Echocardiography was performed as described previously [19 (link)]. Briefly, mice were anesthetized with 1.5% isoflurane by inhalation. End-diastolic (d) and end-systolic (s) LV internal diameters (LVID), interventricular septum thickness (IVS), and posterior wall thickness (PW) [20 (link)] were measured by using two-dimensional guided M-Mode echocardiography with a Vevo 770 Imaging System (VisualSonics, Toronto, Canada) at the papillary muscle level. Fractional shortening was calculated as (LVIDd-LVIDs)/LVIDd*100 and relative wall thickness (RWT) was defined by the ratio of PW plus IVS thickness to LVIDd [21 ]. Once measurements were completed, animals were euthanized with a 100 mg/kg sodium pentobarbital intraperitoneal injection (Sigma, St. Louis, MO). The heart was rapidly excised, weighed, rinsed with ice-cold PBS, and then sectioned. The hearts were then either snap-frozen in liquid nitrogen for biochemical analysis or fixed for histological staining.
Sodium pentobarbital intraperitoneal injection
Manufactured by Merck Group
Sourced in United Kingdom
Sodium pentobarbital intraperitoneal injection is a laboratory product used for the administration of sodium pentobarbital via the intraperitoneal route. It is a barbiturate drug commonly used in research settings.
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2 protocols using sodium pentobarbital intraperitoneal injection
1
Cardiac Function Evaluation in Mice
2
Perfusion and Cryosectioning Protocol for Brainstem
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The rats were anesthetized with 75 mg/kg of sodium pentobarbital intraperitoneal injection (Sigma-Aldrich, UK). After deep anesthesia, a midline sternal incision was made to expose the heart, which was then perfused through the left ventricle. First, a quick infusion of normal saline 200 mL, and then 200 mL of 4% paraformaldehyde (Damao Chemical Reagent Factory, China) was perfused, first fast and then slow. The brain was completely removed and fixed with 4% paraformaldehyde for 2 h and transferred in 10%, 20%, and 30% sucrose solution in order of gradient transfer. After being completely dehydrated, the tissue was moved to cryostats (SLEE, Germany) at −20°C and then frozen in an OCT matrix (TissueTek, Sakura Finetek, UK) in sample preparation positions. Coronal sections of DRN (28 μm) (AP: −6.84~−8.40 mm) were placed into frozen stock solution (equal amounts of 0.01 M PBS and glycerin (Sinopharm Chemical Reagent Co., Ltd, China)) and stored at −20°C.
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