Hæmodynamisk Karakterisering af gnavermodeller af pulmonal arteriel hypertension
Summary
Pulmonary arterial hypertension (PAH) is a disease of pulmonary arterioles that leads to their obliteration and the development of right ventricular failure. Rodent models of PAH are critical in understanding the pathophysiology of PAH. Here we demonstrate hemodynamic characterization, with right heart catheterization and echocardiography, in the mouse and rat.
Abstract
Pulmonary arterial hypertension (PAH) is a rare disease of the pulmonary vasculature characterized by endothelial cell apoptosis, smooth muscle proliferation and obliteration of pulmonary arterioles. This in turn results in right ventricular (RV) failure, with significant morbidity and mortality. Rodent models of PAH, in the mouse and the rat, are important for understanding the pathophysiology underlying this rare disease. Notably, different models of PAH may be associated with different degrees of pulmonary hypertension, RV hypertrophy and RV failure. Therefore, a complete hemodynamic characterization of mice and rats with PAH is critical in determining the effects of drugs or genetic modifications on the disease.
Here we demonstrate standard procedures for assessment of right ventricular function and hemodynamics in both rat and mouse PAH models. Echocardiography is useful in determining RV function in rats, although obtaining standard views of the right ventricle is challenging in the awake mouse. Access for right heart catheterization is obtained by the internal jugular vein in closed-chest mice and rats. Pressures can be measured using polyethylene tubing with a fluid pressure transducer or a miniature micromanometer pressure catheter. Pressure-volume loop analysis can be performed in the open chest. After obtaining hemodynamics, the rodent is euthanized. The heart can be dissected to separate the RV free wall from the left ventricle (LV) and septum, allowing an assessment of RV hypertrophy using the Fulton index (RV/(LV+S)). Then samples can be harvested from the heart, lungs and other tissues as needed.
Introduction
Pulmonal arteriel hypertension (PAH) er en sygdom af den pulmonale vaskulatur associeret med inflammatorisk celleinfiltration, muskel proliferation glat og endotelcelle apoptose. Disse ændringer resulterer i udslettelse af pulmonale arterioler, der efterfølges af højre ventrikel (RV) dysfunktion og hjertesvigt. For at forstå patofysiologien bag PAH og RV fiasko i PAH, har en række forskellige modeller, herunder genetiske og farmakologiske modeller, for at studere denne sygdom blevet udviklet (revideret andetsteds 1,2).
Af disse modeller, de mest populære er hypoxi-induceret (Hx) PAH i musen og monocrotaline (MCT) og SU5416-hypoxi (SuHx) modeller i rotten. I muse Hx model musene udsat for 4 ugers hypoxi (enten normobar eller lavtryksforhold, svarende til en højde på 18.000 fod med en FiO2 på 0,10), med den resulterende udvikling af medial proliferation, forøget RV systOlic pres og udvikling af RV hypertrofi 3. MCT ved en enkelt dosis på 60 mg / kg resulterer i skade på pulmonale endotelceller via en uklar mekanisme, der derefter resulterer i udviklingen af PAH 4. SU5416 er en inhibitor af de vaskulære endotelvækstfaktor receptorer (VEGFR) 1 og 2 blokker, og behandling med en enkelt subkutan injektion af 60 mg / kg efterfulgt af udsættelse for kronisk hypoxi i 3 uger resulterer i permanent pulmonal hypertension med lignende patologiske forandringer den, der ses i den humane sygdom, med dannelsen af obliterativ vaskulære læsioner 5. I de seneste år har flere transgene musemodeller for pulmonal hypertension blevet udviklet. Disse omfatter knockout og mutationer af knoglemorfogenetisk protein receptor 2 (BMPR2), som BMPR2 genmutationer findes i både familiære og idiopatiske former af PAH, hæm oxygenase-1 knockout og IL-6-overekspression (revideret andetsteds 1,2).
Disse forskellige gnaver modeller af PH har forskellige niveauer af pulmonal hypertension, RV hypertrofi og RV fiasko. Mens hypoxi og forskellige transgene musemodeller resultere i langt mildere PAH end den enten rotte model 1, det tillader afprøvning af forskellige genetiske mutationer og deres tilknyttede molekylære signalveje. MCT-modellen medfører alvorlig PAH, selvom MCT synes at være giftige for endotelceller i flere væv 4. Den SuHx model er kendetegnet ved vaskulære forandringer mere ligner den, der ses ved idiopatisk PAH i mennesker, selvom kræver både farmakologisk manipulation og hypoxi eksponering. Øvrigt i alle disse modeller, kan der være en afbrydelse mellem de histopatologiske ændringer, pulmonale tryk og RV funktion er forbundet med udviklingen af PAH. Dette er i modsætning til den humane sygdom, hvor der som regel en forholdsmæssig forhold mellem histopatologiske ændringer, sværhedsgraden af pulmonAry hypertension og graden af RV svigt. Således er en omfattende karakterisering af disse gnavere modeller af PH påkrævet, og ved vurderingen af RV funktion (typisk ved ekkokardiografi), hæmodynamik (ved hjertekateterisation) og histopatologi af hjertet og lungerne (fra væv høst).
