 9 weeks post-MI(Fig. 2B). At 9 weeks post-MI, we measured spleen mass to accu-rately assess splenomegaly and splenic masses were increased ascompared with sham (Table 2), confirming the presence of MI-induced splenomegaly. Splenic CSA and masses of 9 weeks post-surgical intervention animals were normalized to tibial length, toaccount for any differences in growth. Splenic CSA and mass,normalized to tibial length, increased 9 weeks post-MI as com-pared with sham (Figs. 2C and 2D; Table 2). Thus, splenomegalyoccurs by 5 weeks during post-MI progression, which can be accu-rately assessed noninvasively by ultrasound.

As a noninvasive index of renal size, we measured renal CSA at2, 5, and 9 weeks post-MI as compared with sham and renal mass9 weeks post-MI as compared with sham (Figs. 2E–2H). Renal CSAdid not change at any time point (Fig. 2F). However, renal CSA and

renal mass, normalized to tibial length, was significantly lower at9 weeks post-MI (Figs. 2G and 2H; Table 2). These data show thatrenal size is reduced during post-MI progression, suggesting thepresence of renal dysfunction (Kim 2010), but is not well deter-mined by noninvasive imaging alone without accounting forwhole body growth.

Progressive decline of cardiac output shunts blood flowpreferentially to the spleen and brain and away from kidneyand lower limbs

After MI, renal, cerebral, and skeletal muscle blood flow de-creases, resulting in end-organ dysfunction (Wilson et al. 1984)and increase in mortality (Dries et al. 2000; Loncar et al. 2011).Splenic blood flow was similar between sham and MI animals(Fig. 3A.I), even when normalized to tibial length to account for

Fig. 2. Myocardial infarction (MI) reduced cardiac output and renal mass and caused splenomegaly. (A) Representative gross appearance ofspleens 9 weeks post-MI or sham surgery. (B) Splenic cross-sectional area (CSA) 2, 5, and 9 weeks post-MI or sham surgery. (C) Spleen CSA totibial length (TL) ratio 9 weeks post-MI (n = 4) or sham (n = 8) surgery. (D) Spleen mass to TL ratio 9 weeks post-MI (n = 9) or sham surgery (n = 9).(E) Representative gross appearance of kidneys 9 weeks post-MI or sham surgery. (F) Renal CSA 2, 5, and 9 weeks post-MI or sham surgery.(G) Renal CSA to TL ratio 9 weeks post-MI (n = 6) or sham (n = 9) surgery. (H) Renal mass to TL ratio 9 weeks post-MI (n = 9) or sham surgery (n = 9).Remaining n values are reported in Table A1. *, Statistical significance from age-matched sham (P < 0.05). [Colour online.]

0.0

0.5

1.0

1.5 SHAMMI

0

1

2

3

4 SHAMMI

D7000

KIDNEY MORPHOMETRICS

0

10

20

30

40

50

Sple

enW

eigh

t/ TL

(mg/

mm

)

SHAMMI

0

10

20

30

40

50

Ren

alW

eigh

t/TL

(mg/

mm

)

SHAMMI

D7000

MISHAM

SHAM MI

E. G.F.

A.

SPLEEN MORPHOMETRICS

C.

10mm

10mm

B.

0 2 4 6 8 1025

30

35

40

Weeks Post Surgery

SHAMMI

**

*

*

0 2 4 6 8 1060

80

100

120

Weeks Post Surgery

SHAMMI

*

*

D.

H.

Sple

nic

CSA

/TL

Rena

l CSA

/TL

Sple

nic

CSA

(mm

2 )Re

nal C

SA (m

m2 )

Table 2. Morphometric measurements of the spleen and kidney following sham and myocardial infarction (MI)surgery.

Morphometrics

TL (mm) BM (g) SM (g) KM (g) SM/TL (mg/mm) KM/TL (mg/mm)

Sham (n=10) 39.5±0.4 442±12 1.09±0.03 1.24±0.03 27.7±0.7 31.5±0.8MI (n=9) 41.5±0.4* 460±15 1.32±0.06* 1.18±0.03* 31.5±1.4* 28.0±0.7*

Note: Data is expressed as mean ± SE. TL, tibial length; BM, body mass; SM, spleen mass; KM, kidney mass.*Significant difference from age-matched sham (P < 0.05).

