TECHNICAL NOTES Open Access
T ans ho acic echoca diog aphy e e ence alues
in ju enile and adul 129/S mice
Mau ícia Vinhas
1
, Ana Ca olina A aújo
1,2
, Sónia Ribei o
3
, Luís B ás Rosá io
3
and José An ónio Belo
1,2*
Abs ac
Backg ound: In he ecen yea s, he use o Dopple -echoca diog aphy has become a s anda d non-in asi e
echnique in he analysis o ca diac mal o ma ions in gene ically modi ied mice. The e o e, no mal alues ha e o
be es ablished o he mos commonly used inb ed s ains in whose gene ic backg ound hose mu a ions a e
gene a ed. He e we p o ide e e ence alues o ans ho acic echoca diog aphy measu emen s in ju enile
(3 weeks) and adul (8 weeks) 129/S mice.
Me hods: Echoca diog aphic measu emen s we e pe o med using B-mode, M-mode and Dopple -mode in 15
ju enile (3 weeks) and 15 adul (8 weeks) mice, du ing iso lu ane anes hesia. M-mode measu emen s a iabili y o
le en icle (LV) was de e mined.
Resul s: Se e al echoca diog aphic measu emen s signi ican ly di e be ween ju enile and adul mice. Mos o
hese measu emen s a e ela ed wi h ca diac dimensions. All B-mode measu emen s we e di e en be ween
ju eniles and adul s (highe in he adul s), excep o ac ional a ea change (FAC). Ejec ion ac ion (EF) and
ac ional sho ening (FS), calcula ed om M-mode pa ame e s, do no di e be ween ju enile and adul mice.
S oke olume (SV) and ca diac ou pu (CO) we e signi ican ly di e en be ween ju enile and adul mice. SV was
31.93 ± 8.67 μl in ju eniles s 70.61 ± 24.66 μl in adul s, ρ< 0.001. CO was 12.06 ± 4.05 ml/min in ju eniles s
29.71 ± 10.13 ml/min in adul s, ρ< 0.001. No di e ence was ound in mi al al e (MV) and icuspid al e (TV)
ela ed pa ame e s be ween ju enile and adul mice. I was demons a ed ha a iabili y o M-mode measu emen s
o LV is minimal.
Conclusions: This s udy sugges s ha di e ences in ca diac dimensions, as wells as in pulmona y and ao a
ou low pa ame e s, we e ound be ween ju enile and adul mice. Howe e , mi al and icuspid in low pa ame e s
seem o be simila be ween 3 weeks and 8 weeks mice. The e e ence alues es ablished in his s udy would
con ibu e as a basis o u u e s udies in pos -na al ca dio ascula de elopmen and diagnosing ca dio ascula
diso de s in gene ically modi ied mouse mu an lines.
Keywo ds: Echoca diog aphy, Ju enile, Adul , 129/S mouse, Re e ence alues, Dopple
Backg ound
Ca diac ul asound, also known as echoca diog aphy, is
one o he mos commonly used diagnos ic echniques
in human ca diology o possible pa hology o lesion.
This echnique uses high equency ul asound wa es o
isualizing he hea and can p o ide in o ma ion on he
hea ana omy, blood low pa e n and unc ion o hea
muscle, essels and al es. Un il ecen ly, echoca dio-
g aphic applica ion in animals was limi ed p ima ily o
la ge , non- oden species. Due o ad ances in ul a-
sound imaging echnology, ul asound sys ems ha e now
he spa ial and empo al esolu ion o ob ain accu a e
and eliable images o mouse hea s [1]. As a esul , i
has become a aluable non-in asi e imaging ool o
isualize and e alua e ca diac mo phology and unc ion
in i o o mice. I was demons a ed by o he s ha
echoca diog aphy is becoming a use ul echnique o
s udying ca dio ascula de elopmen and diagnosing
ca dio ascula diso de s in small animals [2].
* Co espondence: [email p o ec ed]
1
Regene a i e Medicine P og am, Depa men o Biomedical Sciences and
Medicine (DCBM), Uni e si y o Alga e (UALG), Campus o Gambelas, Ed .8,
8005-139, Fa o, Po ugal
2
Ins i u e o Bio echnology and Bioenginee ing, Cen e o Molecula and
S uc u al Biomedicine (CBME), Uni e si y o Alga e, Campus o Gambelas,
Ed .8, 8005-139, Fa o, Po ugal
Full lis o au ho in o ma ion is a ailable a he end o he a icle
CARDIOVASCULAR
ULTRASOUND
© 2013 Vinhas e al.; licensee BioMed Cen al L d. This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e
Commons A ibu ion License (h p://c ea i ecommons.o g/licenses/by/2.0), which pe mi s un es ic ed use, dis ibu ion, and
ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed.
Vinhas e al. Ca dio ascula Ul asound 2013, 11:12
h p://www.ca dio ascula ul asound.com/con en /11/1/12
Knowledge o indings in heal hy animals is impo an
o in e p e a ion o esul s in gene ically modi ied and su -
gical animals. Mos o he a ailable mouse ES cell lines ha e
been gene a ed using he 129/S s ain. The e o e, he pu -
pose o his s udy is o p o ide e e ence alues o ans-
ho acic echoca diog aphy measu emen s and calcula ed
pa ame e s using B-mode, M-mode and Dopple -mode in
ju enile (3 weeks) and adul (8 weeks) 129/S mice,
ob ained du ing iso lu ane anes hesia.