I denne protokol, beskriver vi de grundlæggende teknikker, der anvendes til hæmodynamisk karakterisering af PAH-modeller i rotter og mus. Disse generelle teknikker kan anvendes på enhver undersøgelse af den højre ventrikel og pulmonal vaskulatur og er ikke begrænset til modeller af PAH. Visualisere RV ved ekkokardiografi er relativt ligetil i rotter, men er mere udfordrende i mus på grund af deres størrelse og komplekse geometri af RV. Desuden er nogle surrogater, der anvendes til at kvantificere RV funktion, såsom TAPSE, pulmonal (PA) accelerationstid og PA Doppler bølgeform udhugning, ikke godt valideret i mennesker og korrelerer kun svagt med vurdering af pulmonary hypertension og RV funktion ved invasive hæmodynamik. Bestemmelse af RV hæmodynamik gøres bedst med en lukket kiste, for at opretholde virkningerne af en negativ intrathoracic tryk med inspiration, selvom åben bryst kateterisation med en impedans kateter tillader bestemmelse af tryk-volumen (PV) loops og en mere detaljeret hæmodynamisk karakterisering . Som med enhver procedure, udvikle erfaringer med de procedurer er afgørende for eksperimentel succes.
Protocol
Representative Results
Discussion
The protocols outlined here describe a comprehensive characterization of hemodynamics and right ventricular function in rodent models of pulmonary hypertension. While right heart catheterization as described here is a terminal procedure, the mortality associated with echocardiography is minimal, which allows for screening and follow-up of disease progression. However, similar to patients with PH having markedly increased mortality with anesthesia17, in our experience, rats with severe PH do not tolerate anesth…
Disclosures
The authors have nothing to disclose.
Acknowledgements
SR is supported by NIH K08HL114643, Gilead Research Scholars in Pulmonary Arterial Hypertension and a Burroughs Wellcome Fund Career Award for Medical Scientists.
Materials
| Vevo 2100 Imaging System (120V) | VisualSonics, inc. | VS-11945 | |
| Vevo 2100 Imaging Station | VisualSonics, inc. | ||
| High-frequency Mechanical Transducers | VisualSonics, inc. | MS250, MS550D, MS400 | |
| Ultrasound Gel Parker | Laboratories Inc. | 01-08 | |
| PowerLab 4/35 | ADInstruments | ML765 | |
| Labchart 8 | ADInstruments | ||
| BP transducer with stopcock and cable | ADInstruments | MLT1199 | |
| BP transducer calibration kit | ADInstruments | MLA1052 | |
| Mikro-Tip Pressure Catheter for mouse | Millar | SPR-1000 | Alternative catheter available from Scisense FT111B (mouse) and FT211B (rat) |
| Mikro-Tip Pressure Catheter for rat | Millar | SPR-513 | Alternative catheter available from Scisense FT111B (mouse) and FT211B (rat) |
| Millar Mikro-Tip ultra-miniature PV loop catheter for mice | Millar | PVR-1035 | Alternative catheter available from Scisense FT112 (mouse) |
| Millar Mikro-Tip ultra miniature PV loop catheter for rats | Millar | SPR-869 | Alternative catheter available from Scisense FT112 (mouse) |
| Millar PV system MPVS-300 | Millar | MPVS-300 | |
| 4-0 Silk Black Braid 100 Yard Spool | Roboz Surgical | SUT-15-2 | |
| 6-0 Silk Black Braid 100 Yard Spool | Roboz Surgical | SUT-14-1 | |
| Iris Scissors, Delicate, Integra Miltex | VWR | 21909-248 | |
| VWR Dissecting Scissors, Sharp/Blunt Tip | VWR | 82027-588 | |
| VWR Delicate Scissors, 4 1/2" | VWR | 82027-582 | |
| Two star Hemostats, Excelta | VWR | 63042-090 | |
| Neutral-buffered formalin | VWR | 89370-094 | |
| Crotaline | Sigma | C2401 | |
| SU5416 | Tocris Biosciences | 3037 | |
| 3.5X-45X Boom Stand Trinocular Zoom Stereo Microscope | AmScope | SM-3BX | |
| PE (Polyethylene Tubing)-10 | Braintree Scientific Inc | PE10 36 FT | |
| PE (Polyethylene Tubing)-50 | Braintree Scientific Inc | PE50 36 FT | |
| PE (Polyethylene Tubing)-60 | Braintree Scientific Inc | PE60 36 FT | |
| Tabletop Isoflurane Anesthesia Unit | Kent Scientific | ACV-1205S | |
| Surgisuite multi-functional surgical platform | Kent Scientific | Surgisuite | |
| Retractor set | Kent Scientific | SURGI-5002 | |
| Anesthesia induction chamber | VetEquip | 941443 | |
| Anesthesia Gas filter canister | Kent Scientific | ACV-2001 | |
| Rodent nose cone | VetEquip | 921431 |
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