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changes in growth or splenic CSA or mass, suggesting flow ismatched to the change in organ size following MI (Fig. 3A.II).Renal blood flow at 2 and 5 weeks post-MI was unchanged ascompared with sham but was significantly reduced at 9 weekspost-MI (Fig. 3B.I). The reduction in renal blood flow at 9 weekswas persistent when normalized to tibial length and renal CSA in9 weeks post-MI animals as compared with sham but not kidneymass (Fig. 3B.II). Thus, the reduction in renal blood flow is partlyattributable to reduced organ mass rather than shunting per se.Common carotid artery blood flow was unchanged at all timepoints post-MI (Fig. 3C.I) even when normalized to tibial length in9 weeks post-MI animals as compared with sham (Fig. 3C.II). Fem-oral blood flow, although initially unchanged at 2 weeks, decreasedat 5 and 9 weeks post-MI, as compared with sham (Fig. 3D.I). Whennormalized to tibial length in 9 weeks post-MI animals, femoralblood was again decreased compared with sham (Fig. 3D.II).

Although we did not find an increase in mean splenic bloodflow post-MI, this does not preclude the possibility of slight in-creases in splenic blood flow being present with splenomegalypost-MI. Thus, to further investigate the relationship between or-gan blood flow and organ size, linear regression analysis was per-formed. Measuring organ CSA is advantageous as it is noninvasiveand, therefore, provides a means to assess organ size longitudi-nally without sacrificing the animal. Thus, we were able to in-crease our statistical power, while reducing the number ofsacrificed animals using a noninvasive method to approximateorgan size. In the sham animals, there was no significant correla-tion between blood flow and CSA (Fig. 4A, left). In contrast,post-MI animals were positively correlated with splenic CSA(Fig. 4A, right). Splenic CSA and splenic mass showed a positivelinear correlation at 9 weeks post-surgery, indicating that splenicCSA is an accurate estimation of spleen size (Fig. 4B). Thus, modestincreases in splenic blood flow occur in parallel with splenomeg-aly post-MI, not because of splenomegaly. Renal blood flow andCSA show a positive linear correlation in both sham (Fig. 4C, left)and post-MI animals (Fig. 4C, right). However, no correlation wasdetermined between renal CSA and mass (Fig. 4D), suggesting thatrenal size does not change with mass (Fig. 4D). This indicates thatrenal CSA does not accurately predict renal mass. Nonetheless,the positive correlation between renal blood flow and CSA, butnot mass, indicates that increased size results in increased renalblood flow in both sham and post-MI animals. Taken together,prioritization of blood flow with a progressive declining cardiacoutput following MI is preferential to the spleen.

DiscussionOur data show that following MI, there is preferred blood shunt-

ing to the spleen and brain, with reduced kidney mass and aproportional reduction in blood flow to the kidney and skeletalmuscle. Further, we were surprised by the progressive splenomeg-aly with the development of heart failure as organs generallydecline in size, not increase. These findings suggest that theremay be more salient pathophysiological mechanisms at work inthe cardiosplenic axis that remains to be fully understood.

SplenomegalyHere, we show modest splenomegaly occurs with MI. Previous

studies investigating heart failure and splenomegaly have pro-

duced varying results. A retrospective study of 2505 heart failurepatients diagnosed with splenomegaly between 1913 and 1995found that splenomegaly was associated with congestive heartfailure (O’Reilly 1998). In contrast, in 1951, Ibrahim and colleaguesreviewed 206 cases in Egypt of congestive heart failure with andwithout rheumatic infection to determine whether there was anincreased incidence of splenomegaly (Ibrahim et al. 1951). Only5 out of 206 patients showed splenomegaly and these cases wereall attributed to unrelated complications (e.g., liver cirrhosis, in-fection, hepatic congestion) (Ibrahim et al. 1951). This conflict maybe explained methodologically. Specifically, in the Ibrahim et al.study, spleen enlargement was assessed by palpation (Ibrahimet al. 1951). This method is highly susceptible to human interpre-tation (i.e., physician variability) and is strictly qualitative. In con-trast, we were able to obtain an accurate spleen mass and confirmthese findings with ultrasound CSA measurements. It is possiblethat human studies investigating splenomegaly missed subtle in-creases in spleen mass (due to length or width variation) that wereeasily detectable with the methods we employed. Indeed, 67% ofpatients that were diagnosed with splenomegaly and heart failuredid not present with a palpable spleen, but splenomegaly wasdiscovered in autopsy (O’Reilly 1998). Advanced imaging methodsshould be used to diagnose splenomegaly quantitatively to con-firm our findings in patients.