Me hods
E hics s a emen
All animal wo k pe o med in his s udy was conduc ed
complian wi h he Po uguese law and app o ed by
he Consul i e Commission o he Ve e ina y Agency
(Po uguese Minis y o Ag icul u e), he sole Agency/
Commi ee in Po ugal esponsible o issue he e hical
app o al o hese ype o s udies, ollowing he EU
guidelines o animal esea ch and wel a e.
Animals
30 wild ype male 129/S mice (Ha lan Labo a o ies)
di ided by wo g oups, 15 ju enile mice (3 weeks) and
15 adul mice (8 weeks), we e s udied. The mice we e
housed in ou animal acili y in a con olled en i on-
men , a 22°C wi h a i icial 12 hou s o ligh /da k cycle,
s anda d die and ee access o wa e we e supplied.
The body weigh (BW) o each mouse was eco ded
p io o ca diac examina ion.
Echoca diog aphy
Mice we e con inuously anes he ized by 1.5-2% o
iso lu ane inhalan mixed wi h 1 L/min 100% O
2
o
main ain a ligh seda ion le el h oughou he p oced-
u e. They we e immobilized on a hea ing pla o m en-
al side up o main ain he body empe a u e a 37°C ±
0.5°C. Hea a e (HR) and espi a o y physiology we e
con inuously moni o ed by ECG elec odes. Mice ches s
we e sha ed and wa med ul asound gel was applied o
he a ea o in e es . T ans ho acic echoca diog aphy was
pe o med using a Ve o 2100 sys em (VisualSonics,
To on o, Canada) wi h a 40-MHz ansduce . Ca e was
aken o a oid excessi e p essu e o e he s e num,
which can dis o he signal. Images we e cap u ed on
cine loops a he ime o he s udy and a e wa d mea-
su emen s we e done o -line.
Measu emen s
The hea was i s imaged in B-mode in he pa as e nal
long axis iew o examine he le en icle (LV). The
measu emen s included LV endoca dial and epica dial
leng h (LVEndoL and LVEpiL, espec i ely) in dias ole
and sys ole. LV leng hs we e measu ed om he ao ic
annulus o he apex le el. Fo long axis iew in B-mode,
image dep h was 11 mm and image wid h was 12.08
mm. Mo eo e , pa as e nal sho axis iew was ob ained
a he le el o papilla y muscles o measu e LV endoca -
dial and epica dial a ea (LVEndoA and LVEpiA, espec -
i ely) in dias ole and sys ole. These measu emen s we e
ob ained by acing he endoca dial and epica dial
bo de o he LV, whe e he papilla y muscles we e
excluded om he endoca dial acings. In o de o es i-
ma e LV mass (LVM) and LV olume (LVV), he a ea-
leng h me hod was used [3,4]. LVM was no malized o
BW and ep esen ed as LVM index (LVMi). Endoca dial
a ea change (EAC) and ac ional a ea change (FAC)
we e also calcula ed. Fo sho axis iew in B-mode,
image dep h was 10 mm and image wid h was 9.08 mm.
In o de o acqui e accu a e measu emen s o ca diac
dimensions, M-mode images we e ob ained om long
axis and sho axis B-mode images by placing he M-
mode sample ga e pe pendicula o he in e en icula
sep um (IVS) and LV walls, espec i ely, a he le el o
papilla y muscles. M-mode sample ga e dep h, leng h
and angle o long axis iew we e 8–11 mm, 4.6-7.6 mm
and 0 dg, espec i ely. Fo sho axis iew, M-mode
sample ga e dep h, leng h and angle we e 8–10 mm,
4.6-7.6 mm and 0 dg, espec i ely. M-mode om long
axis iew was pe o med o measu e IVS hickness,
while M-mode om sho axis iew was pe o med o
measu e hickness o LV an e io wall (LVAW), LV pos-
e io wall (LVPW) and LV in e nal diame e (LVID). All
M-mode measu emen s we e pe o med in end-dias ole
(−d) and end-sys ole (−s) acco ding o he leading-edge
me hod o he Ame ican Socie y o Echoca diog aphy
[5]. End-dias olic and end-sys olic measu emen s we e
ob ained a he ime o maximal in e nal chambe di-
mensions and a he minimal in e nal chambe dimen-
sions, espec i ely [6]. The LV s uc u al pa ame e s
measu ed om sho axis iew in M-mode we e used in
he calcula ion o LV ejec ion ac ion (EF) and LV ac-
ional sho ening (FS). M-mode om igh pa as e nal
long axis iew was pe o med o e alua e he igh en-
icle in e nal diame e (RVID) and hickness o igh
en icle an e io wall (RVAW). M-mode sample ga e
dep h, leng h and angle o he igh en icle (RV) iew
we e 7–9 mm, 2.6-4.6 mm and 0 dg, espec i ely.
Blood low was assessed using PW Dopple -mode, by
posi ioning he Dopple sample olume pa allel o low
di ec ion, which was assis ed by Colo Dopple -mode.
