La ideología y la violencia paramilitar a través del romance

3.5.1. Langendorff preparation of heart samples

A Langendorff perfusion setup was used to prepare heart samples to assess ATP levels and permit investigation of the effects of myocardial ischaemia. A basic Langendorff constant pressure perfusion system was used to perfuse the hearts and initiate ischaemia. A detailed description of this model is beyond the scope of this thesis; a simplified overview is illustrated in Figure 3.15 and reviewed by Bell et al. (2011).

Mice were anaesthetised with ketamine (100 mg/kg [Vetlar, Bioniche Animal Health, Canada]), xylazine (20 mg/kg [Rompun, Bayer, UK]) and atropine (0.6 mg/kg [Sigma- Aldrich, UK]) administered as a single intraperitoneal dose. Upon surgical anaesthesia, hearts were extracted and rapidly cannulated onto a 21 G standard perfusion cannula as described above (see 3.2.3). The ideal time to perfusion was less than 3 minutes although this rarely took more than 2 minutes for the experiments presented here. Langendorff perfusion chambers were filled with Krebs-Henseleit buffer (KHB) containing (in mmol/L): NaCl (118), glucose (11), NaHCO3 (25), KCl (4.7); CaCl2 (1.8),

MgSO4.7H2O (1.2) and pyruvate (0.5), equilibrated with 95% oxygen and 5% carbon

dioxide to pH 7.4 and perfused at 80 mmHg. Physiological temperature of the heart was maintained by warming of the perfusion buffer and monitored by insertion of a fine tipped temperature probe into the LV wall (via Powerlab coupled to Chart 7 software, AD

Figure 3.15: Overview of Langendorff perfusion system for preparation of heart samples Simplified diagram of the constant perfusion pressure Langendorff system used to prepare control and ischaemic heart samples. Hearts were perfused with warmed Krebs-Henseleit buffer (KHB; indicated in pale blue). Buffer was gassed with 95% oxygen and 5% carbon dioxide (gassing rod shown in dark blue) and warmed by a system of heated jackets (indicated in faint red). A bubble trap system was used to ensure that no air bubbles could enter the perfusate. The heart was perfused in a retrograde manner via the aorta whilst submerged in a warmed organ bath. Buffer flow was regulated by opening and closing the flow regulator tap (iii) to allow perfusion (for stabilisation) and cessation of perfusion (for simulated ischaemia).

Immediately following cannulation of the heart, it was connected to the Langendorff apparatus and perfused at approximately 80 mmHg. Hearts were seen to contract rapidly upon perfusion and good function was confirmed prior to starting the experiment. Cardiac function was not formally assessed given the complications of inserting the pressure transducing balloon and often unreliable measurements using this system obtained with this setup in our laboratory. Perfusion of the heart was controlled by means of the flow regulator tap (see Figure 3.15) which remained in the open position for perfusion. Simulated ischaemia was induced by closure of the flow regulator tap and i. Buffer chamber

Reservoir of Krebs-Henseleit buffer (KHB) Supplied with gas (dark blue tube) Heated by water jacket (faint red colour)

KHB filled to set level to give perfusion pressure of approximately 80 mmHg

Equilibrated with 95% oxygen, 5% carbon dioxide via gas inlet

Heated water jacket (approximately 40ºC)

(Indicated in faint red colour)

Bubble trap

(To prevent bubbles entering perfusate supplying the heart)

Bubble trap regulator (Closed during experiment)

Perfusion cannula (21 G) Temperature probe

(Connected to Powerlab) ii. Heated perfusion coil

Heat exchange coil to evenly warm perfusion buffer

iii. FLOW REGULATOR TAP Open for perfusion of the heart Closed for simulated ischaemia

Heated organ bath iv. Connection to cannulated heart

thus cessation of perfusion and confirmed by the lack of effluent from the heart and the relatively rapid onset of dramatically reduced cardiac contractility.

Hearts were randomised to control and ischaemic groups, where control hearts were subjected to 30 minutes stabilisation and ischaemic hearts to 10 minutes stabilisation and 20 minutes global ischaemia (Figure 3.16). Upon completion of this protocol, hearts were removed from the Langendorff apparatus and the left ventricles dissected and immediately frozen by submersion into liquid nitrogen and then stored at -80ºC.

