Transthoracic echocardiography reference values in juvenile and adult 129/Sv mice

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 Vinhas e al. Ca dio ascula Ul asound 2013, 11:12 Page 2 o 10 h p://www.ca dio ascula ul asound.com/con en /11/1/12 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. Vinhas e al. Ca dio ascula Ul asound 2013, 11:12 Page 3 o 10 h p://www.ca dio ascula ul asound.com/con en /11/1/12 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 . Vinhas e al. Ca dio ascula Ul asound 2013, 11:12 Page 4 o 10 h p://www.ca dio ascula ul asound.com/con en /11/1/12 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. Vinhas e al. Ca dio ascula Ul asound 2013, 11:12 Page 5 o 10 h p://www.ca dio ascula ul asound.com/con en /11/1/12 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. Vinhas e al. Ca dio ascula Ul asound 2013, 11:12 Page 6 o 10 h p://www.ca dio ascula ul asound.com/con en /11/1/12 (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. Vinhas e al. Ca dio ascula Ul asound 2013, 11:12 Page 7 o 10 h p://www.ca dio ascula ul asound.com/con en /11/1/12 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. Vinhas e al. Ca dio ascula Ul asound 2013, 11:12 Page 8 o 10 h p://www.ca dio ascula ul asound.com/con en /11/1/12 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. Vinhas e al. Ca dio ascula Ul asound 2013, 11:12 Page 9 o 10 h p://www.ca dio ascula ul asound.com/con en /11/1/12