F om a modi ied sho axis iew o he pulmona y al e
(PV) we measu ed pulmona y a e y diame e (PAD)
and PV peak eloci y (PVPV). PV peak p essu e g adien
(PVPPG) was calcula ed. Fo pulmona y a e y iew in
B-mode, image dep h was 10–11 mm and image wid h
was 9.08 mm. Fo Dopple mode o pulmona y a e y
iew, Dopple sample olume dep h, size and angle we e
5–8 mm, 0.22 mm and 5–30 dg, espec i ely. Ascending
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ao a al e (AoV) low was ob ained om a sup as e nal
iew o measu e AoV peak eloci y (AoVPV). The ao ic
a ch iew was pe o med o measu e he ascending ao a
diame e (AoD) and he descending ao a peak eloci y
(DAoPV). AoV peak p essu e g adien (AoVPPG) was
calcula ed. All a e ial diame e s we e measu ed in sys-
ole, a he ime o maximal a e y diame e . Fo ao a
iew in B-mode, image dep h was 8–12 mm and image
wid h was 6–11 mm. Dopple sample olume dep h, size
and angle o ascending ao a we e 6–10 mm, 0.27 mm
and 20–55 dg, espec i ely. Fo descending ao a, Dop-
ple sample olume dep h, size and angle we e 6–9 mm,
0.27 mm and 5–30 dg, espec i ely. S oke olume (SV)
and ca diac ou pu (CO) we e calcula ed using ao ic
ou low as p e iously desc ibed by o he s [7]. CO was
no malized o BW and ep esen ed as CO index
(COi). HR was de e mined om spec al Dopple
acings o he pulmona y a e y and ascending ao a
low.
Mi al al e (MV) and icuspid al e (TV) in low
we e assessed om he apical 4 chambe iew. The MV
measu emen s pe o med we e he ollowing: MV ea ly
wa e peak (MVE), MV a ial wa e peak (MVA), no low
ime (NFT), ao ic ejec ion ime (AET), iso olumic
elaxa ion ime (IVRT), iso olumic con ac ion ime
(IVCT) and MV ejec ion ime (MVET). The MV peak
p essu e g adien (MVPPG), LV myoca dial pe o mance
index (LVMPI) and MVE/A a io we e calcula ed. Fo
mi al al e iew, Dopple sample olume dep h, size
and angle we e 7–11 mm, 0.22 mm and 5–30 dg,
espec i ely. TV measu emen s included TV ea ly wa e
peak (TVE) and TV a ial wa e peak (TVA). The TV
peak p essu e g adien (TVPPG) and TVE/A a io we e
calcula ed. Fo icuspid al e iew, Dopple sample
olume dep h, size and angle we e 6–12 mm, 0.29 mm
and 5–50 dg, espec i ely.
All measu emen s we e pe o med excluding he espi -
a ion peaks and ob ained in iplica e; he mean o median
alue was used o da a analysis. All calcula ed pa ame e s
we e au oma ically compu ed by he Ve o 2100 s anda d
measu emen package. The equa ions used by he sys em
a e shown in de ail (see Addi ional ile 1).
In a- and in e -obse e a iabili y
The a iabili y o LV M-mode measu emen s was
de e mined. Fo in a-obse e a iabili y, one examine
analyzed all animals wice, in di e en occasions. Fo
in e -obse e a iabili y, all animals we e e-analyzed
by a blinded examine . The pe cen age o e o and he
obse e a ia ion we e calcula ed. The pe cen age o
e o is he di e ence be ween wo obse a ions di ided
by he mean and exp essed as pe cen ages, while he
obse e a ia ion is he di e ence be ween he wo
measu emen s.
S a is ical analysis
S a is ical analysis was pe o med using SPSS so wa e
(Ve sion 20). Shapi o-Wilk es was used o assess no mal-
i y o da a. The ollowing pa ame e s we e no no mally
dis ibu ed: AoVPV, DAoPV, AoVPPG and RVIDs om ju-
enile da a and LVEndoAs, LVEndoLd and LVEpiLd om
adul da a. Da a a e p esen ed as mean ± s anda d de i-
a ion o median wi h in e qua ile ange (IQR), whene e
app op ia e. To compa e esul s be ween he wo g oups,
ju enile and adul mice, S uden ’sunpai ed - es was used
o he no mally dis ibu ed da a, while Mann–Whi ney
U es was used o no no mally dis ibu ed da a. Pai ed
- es was used o wi hin g oup compa ison. Bland-
Al man analysis was pe o med o assess ag eemen
be ween measu emen s o in a- and in e -obse e mea-
su emen s a iabili y; in addi ion Pea son’s co ela ion was
used. Fo BW and HR associa ion wi h all he measu ed
pa ame e s, Pea son’s co ela ion o Spea man’s co ela-
ion we e used depending i da a ollowed a linea model
and i i was no mally dis ibu ed o no , espec i ely. A
p- alue < 0.05 was conside ed signi ican .
Resul s
Sa is ac o y images could be cap u ed in all animals. Ca -
diac examina ion ime was a ound 30 min, in all animals.
All echoca diog aphic measu emen s pe o med in ju en-
ile and adul mice we e summa ized (Tables 1, 2 and 3).