The period of stabilisation was started once the heart was securely attached to the Langendorff apparatus and good cardiac contractility had resumed. If the heart did not start contracting sufficiently it was discarded and the experiment started again to avoid any confounding effects on the final ATP assay conducted on this tissue. Two simultaneous perfusion experiments were conducted using this Langendorff system and the condition (control or ischaemia) randomised between hearts and perfusion rigs.

Figure 3.16: Langendorff perfusion protocol for the preparation of hearts for assay of ATP Hearts were extracted from mice and cannulated to allow Langendorff perfusion. A) Control: 30 minutes stabilisation. B) Ischaemia: 10 minutes stabilisation and 20 minutes global ischaemia by cessation of perfusion. Left ventricles were then immediately frozen for subsequent processing.

Frozen heart samples were subsequently homogenised in ice-cold conditions in the standard homogenisation buffer described previously and the protein concentration of each sample assessed using a BCA assay, described above (see 3.4.1).

Extract 30 min -3 0 Stabilisation 30 min Extract Perfuse on Langendorff

Remove heart for homogenisation 30 min Perfuse on Langendorff Stop perfusion

Remove heart for homogenisation A. Control B. Ischaemia -3 0 Stabilisation 30 min Stabilisation 10 min Ischaemia 20 min 10

3.5.2. ATP luminescence assay

Myocardial ATP levels were measured using a commercial ATP assay kit (ATP Bioluminescent Assay Kit, Sigma-Aldrich, UK). This is a luciferase based assay whereby luciferin (the assay substrate) is converted to oxyluciferin by the luciferase enzyme in an ATP-dependent manner. When ATP is the limiting reagent, the intensity of the luminescence is proportional to the amount of ATP present (summarised in Figure 3.17).

Figure 3.17: Molecular basis of luciferase assay quantification of tissue ATP levels The chemical basis of this reaction is described in the datasheet by Sigma-Aldrich (2009).

This ATP assay was conducted in accordance with manufacturer’s instructions (detailed by Sigma-Aldrich, 2009). This kit supplied an ATP assay mix (containing luciferase, luciferin, MgSO4, dithiothreitol [DDT], BSA and tricine buffer salts) and ATP dilution

buffer (containing MgSO4, DDT, EDTA, BSA and tricine buffer salts).

Sample plate: Heart homogenates, prepared as described above, were diluted ten-fold in TAE buffer (buffer as above) and then incubated at 85ºC for 30 seconds. These heart samples were then transferred to a sample plate (standard clear 96-well microplate) to allow rapid transfer of samples when required, as detailed below.

Assay plate: The required volume of ATP assay mix was diluted 25-fold in the supplied ATP assay dilution buffer and vigorously mixed and rapidly transferred to the assay plate (white, clear bottom 96-well microplates; 100 µl diluted assay mix per well). The assay plate was swirled vigorously and incubated for 3 minutes to allow the signal from any endogenous ATP to be quenched to reduce any background luminescence signal. Immediately following the 3 minute assay plate incubation period, the pre-prepared diluted heart samples were then rapidly added to the assay plate using a multi-channel pipette (100 µl heart sample per well) to ensure simultaneous addition of the heart samples to the assay plate for consistency of the luciferin reaction. The assay plate was then vigorously swirled and rapidly transferred to the microplate reader for assessment of luminescence every 5 seconds for 2 minutes using the FLUOstar Omega microplate reader (BMG Labtech, USA).

Assay substrate

Luciferin Adenyl-luciferin Oxyluciferin

Luminescence

Detected by spectrophotometer mircroplate reader

ATP

Statistical analysis of ATP levels: Luminescence values recorded during the first 20

seconds were used for all statistical analyses. These values were normalised to the average value recorded for wildtype control samples and expressed as a percentage of control ± standard error of mean (SEM). Data were analysed by one-way ANOVA, followed by Bonferroni test comparing relevant columns of data using GraphPad Prism® version 5.0 (GraphPad Software, USA). Statistical significance was reported where P<0.05 using standard significance coding.