Splenomegaly, developing secondary to disease (e.g., heart fail-ure, cirrhosis of the liver, cancer, hemolytic anemia), is character-ized by the underlying cause of enlargement (e.g., congestion,infiltrative, immune, and neoplastic) (Chapman and Azevedo2018). For example, in chronic heart failure, venous congestion,owing to right-sided ventricle failure (Bolognesi et al. 2012), con-tributes to the development of splenic enlargement. Acutely, inresponse to sterile injury (e.g., MI), enlargement of the spleen iscaused, at least in part, by the hyperplasia of the immune cellswithin the spleen (Ismahil et al. 2014). Clinically, splenomegalymay be the result of a combination of factors. Patients with heartfailure typically have a number of comorbidities that could poten-tially contribute to the development of splenomegaly including,but not limited to, ischemic liver disease (Alvarez and Mukherjee2011), obesity (Gotoh et al. 2012), and chronic kidney disease(Gotoh et al. 2013). In some patients, splenomegaly may be unre-lated to heart failure itself but secondary to liver injury. Indeed,numerous drugs, herbal medications, and dietary supplementsare associated with drug-induced liver injury, the most commoncause of acute liver failure. Some of the more commonly pre-scribed therapies for patients with heart failure (e.g., aspirin, st-atins, angiotensin-converting enzyme) cause drug-induced liverinjury. Thus, for these patients, inflammation of the liver andsecondary portal congestion could cause and (or) further exacer-bate enlargement of the spleen.

Whether splenomegaly indicates the spleen is under stress, orwhether the spleen is simply increasing functional capacity re-mains to be determined. Following MI, splenic leukocytes contrib-ute to the resolution of the inflammatory response with theproduction of special pro-resolving mediators and expansion ofimmune cell populations within the spleen (Halade et al. 2018),suggesting splenomegaly post-MI is a critical compensatory re-sponse. However, long-term alterations in splenic immune cellprofiles contribute to adverse cardiac remodeling. Future studies

Fig. 3. Progressive decline of cardiac output shunted blood flow (BF) preferentially to the spleen and brain and away from renal and lowerlimbs. (A) (I) Splenic BF 2, 5, and 9 weeks post myocardial infarction (MI) or sham surgery. (II) Splenic BF 9 weeks post-MI or sham surgerynormalized to tibial length (n = 5,8), splenic cross-sectional area (CSA) (n = 23,25) or spleen mass (n = 4,8); (B) (I) Renal BF 2, 5, and 9 weekspost-MI or sham surgery. (II) Renal BF 9 weeks post-MI or sham surgery normalized to tibial length (n = 6,8), renal CSA (n = 28,23), or renalmass (n = 6,9); (C) (I) Cerebral blood flow 2, 5, and 9 weeks post-MI or sham surgery. (II) Cerebral BF 9 weeks post-MI or sham surgerynormalized to tibial length (n = 5,8); (D) (I) Femoral BF 2, 5, and 9 weeks post-MI or sham surgery. (II) Femoral BF 9 weeks post-MI or shamsurgery normalized to tibial length (n = 6,9). *, Statistical significance from age-matched sham (P < 0.05). [Colour online.]

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investigating cardiovascular diseases must consider the underly-ing cause of splenomegaly to gain a better understanding ofwhether the response is benign or playing a compensatory role.

Immune function and the spleenSplenic blood flow is preserved post-MI. Preserving organ blood

flow is critical for maintaining healthy organ function. Previousfindings show that maintaining splenic blood flow during diseasestates is important for preserving the immune function of thespleen. Following hemorrhagic trauma, splenic blood flow (Wanget al. 1992) and splenic immune function (Kawasaki et al. 2006) arereduced. When splenic blood flow is experimentally increased (viaL-arginine infusion), the ability of cultured spleen cells to produceand release interleukin 2 and 3 (i.e., cytokines that regulate im-munity) (Boyman and Sprent 2012; Lantz et al. 1998) is restored(Angele et al. 1999). The rate of pneumococcal clearance, an indexof the spleen’s ability to rid the blood stream of bacteria (Brownet al. 1981), was compared in rabbits during various splenic condi-tions (Horton et al. 1982). Interestingly, bacterial clearance wasimpaired when splenic blood flow was reduced, independent ofsplenic mass (Horton et al. 1982). These findings confirm thatsplenic blood flow was critical to the performance of splenic im-mune functions. These studies also established a direct link be-tween splenic blood flow and immune function. In the presentstudy, we found that splenic blood flow increases following MI.Given that splenic damage results in adverse cardiac effects(Robinette and Fraumeni 1977; Tsai et al. 2015) and that reducedsplenic blood flow results in impaired splenic immune function(Angele et al. 1999; Horton et al. 1982), we interpret this to be thatmaintenance of splenic blood flow post-MI is critical for beneficialimmune-meditated cardiac remodeling. For example, it may be