Table 1 Echoca diog aphic measu emen s om B-mode
images
Measu emen Ju enile n Adul n p
LVEndoLd (mm) 6.18 (0.57) 15 7.40 (0.58) 15 < 0.001
LVEndoLs (mm) 4.99 ± 0.38 15 6.58 ± 0.45 15 < 0.001
LVEpiLd (mm) 6.38 (0.56) 15 7.65 (0.67) 15 < 0.001
LVEpiLs (mm) 5.35 ± 0.34 15 7.03 ± 0.44 15 < 0.001
LVEndoAd (mm
2
) 6.06 ± 0.87 15 9.87 ± 1.20 15 < 0.001
LVEndoAs (mm
2
) 3.06 (0.98) 15 4.25 (1.12) 15 < 0.001
LVEpiAd (mm
2
) 11.85 ± 1.73 15 19.47 ± 1.71 15 < 0.001
LVEpiAs (mm
2
) 8.84 ± 1.83 15 14.46 ± 1.57 15 < 0.001
EAC (mm
2
) 3.22 ± 0.81 15 5.62 ± 1.16 15 < 0.001
FAC (%) 52.89 ± 10.07 15 56.87 ± 7.80 15 ns
LVVd (μl) 29.57 ± 6.04 15 59.90 ± 8.78 15 < 0.001
LVVs (μl) 11.85 ± 3.19 15 23.32 ± 5.31 15 < 0.001
LVM (mg) 39.68 ± 8.64 15 81.90 ± 9.69 15 < 0.001
LVMi (mg/g) 5.26 ± 1.27 15 4.16 ± 0.42 15 0.005
AoD (mm) 0.93 ± 0.09 15 1.27 ± 0.13 15 < 0.001
PAD (mm) 1.34 ± 0.11 15 1.59 ± 0.13 15 < 0.001
Values a e mean ± SD o median (IQR), as app op ia e. p= s a is ical
signi icance; ns = no signi ican . n = 15 o all measu emen s.
LVEndoL = Le en icle endoca dial leng h. LVEpiL = Le en icle epica dial
leng h. LVEndoA = Le en icle endoca dial a ea. LVEpiA = Le en icle
epica dial a ea. EAC = Endoca dial a ea change. FAC = F ac ional a ea change.
LVV = Le en icle olume. LVM = Le en icle mass. LVMi = LVM index. AoD
= Ascending ao a diame e . PAD = Pulmona y a e y diame e . -d = In dias ole.
-s = In sys ole.
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The RV could only be assessed in 7 ju enile and in 11
adul mice. The TV in low could only be assessed in 12
ju enile and in 6 adul mice.
BW was signi ican ly lowe in he ju enile (7.68 ± 1.33 g)
as compa ed o adul mice (19.84 ± 2.84 g; p<0.001). In
he same way, HR was signi ican ly lowe in he ju enile
(387.34 ± 44.12 bpm) as compa ed o adul mice (421.82 ±
25.34 bpm; p=0.015).
Table 1 summa izes B-mode echoca diog aphic mea-
su emen s. This able shows ha all pa ame e s ha e
highe alues in he adul mice han ju enile, excep o
FAC and LVMi. Table 2 summa izes M-mode echoca -
diog aphic measu emen s. This able shows ha EF and
FS a e conside ed he same in he wo g oups. Rep esen-
a i e images and measu emen s o echoca diog aphic
B-mode and M-mode a e shown in Figu e 1. Table 3
summa izes Dopple echoca diog aphic measu emen s.
SV and CO we e lowe in he ju enile as compa ed o
adul mice. No signi ican di e ence was ound in MV
and TV ela ed da a be ween ju enile and adul mice,
excep o IVRT. Rep esen a i e images o echoca dio-
g aphic PW Dopple -mode a e shown in Figu e 2. The
mos impo an alues o Tables 1, 2 and 3 a e also sum-
ma ized in Figu e 3.
We also ound ha some o he s uc u al pa ame e s an-
alyzed, such as AoD, LVEpiLs andLVEndoLd,hadas ong
posi i e co ela ion wi h BW, bo h in ju enile and adul
mice (see Addi ional ile 2 and Addi ional ile 3). Bo h
CO and SV co ela ed s ongly wi h BW in ju eniles and
adul s (p< 0.01). In 3 weeks mice, NFT had a mode a ely
s ong nega i e co ela ion wi h HR (p<0.01) (see
Addi ional ile 2). In 8 weeks mice, also NFT and AET had
a s ong nega i e co ela ion wi h HR (p<0.01) (see
Addi ional ile 3).
Table 4 summa izes in a- and in e -obse e a iabil-
i y o LV M-mode measu emen s. No signi ican di e -
ences be ween measu emen s we e ound, excep o
in a-obse e measu emen o LVPWd om adul mice
(p< 0.05 by pai ed - es ). Howe e , Bland-Al man ana-
lysis showed a good ag eemen o bo h measu emen s
(Figu e 4), linea eg ession LVPWd (in a_Obs.1) =
0.885 × LVPWd (in a_Obs.2) + 0.107,
2
= 0.89, Pea son’s
co ela ion 0.94, p< 0.01. The ag eemen be ween in a-
and in e -obse e measu emen s was conside ed high,
as illus a ed in Bland-Al man analysis (Table 5).
Table 2 Echoca diog aphic measu emen s om M-mode
images
Measu emen Ju enile n Adul n p
LVAWd (mm) 0.61 ± 0.09 15 0.74 ± 0.14 15 0.005
LVAWs (mm) 0.85 ± 0.13 15 1.08 ± 0.18 15 < 0.001
LVIDd (mm) 2.84 ± 0.20 15 3.56 ± 0.19 15 < 0.001
LVIDs (mm) 1.96 ± 0.21 15 2.44 ± 0.28 15 < 0.001
LVPWd (mm) 0.59 ± 0.15 15 0.64 ± 0.13 15 ns
LVPWs (mm) 0.81 ± 0.17 15 0.88 ± 0.19 15 ns
EF (%) 59.63 ± 9.05 15 59.91 ± 8.15 15 ns
FS (%) 30.76 ± 6.15 15 31.45 ± 5.63 15 ns
IVSd (mm) 0.41 ± 0.08 15 0.47 ± 0.15 15 ns
IVSs (mm) 0.50 ± 0.13 15 0.62 ± 0.25 15 ns
RVIDd (mm) 1.08 ± 0.15 7 1.42 ± 0.19 11 0.001
RVIDs (mm) 0.65 (0.31) 7 0.89 (0.21) 11 ns
RVAWd (mm) 0.50 ± 0.11 7 0.32 ± 0.08 11 0.001
RVAWs (mm) 0.66 ± 0.16 7 0.57 ± 0.13 11 ns
Values a e mean ± SD o median (IQR), as app op ia e. p= s a is ical
signi icance. ns = no signi ican . n = 15 o all measu emen s, excep o RV
measu emen s (n = 7 o he ju eniles and n = 11 o he adul s).
