Korean Circ J.  2017 Sep;47(5):714-726. 10.4070/kcj.2017.0092.

Remote Ischemic Conditioning by Effluent Collected from a Novel Isolated Hindlimb Model Reduces Infarct Size in an Isolated Heart Model

• Youn YJ
• Yoo BS
• Son JW
• Lee JW
• Ahn MS
• Ahn SG
• Kim JY
• Lee SH
• Yoon J
• Eom YW
• Oh JE
• Choi SK

• KMID: 2389604
• DOI: http://doi.org/10.4070/kcj.2017.0092

Abstract

BACKGROUND AND OBJECTIVES
Experimental protocols for remote ischemic conditioning (RIC) utilize models in which a tourniquet is placed around the hindlimb or effluent is collected from an isolated heart. In analyzing the humoral factors that act as signal transducers in these models, sampled blood can be influenced by systemic responses, while the effluent from an isolated heart might differ from that of the hindlimb. Thus, we designed a new isolated hindlimb model for RIC and tested whether the effluent from this model could affect ischemia/reperfusion (IR) injury and if the reperfusion injury salvage kinase (RISK) and survivor activating factor enhancement (SAFE) pathways are involved in RIC.
MATERIALS AND METHODS
After positioning needles into the right iliac artery and vein of rats, Krebs-Henseleit buffer was perfused using a Langendorff apparatus, and effluent was collected. The RIC protocol consisted of 3 cycles of IR for 5 minutes. In the RIC effluent group, collected effluent was perfused in an isolated heart for 10 minutes before initiating IR injury.
RESULTS
Compared with the control group, the infarct area in the RIC effluent group was significantly smaller (31.2%±3.8% vs. 20.6%±1.8%, p<0.050), while phosphorylation of signal transducer and activation of transcription-3 (STAT-3) was significantly increased. However, there was a trend of increased phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 in this group.
CONCLUSION
This is the first study to investigate the effect of effluent from a new isolated hindlimb model after RIC on IR injury in an isolated heart model. The RIC effluent was effective in reducing the IR injury, and the cardioprotective effect was associated with activation of the SAFE pathway.

Keyword

Models, Animal; ERK 1/2; Rats; Remote ischemic conditioning; STAT-3

MeSH Terms

Animals
Heart*
Hindlimb*
Humans
Iliac Artery
Models, Animal
Needles
Phosphorylation
Phosphotransferases
Rats
Reperfusion Injury
Survivors
Tourniquets
Transducers
Veins
Phosphotransferases

Figure

• 

  Figure 1 Schema of the perfusion system for the RIC model (A) and isolated heart model for IR injury (B). (A) After exposing the abdominal aorta, the infrarenal aorta and IVC were cannulated with 26-gauge plastic needles that were advanced to the right CIA and CIV, respectively. The aorta and IVC above the cannulated site, right CIA and right CIV above the tip of the needles, and left CIA and left CIV were tied with 5-0 silk to isolate right hindlimb circulation from systemic circulation. Heparin (200 IU) was given via venous cannulation. KHB was perfused through the arterial cannulation, and effluent from venous cannulation was collected. (B) The heart was rapidly excised via clamshell thoracotomy and placed in ice-cold KHB before being mounted on a Langendorff apparatus. KHB was used for retrograde perfusion at a constant pressure (60–80 mmHg). The KHB temperature was kept at a constant 37.0°C using a heat exchanger. A water-filled latex balloon, connected to a hydrostatic pressure transducer and coupled to a high-performance data acquisition system, was inserted into the LV and inflated to an end-diastolic pressure of 5–15 mmHg. CF was measured by timed collection of effluent over 1 minute. CF = coronary flow; CIA = common iliac artery; CIV = common iliac vein; IR = ischemia/reperfusion; IVC = inferior vena cava; KHB = Krebs-Henseleit buffer; LV = left ventricle; RIC = remote ischemic conditioning; Rt = right.

• 

  Figure 2 Design of experimental protocols. (A) RIC procedures. Anesthetized rats were subjected to either 1) a sham procedure, or 2) remote ischemia (3×5 minutes hindlimb IR using a Langendorff system). During the procedures, effluents were collected. (B) The heart from another rat was excised and perfused on a Langendorff system before being subjected to the IR protocol (30-minute regional ischemia and 60-minute reperfusion): 1) control group, 2) IPC group: prior to the IR protocol, IPC (3×1 minute LAD IR) was applied, 3) sham effluent group: effluent after sham procedure was perfused for 10 minutes before the IR protocol, and 4) RIC effluent group: effluent after RIC procedure was perfused for 10 minutes before the IR protocol. Following each Langendorff experiment, infarct size was determined using TTC staining. IPC = ischemic preconditioning; IR = ischemia/reperfusion; LAD = left anterior descending coronary artery; RIC = remote ischemic conditioning; TTC = triphenyl-tetrazolium-chloride.

• 

  Figure 3 Representative sections of TTC-stained hearts following ischemia for 30-minute and reperfusion for 60-minute. (A) Bar graph showing the AAR expressed as a percentage of total left ventricular area (B) and IA as a percentage of AAR (C). All data is expressed as mean±SEM. n=3 in each group. AAR = area at risk; IA = infarct area; IPC = ischemic preconditioning; RIC = remote ischemic conditioning; SEM = standard error of mean; TTC = triphenyl-tetrazolium-chloride. *p<0.050 vs. control group.

• 

  Figure 4 Western blot analysis of ERK 1/2 (A) and STAT-3 (B) phosphorylation in rat hearts subjected to ischemia-reperfusion. Top: representative immunoblots of p-ERK 1/2 and total ERK 1/2 (A) and p-STAT-3 and total STAT-3 (B) in LV homogenates from hearts subjected to ischemia-reperfusion. Bottom: bar graphs show mean±SEM of the densitometry of p-ERK to ERK ratio (A) and p-STAT-3 to STAT-3 ratio (B). Data are expressed as the mean±SEM. n=3 in each group. AU = arbitrary units; ERK = extracellular signal-regulated kinase; IPC = ischemic preconditioning; LV = left ventricle; p-ERK = phosphorylated ERK; p-STAT-3 = phosphorylated STAT-3; RIC = remote ischemic conditioning; SEM = standard error of mean; STAT-3 = signal transducer and activation of transcription-3. *p<0.050 vs. control, †p<0.050 vs. IPC.

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