essential to maintain spleen blood flow to support monocyte ex-trusion for repair and remodeling. During sterile ischemic injurysuch as an MI, the spleen mobilizes the monocyte reservoir to aidin clearance of debris from the area of infarction (Swirski et al.2009). After the debris is cleared, these monocytes help resolvethis initial inflammatory reaction (van der Laan et al. 2014). Fol-lowing MI, the splenic leukocyte reservoir mobilizes to the infarctzone to aid in the resolution of inflammation, preventing furthermyocardial damage (Halade et al. 2018). What is less clear is whenthis important function abates. An infarct scar is formed to sus-tain the structural integrity for the pumping force of the heartmuscle and low-level chronic inflammation ensues (Dick andEpelman 2016). Unfortunately, little is known about the chronicinflammation or the spleen’s role in cardiovascular disease gen-erally. However, the spleen’s role following a stroke, an ischemicevent similar to MI, provides some insight. Two months aftermiddle cerebral artery occlusion, bone marrow stem cells that aretransfused into rats preferentially migrate towards the spleenrather than the brain (Acosta et al. 2015). Further, bone marrowstem cell injection has been shown to decrease cerebral infarctsize following a stroke and resolve chronic cerebral inflammation(Acosta et al. 2015). Recently, the spleen was also shown to be animportant contributor in the production of pro-resolving lipidmediators that modulate inflammation in aging (Kain et al. 2018).These findings indicate that the spleen plays a key role in resolv-ing chronic inflammation, possibly through medullary traffickingor differentiation.

Importance of spleen to volume regulationRAAS activation increases blood volume while atrial natriuretic

peptide reduces blood volume (de Bold 1985). The hypervolemic

Fig. 4. Linear regression between organ size and blood flow demonstrated preferential blood flow to the spleen. Time points are identified asfollows: 2 weeks (red), 5 weeks (black), 9 weeks (blue); (A) Splenic cross-sectional area (CSA) vs. splenic blood flow (grouped time points) insham (left) and myocardial infarction (MI, right) animals. All individual time points showed no significant correlations for both MI and sham.(B) Splenic CSA vs. spleen mass 9 weeks post-surgery (sham and MI grouped). (C) Renal CSA vs. renal blood flow (grouped time points) in sham(left) and MI (right) animals. All individual time points for both MI and sham showed no significant correlations. (D) Renal CSA vs. renal massat 9 weeks post-surgery (sham and MI grouped). *, Significant correlation (P < 0.05). [Colour online.]

A.

SPLEEN

KIDNEY

B.

C. D.

0.8 1.0 1.2 1.4 1.670

80

90

100

110

120

130

Renal Weight (g)

Ren

alC

SA(m

m2 )

Y = 26.78X + 68.04r2=0.1225p=0.2010

0.5 1.0 1.5 2.010

20

30

40

50

Splenic Weight (g)

Sple

nic

CSA

(mm

2 )

Y = 24.30X +3.838r2=0.6564p=0.0014*

10 20 30 40 50 600.0

0.5

1.0

1.5

2.0

2.5

Splenic CSA (mm2)

Blo

odFl

ow(m

l/min

)

Y = 0.002543X + 1.064r2 = 0.001656p = 0.7235

10 20 30 40 50 600.0

0.5

1.0

1.5

2.0

2.5

Splenic CSA (mm2)

Blo

odFl

ow(m

l/min

)

Y = 0.01605X + 0.5608r2 = 0.06467p = 0.0288*

50 100 1500

5

10

15

20

Renal CSA (mm2)

Blo

odFl

ow(m

l/min

)

Y = 0.08810X + 0.8617r2 = 0.1520p = 0.0005*

50 100 1500

5

10

15

20

Renal CSA (mm2)