LVAW = Le en icle an e io wall. LVID = Le en icle in e nal diame e .
LVPW = Le en icle pos e io wall. EF = Ejec ion ac ion. FS = F ac ional
sho ening. IVS = In e en icula sep um. RVID = Righ en icle in e nal
diame e . RVAW = Righ en icle an e io wall. -d = In dias ole. -s = In sys ole.
Table 3 Echoca diog aphic measu emen s om Dopple
images
Measu emen Ju enile n Adul n p
AoVPV (mm/s) 1055.28 (327.21) 15 1557.67 (693.61) 15 0.003
AET (ms) 50.37 ± 4.56 15 49.59 ± 4.50 15 ns
AoVPPG (mmHg) 4.45 (3.13) 15 9.76 (9.46) 15 0.003
DAoPV (mm/s) 747.59 (162.89) 15 1049.88 (321.15) 15 0.002
SV (μl) 31.93 ± 8.67 15 70.61 ± 24.66 15 < 0.001
CO (ml/min) 12.06 ± 4.05 15 29.71 ± 10.13 15 < 0.001
COi (ml/min.g) 1.54 ± 0.36 15 1.48 ± 0.39 15 ns
PVPV (mm/s) 663.34 ± 141.08 15 810.62 ± 149.55 15 0.01
PVPPG (mmHg) 1.83 ± 0.77 15 2.71 ± 0.96 15 0.01
MVE (mm/s) 673.53 ± 149.31 15 747.08 ± 92.31 15 ns
MVA (mm/s) 456.51 ± 73.29 15 500.17 ± 85.14 15 ns
IVCT (ms) 10.90 ± 2.76 15 12.91 ± 3.45 15 ns
IVRT (ms) 16.52 ± 3.29 15 12.48 ± 2.49 15 0.001
MVET (ms) 61.05 ± 9.74 15 59.24 ± 8.34 15 ns
NFT (ms) 77.45 ± 5.66 15 75.66 ± 7.57 15 ns
MVPPG (mmHg) 1.98 ± 0.76 15 2.30 ± 0.52 15 ns
MVE/A 1.50 ± 0.37 15 1.53 ± 0.27 15 ns
LVMPI 0.55 ± 0.14 15 0.51 ± 0.07 15 ns
TVE (mm/s) 241.72 ± 30.57 12 252.83 ± 77.42 6 ns
TVA (mm/s) 400.90 ± 47.96 12 386.53 ± 123.06 6 ns
TVPPG (mmHg) 0.74 ± 0.21 12 0.68 ± 0.39 6 ns
TVE/A 0.60 ± 0.06 12 0.66 ± 0.13 6 ns
Values a e mean ± SD o median (IQR), as app op ia e. p= s a is ical
signi icance. ns = no signi ican . n = 15 o all measu emen s, excep o TV
measu emen s (n = 12 o he ju eniles and n = 6 o he adul s).
AoVPV = Ascending ao a al e peak eloci y. AET = Ao ic ejec ion ime.
AoVPPG = Ascending ao a al e peak p essu e g adien . DAoPV = Descending
ao a peak eloci y. SV = S oke olume. CO = Ca diac ou pu . COi = CO index.
PVPV = Pulmona y al e peak eloci y. PVPPG = Pulmona y al e peak p essu e
g adien . MVE = Mi al al e ea ly wa e peak. MVA = Mi al al e a ial wa e
peak. IVCT = Iso olumic con ac ion ime. IVRT = Iso olumic elaxa ion ime.
MVET = Mi al al e ejec ion ime. NFT = No Flow Time. MVPPG = Mi al al e
peak p essu e g adien . LVMPI = Le en icle myoca dial pe o mance index.
TVE = T icuspid al e ea ly wa e peak. TVA = T icuspid al e a ial wa e peak.
TVPPG = T icuspid al e peak p essu e g adien .
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Discussion
In o de o unde s and and analyze s udies ha use ans-
genic animals o animals ha unde go su gical p ocedu es,
he ca diac cha ac e iza ion o no mal/wild ype and
heal hy animals is conside ed ex emely impo an . To ou
bes knowledge, echoca diog aphic e alua ion o e e ence
alues o B-mode, M-mode and Dopple -mode in ju enile
(3 weeks) and adul (8 weeks) 129/S wild- ype mice has
no been epo ed. In he p esen s udy, ca diac dimen-
sions we e signi ican ly di e en be ween ju enile and
adul mice, as expec ed. Dias olic unc ion does no di e
be ween ju enile and adul mice. Addi ionally, we demon-
s a e ha he a iabili y o LV measu emen s in M-mode
is minimal, indica ing ha his me hod is eliable.
