Stroke Volume

MRI lesions for acute or subacute volumes were measured on DWI or MTT images. Subacute infarcts on CT images were measured using windows/levels of 80/20 or 30/30 for Houndsfield units2 (link). Absolute infarct were measured by E.S.R. using Alice software (Parexel Corp.). DWI and MTT lesions volumes were measured by P.W.A. using Analyze 7.0 software (Analyze Direct, KS). The ischemic ROIs were visually segmented to determine the volume. Stroke volumes ranged over 3 orders of magnitude from 0.25 to 403 cm³ by computerized planimetry. Observers (J.R.S. and L.R.G) blinded to planimetric data measured lesions in three perpendicular axes. The slice with the largest lesion was first selected by eye. The longest lesion axis on this slice was measured with the ruler tool on an AGFA R4 Workstation with Impax Select software (v5205.0.0.1). A second line was drawn perpendicular to the first at the widest dimension. These two measurements were called the x (A) and y (B) axes. A third axis, the z (C) axis, was computed by multiplying the number of slices by slice thickness (Fig. 1). The scan slice for CT was 5mm. MRI thickness ranged from 6-7 mm. Time to perform these three measurements was less than 1 minute.
For analysis of DWI and MTT mismatch, mismatch was defined as, (MTT volume/DWI volume ≥ 1.2. A parameter of 20% mismatch was chosen based on trials using “eyeball estimate” of 20% mismatch and may not be the optimal mismatch1 (link), 25 (link). Absolute volumes measured by planimetry were compared to estimates of ellipsoid ABC/2 (see below) for DWI volume and MTT volume. Euclidean Shapes (Fig. 1)
We tested the ellipsoid model both unadjusted and the adjusted model used for ICH20 (link), as well as sphere, cylinder and bicone. For the hemorrhage-adjusted ellipsoid model according to Kothari et al.20 (link), all slices with lesion volume less than 25% of the slice with the maximum lesion volume were not counted in the z axis. For slices in which the lesion volume was between 25-75%, the slice thickness was multiplied by 0.5, and for slices where the lesion volume was >75%, the slice thickness was multiplied by 1. For all geometric models, π was simplified to 3, for ease of clinical assessment. Formula simplifications for A, B, and C axes are below:
Ellipsoid model: $$\mathrm{V}=4∕3\pi {\mathrm{r}}_{\mathrm{A}}{\mathrm{r}}_{\mathrm{B}}{\mathrm{r}}_{\mathrm{C}}=4∕3\times 3\times (\mathrm{A}∕2)\times (\mathrm{B}∕2)\times (\mathrm{C}∕2)\times =\mathrm{ABC}∕2$$
Where
A= longest dimension in axis x
B= longest perpendicular dimension to axis x (y)
C= total length in z dimension
Sphere model: $$\mathrm{V}=4∕3\pi {\mathrm{r}}^3=4∕3\times 3\times (\mathrm{D}∕2{)}^3=(\mathrm{D}{)}^3∕2={\mathrm{D}}^3∕2$$
Where
D= longest measurement of A, B or C
Cylinder model: $$\mathrm{V}=\mathrm{h}\pi {\mathrm{r}}^2=\mathrm{h}\times 3\times (\mathrm{D}∕2{)}^2=3∕4(\mathrm{D}{)}^2\mathrm{h}=3∕4{\mathrm{D}}^2\mathrm{C}$$
Where
D= longest measurement of A or B
h= C
Bicone model: $$\mathrm{V}=(1∕3\mathrm{h}\pi {\mathrm{r}}^2)2=(1∕3\times 3\times \mathrm{h}\times (\mathrm{D}∕2{)}^2)2=(\mathrm{h}{\mathrm{D}}^2∕4)2\text{or}{\mathrm{D}}^2\mathrm{C}∕4$$
Where
D= longest measurement of A or B
h= C/2

After set duration of hypoxic or normoxic exposure, mice were weighed and anesthetized with Avertin (tribromoethanol) 0.375 mg/g body weight injected intraperitoneally. A tracheostomy was performed with a 22 gauge angiocatheter and secured in place with a 4.0 silk suture. Mice were ventilated with a Harvard Mini-Vent with a stroke volume of 325 µl and rate of 150 stroke/min. Anesthesia was maintained throughout with 1% isoflurane mixed with room air or 10% O₂. After thoracotomy, a 25 gauge needle fitted to a pressure transducer was inserted into the right ventricle. Right ventricular systolic pressure (RVSP) was measured and continuously recorded on a Gould polygraph (model TA-400, Gould instruments, Cleveland, Ohio). Immediately after RVSP measurements were obtained, the mice were sacrificed.
An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org and provides details of all materials, bone marrow transplantation, pulmonary vascular morphometry, Western blot, immunohistochemistry, immunofluorescence and statistical analyses.