Blo

odFl

ow(m

l/min

)

Y = 0.04673X + 3.781r2 = 0.06099p = 0.0327*

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state, which will develop after MI, causes atrial stretch and subse-quent atrial natriuretic peptide release, but the renal response toatrial natriuretic peptide is impaired in heart failure (Ibebuoguet al. 2011). Thus, after MI, not only is the RAAS increasing bloodvolume, but a key mechanism that protects against hypervolemiais impaired, which is reinforced by our findings showing reducedrenal blood flow after MI. Kaufman and colleagues also showedthat in rats, the hypovolemic effects of atrial natriuretic peptidewas abolished following splenectomy, indicating that the spleenpartially mediates the effects of atrial natriuretic peptide inhealth (Kaufman 1992). Although the renal response to atrial na-triuretic peptide is impaired in heart failure (Ibebuogu et al. 2011),the splenic response to atrial natriuretic peptide has not beeninvestigated. It is possible that the spleen still responds to atrialnatriuretic peptide. If this were the case, impairing splenic function(e.g., by reducing splenic blood flow) could facilitate an increase inhypervolemia and exacerbate heart failure after MI. Collectively,these findings support the contention that the spleen is in-volved in maintaining hemodynamic homeostasis. Thus, splenicblood flow could be increased after MI to preserve the blood volumeregulatory function whilst renal blood flow is impaired.

Conclusions and future directionsOur study establishes the importance of evaluating hemody-

namic changes in the context of overall physiology (e.g., bloodflow to various organs) and from a regional standpoint (e.g., bloodflow compared with organ size). We established preservation ofsplenic blood flow and modest splenomegaly following MI.Splenomegaly could be attributed to a number of factors indepen-dent or collectively due to increased congestion, medullary infil-tration, or tissue hyperplasia/hypertrophy. This requires furtherinvestigation to determine whether splenomegaly is always dueto the same mechanism. Also of interest is whether splenomegalyis specific to MI as opposed to other types of cardiac stresses (e.g.,hypertension) and whether this exacerbates with time or resolves.

In the past century, our understanding of the spleen was largelythe result of observational studies. This study reiterates that ourunderstanding of the spleen is still in a rudimentary form.Amongst most organs within the body, the spleen has a uniqueresponse to cardiac damage in that blood flow is preserved, indi-cating the spleen is a critical organ.

Conflict of interestThe authors declare that there is no conflict of interest associ-

ated with this work.

Author contributionsS.A.R., J.S.H., C.M., K.R.B., and J.A.S. conceptualized the study

and designed the experiments. S.A.R. and J.S.H. conducted theexperiments. All authors analyzed and (or) interpreted the exper-imental results. S.A.R., C.M., K.R.B., and J.A.S. drafted the manu-script, critically revised the manuscript for intellectual content,and gave the final approval of the article. All authors read andgave permission for the final draft of the manuscript.

AcknowledgementsThis work was supported by grants from National Science Re-

search Engineering Council (K.R.B and J.A.S.) and a Heart andStroke Foundation of Canada Grant (J.A.S. and K.R.B.). J.A.S. is alsoa New Investigator of the Heart and Stroke Foundation of Canada.We further acknowledge the philanthropic support for cardiovas-cular research from Betty and Jack Southen (London, Ontario,Canada).

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Appendix A

Table A1. Remaining n values for echocardiography, ultrasound, andimmunohistochemical analysis.

Procedure Region Group n

Echocardiography (2, 5, 9 weeks)Cardiac MI 37, 48, 20

Sham 37, 30, 22

Ultrasound (2, 5, 9 weeks)Splenic artery MI 27, 31, 24

Sham 33, 24, 25Femoral artery MI 11, 29, 21

Sham 11, 14, 14Carotid artery MI 10, 26, 20

Sham 11, 16, 12Renal artery MI 31, 49, 38

Sham 30, 33, 27Spleen CSA MI 23, 31, 23

Sham 28, 25, 25Renal CSA MI 24, 27, 24

Sham 24, 24, 23

Skeletal muscle capillary density 9 weeksSoleus MI 3

Sham 3EDL MI 4

Sham 3

Skeletal muscle CSASoleus MI 3

Sham 3EDL MI 4

Sham 3

Note: MI, myocardial infarction; CSA, cross-sectional area; EDL, extensor digi-torum longus.

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