Body weigh and hea a e
The BW was signi ican ly lowe in he ju enile han in
he adul mice, as expec ed. Simila esul s we e
obse ed in o he mice s ains, as C57BL/6 and CD1
mice [6,8,10]. Ne e heless, BW o 3 weeks 129/S mice
was lowe han 3 weeks C57BL/6 mice (7.68g s 10.2g)
[10] and 8 weeks 129/S mice we e lowe han 8 weeks
CD1 mice (19.84g s 32.4g) [6], showing he in luence o
mouse backg ounds in body weigh . In addi ion, HR was
signi ican ly lowe in he younge animals, despi e he
simila iso lu ane concen a ion used o bo h g oups,
which is inconsis en wi h some s udies [6,8,11]. One
s udy shows ha HR in C57BL6 mice is cons an
be ween 1 mon h and 2 mon hs mice and dec eases be-
ween 2 mon hs and 16 mon hs [8], while o he s udies
show ha HR in C57BL6 conscious mice and CD1 mice
dec eases wi h age [6,11]. We ound one s udy in
acco dance wi h ou esul , whe e HR was highe in
old s young C57BL/6 mice [10]. The highe HR ob-
se ed in he adul mice migh be explained possibly
due o a highe ho acic comp ession in he adul
Figu e 1 Rep esen a i e B-mode and M-mode echoca diog aphic images and measu emen s. (A) Pa as e nal long axis iew o le en icle
(LV) in B-mode. (B) Ao ic a ch iew in B-mode. (C) Sho axis iew in 2D (le panel) and M-mode acing ( igh panel) o he LV, a he le el o papilla y
muscle. (D) Righ pa as e nal long axis iew in 2D (le panel) and M-mode acing ( igh panel) o he igh en icle (RV). LVEndoL = Le en icle
endoca dial leng h. AoD = Ascending ao a diame e . LVAW = Le en icle an e io wall. LVID = Le en icle in e nal diame e . LVPW = Le en icle
pos e io wall. RVAW = Righ en icle an e io wall. RVID = Righ en icle in e nal diame e ; -d = In dias ole. -s = In sys ole.
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animals du ing echoca diog aphy, o allow a be e
access o he hea .
Ca diac dimensions pa ame e s
We obse ed a signi ican di e ence in LV leng hs and
LV a eas in 3 weeks mice s 8 weeks mice, which could
be explained by he signi ican ly highe BW o adul
mice, due o he con inuous inc ease o he hea and
body weigh be ween hese ages.
A highe alue o EAC was obse ed in he adul mice,
bu we couldn’ ind any da a o co ela e wi h ou
esul . While FAC, a pa ame e ha ep esen s LV sys-
olic unc ion, does no di e be ween ju enile and
adul . Acco ding o p e ious s udies in C57BL6 mice,
FAC does no di e be ween hese ages, which co ela e
wi h ou esul . Howe e he alue ound is lowe han
in ou s udy, a ound 46% [8]. Toge he , hese esul s
ein o ce he in luence o mice backg ound on ca diac
pa ame e s.
As expec ed, LVV and LVM we e di e en be ween 3
weeks mice and 8 weeks mice, since he animals a e s ill
in g ow h. Howe e , when LVM was no malized o BW
Figu e 2 Rep esen a i e Dopple echoca diog aphic images. (A) Sup as e nal iew (le panel) and PW Dopple acing ( igh panel) o ao ic
ou low. (B) Modi ied sho axis iew (le panel) and PW-Dopple acing ( igh panel) o pulmona y ou low. (C) Apical 4 chambe iew (le panel) and
PW-Dopple acing ( igh panel) o mi al al e (MV) in low. (D) Apical 4 chambe iew (le panel) and PW-Dopple acing ( igh panel) o icuspide
al e (TV) in low.
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(LVMi), we no iced ha LVMi was highe in he ju e-
niles due o he highe BW o he adul mice. These
esul s a e consis en wi h he li e a u e ound o o he
s ains [6,12].
We obse ed no signi ican di e ence in hickness o
IVS and LVPW be ween ju enile and adul mice. A simi-
la esul was ob ained in a p e ious s udy wi h o he
mice s ain be ween 6 and 12 weeks old [9]. LVAW
hickness and LVID we e signi ican ly di e en in he
wo g oups. The li e a u e ound o hese pa ame e s
showed a endency owa d an inc ease o LVAW [6] and
LVID [8,9] wi h age, al hough no s a is ical signi icance
was eached.
Ou da a sugges s ha EF and FS, pa ame e s o
sys olic unc ion, does no di e be ween 3 weeks and 8
weeks mice, which ma ch wi h p e ious esul s ound
o o he mice s ains [6,8,9]. The alues ound o EF in
o he s udies wi h anes he ized animals we e a ound 53
–55% o 129/S mice [13] and a ound 60 –63% o
CD1 mice [6]. EF in conscious animals was a ound 65%
o C57BL6 mice [14] and 84 % o 129/S mice [13]. The
alues ound o FS in o he s udies wi h anes he ized ani-
mals we e a ound 37 –40% o CD1 mice [6], a ound
33 –35% o 129/S mice [13] and a ound 35% o
C57BL6 mice [8]. In ano he s udy, a FS alue o 45% was
obse ed o 6–8 mon hs C57BL6 mice [15]. FS in
conscious animals we e a ound 51% o 129/S mice [13].
Ou low- ela ed pa ame e s
Conce ning he ao a and pulmona y a e y, hei
espec i e diame e s, peak eloci ies and peak p essu es
we e di e en be ween ju enile and adul mice. The
highe a e ial diame e s in he adul s, due o highe
body su ace in olde animals, go along wi h he
Figu e 3 Ba g aphs showing o e iew o e he esul s o ju enile (n = 15) s adul mice (n = 15). (A) Thicknesses o le en icle (LV)
walls and in e en icula sep um (IVS). (B) Diame e s o LV and a e ies. (C) Pe cen ages o ac ional a ea change (FAC), ejec ion ac ion (EF) and
ac ional sho ening (FS). (D) Peak eloci ies o a e ial ou low and mi al al e (MV) in low. * S a is ically signi ican . LVID = Le en icle in e nal
diame e . AoD = Ascending ao a diame e . PAD = Pulmona y a e y diame e . LVAW = Le en icle an e io wall. LVPW = Le en icle pos e io
wall. AoVPV = Ascending ao a al e peak eloci y. DAoPV = Descending ao a peak eloci y. PVPV = Pulmona y al e peak eloci y. MVE = Mi al
al e ea ly wa e peak. MVA = Mi al al e a ial wa e peak. -d = In dias ole. -s = In sys ole.
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li e a u e ound [8]. AET was he only a e ial- ela ed
pa ame e ha did no show a signi ican di e ence
be ween he wo g oups.
SV and CO, calcula ed by he ao ic ou low me hod,
we e signi ican ly highe in 8 weeks mice. On he o he
hand, COi was conside ed he same in he wo g oups
due o BW no maliza ion o CO esul ed om highe
BW in he adul mice compa ed o lowe BW in he ju-
enile mice. Pa ame e s ha a e cons an when indexed
o BW indica e ha hese alues a e ela ed o body size.
Table 4 Va iabili y o LV M-mode measu emen s
In a-Obse e In e -Obse e
Ju enile E o (%) Obse e a ia ion (mm) E o (%) Obse e a ia ion (mm)
LVAWd 1.08 ± 6.66 −0.01 ± 0.04 3.05 ± 11.36 0.02 ± 0.06
LVAWs 3.74 ± 5.11 −0.03 ± 0.04 4.88 ± 12.02 0.04 ± 0.10
LVIDd 1.07 ± 3.30 0.03 ± 0.10 0.32 ± 6.29 0.01 ± 0.18
LVIDs 3.11 ± 7.32 0.06 ± 0.16 1.11 ± 14.40 0.02 ± 0.29
LVPWd 1.78 ± 7.11 −0.01 ± 0.04 13.82 ± 27.87 0.07 ± 0.14
LVPWs 0.78 ± 9.52 0.00 ± 0.08 9.15 ± 30.98 0.06 ± 0.20
IVSd 4.96 ± 11.52 0.02 ± 0.04 8.83 ± 24.40 0.05 ± 0.11
IVSs 2.40 ± 13.61 0.00 ± 0.07 2.07 ± 23.71 0.02 ± 0.13
Adul E o (%) Obse e Va ia ion (mm) E o (%) Obse e Va ia ion (mm)
LVAWd 4.18 ± 8.30 −0.03 ± 0.07 3.24 ± 12.83 0.03 ± 0.10
LVAWs 2.40 ± 8.69 −0.02 ± 0.10 1.70 ± 9.05 0.02 ± 0.10
LVIDd 1.49 ± 3.52 0.05 ± 0.12 −0.43 ± 2.77 −0.02 ± 0.10
LVIDs 1.13 ± 5.56 0.03 ± 0.14 0.57 ± 5.44 0.01 ± 0.15
LVPWd 5.68 ± 7.16 * −0.03 ± 0.04* −5.64 ± 20.58 −0.05 ± 0.19
LVPWs 2.10 ± 6.29 −0.01 ± 0.05 −8.86 ± 21.86 −0.10 ± 0.24
IVSd 2.73 ± 8.55 −0.01 ± 0.04 −2.88 ± 21.07 −0.01 ± 0.09
IVSs 2.04 ± 10.62 0.00 ± 0.06 −0.84 ± 18.54 0.01 ± 0.10
Values a e mean ± SD. p= s a is ical signi icance. All p> 0.05, excep * p= 0.01.
LVAW = Le en icle an e io wall. LVID = Le en icle in e nal diame e . LVPW = Le en icle pos e io wall. IVS = In e en icula sep um. -d = In dias ole.
-s = In sys ole.
Figu e 4 Bland-Al man co ela ion o in a-obse e measu emen s o LVPWd in adul mice. LVPWd = Le en icle pos e io wall in
dias ole. Obs.1 = Measu emen 1 o obse e 1. Obs. 2 = Measu emen 2 o obse e 1.
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Ou CO esul is inconsis en wi h he da a ound o
o he s ain [6], which shows no di e ence be ween di -
e en ages. This could be explained by he olde ages
es ed in he e e ed s udy (be ween 8 and 52 weeks).
The alues ound in o he s udies o CO we e a ound
18 –20 ml/min o 129/S mice [13] and 16 –17 ml/min
o CD1 mice [6].
In low- ela ed pa ame e s
We did no ind any signi ican di e ence in MV and
TV in low pa ame e s be ween he wo g oups, excep
o IVRT ha was signi ican ly lowe in he 8 weeks
mice. E wa e and A wa e a e dias olic pa ame e s ha
depend mainly on myoca dial elaxa ion, LV geome y
and loading condi ions. The e o e du ing ma u a ion o
he LV, om ju enile o adul , hese pa ame e s e ol e
in pa allel, keeping he esul ing E and A wa e cons an .
IVRT is dependen o LV elaxa ion, loading condi ions
and HR. Bea ing in mind he cons an alues o A and E
wa e, IVRT changes could be dependen on in insic
myoca dial elaxa ion. A simila esul was ob ained o
MVE [6,8], MVA [6], MVPPG [6], IVRT [8] and MVE/A
[6,8] o o he mice s ains in p e ious s udies.
Limi a ions
One limi a ion o his s udy is he in luence o anes he ic
agen as dep esso o ca dio ascula unc ion. Howe e
he use o anes hesia du ing echoca diog aphy is c ucial
o acili a e da a acquisi ion, by p o iding seda ion and
immobili y o animals, a oiding he animal s ess du ing
he p ocedu e. We used he iso lu ane, since i ’s one o
he mos common inhalan anes he ics, and has many
ad an ages when compa ed wi h o he anes he ics. I
p oduces minimal ca diac dep ession and has highe
molecula s abili y [16]. Also, iso lu ane was conside ed
he mos ep oducible anes he ic in epea s udies a 12
days [17].
I would ha e been ad an ageous o conside mo e
han wo ime poin s, in o de o analyze da a du ing
he en i e li e span o hese animals.
Also, i would be in e es ing o analyze, in he u u e,
global and egional s ain da a o de o ma ions e e -
ence alues.
Conclusions
Since mice a e a common model sys em o s udying
ca diac de elopmen and disease, ca diac cha ac e iza ion
o no mal/wild ype and heal hy animals is conside ed ex-
emely impo an o in e p e a ion o esul s in ans-
genic and su gical animals. And so, his s udy de ines
e e ence echoca diog aphic measu emen s and calcula ed
pa ame e s o ju enile (3 weeks) and adul (8 weeks)
129/S wild ype mice.
The wo k p esen ed he e sugges s a signi ican di e -
ence o almos all ca diac dimensions as well as ou low-
ela ed pa ame e s (pulmona y and ao a low) o 3
weeks s 8 weeks 129/S wild ype mice. In e es ingly,
in low- ela ed pa ame e s (mi al and icuspid low) do
Table 5 Ag eemen be ween measu emen s o in a- and in e -obse e a iabili y
In a-Obse e In e -Obse e
Ju enile Mean di e ence ± SD Limi s o ag eemen p*Mean di e ence ± SD Limi s o ag eemen p*
LVAWd −0.01 ± 0.04 −0.09 o 0.08 0.91 <0.01 0.02 ± 0.06 −0.11 o 0.14 0.82 <0.01
LVAWs −0.03 ± 0.04 −0.12 o 0.06 0.94 <0.01 0.04 ± 0.10 −0.16 o 0.24 0.72 <0.01
LVIDd 0.03 ± 0.10 −0.17 o 0.23 0.89 <0.01 0.01 ± 0.18 −0.36 o 0.38 0.64 <0.05
LVIDs 0.06 ± 0.16 −0.25 o 0.38 0.77 <0.01 0.02 ± 0.29 −0.55 o 0.60 0.20 ns
LVPWd −0.01 ± 0.04 −0.08 o 0.07 0.97 <0.01 0.07 ± 0.14 −0.21 o 0.36 0.50 ns
LVPWs 0.00 ± 0.08 −0.16 o 0.15 0.89 <0.01 0.06 ± 0.20 −0.34 o 0.46 0.42 ns
IVSd 0.02 ± 0.04 −0.07 o 0.11 0.83 <0.01 0.05 ± 0.11 −0.17 o 0.26 0.39 ns
IVSs 0.00 ± 0.07 −0.13 o 0.14 0.91 <0.01 0.02 ± 0.13 −0.24 o 0.27 0.57 <0.05
Adul Mean di e ence ± SD Limi s o ag eemen p*Mean di e ence ± SD Limi s o ag eemen p*
LVAWd −0.03 ± 0.07 −0.17 o 0.10 0.90 <0.01 0.03 ± 0.10 −0.18 o 0.23 0.70 <0.01
LVAWs −0.02 ± 0.10 −0.22 o 0.17 0.84 <0.01 0.02 ± 0.10 −0.19 o 0.23 0.82 <0.01
LVIDd 0.05 ± 0.12 −0.18 o 0.28 0.87 <0.01 −0.02 ± 0.10 −0.22 o 0.18 0.91 <0.01
LVIDs 0.03 ± 0.14 −0.24 o 0.30 0.89 <0.01 0.01 ± 0.15 −0.29 o 0.30 0.91 <0.01
LVPWd −0.03 ± 0.04 −0.12 o 0.05 0.94 <0.01 −0.05 ± 0.19 −0.42 o 0.32 0.47 ns
LVPWs −0.01 ± 0.05 −0.12 o 0.10 0.96 <0.01 −0.10 ± 0.24 −0.57 o 0.38 0.49 ns
IVSd −
0.01 ± 0.04 −0.08 o 0.06 0.97 <0.01 −0.01 ± 0.09 −0.18 o 0.17 0.83 <0.01
IVSs 0.00 ± 0.06 −0.11 o 0.11 0.98 <0.01 0.01 ± 0.10 −0.18 o 0.21 0.93 <0.01
= Pea son’s coe icien . *p- alue o co ela ion coe icien .
LVAW = Le en icle an e io wall. LVID = Le en icle in e nal diame e . LVPW = Le en icle pos e io wall. IVS = In e en icula sep um. -d = In dias ole.
-s = In sys ole.
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