Preclinical model of type 1 diabetes and myocardial ischemia/reperfusion injury in conscious rabbits—demonstration of cardioprotection with rapamycin

Figures

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  Figure 1. Major surgical tools (A–L) (A) Metzenbaum Scissors-RS-6956, (B) Micro Dissecting Scissors-ROBOZ RS-5983, (C) Moloney Forceps-ROBOZ RS-8254, (D) Mayo Scissors, (E) KELLY Hemostatic forceps, (F) Kelly Forceps-ROBOZ RS-7131, (G) SEMKEN Tissue Forceps, RS-5240, (H) Thumb Dressing Forceps, RS-8100, (I) Weitlaner Retractor - ROBOZ RS-8612, (J) Micro Dissecting Scissors, RS-5983, (K) Miltex Adair Tissue Holding Tenaculum Forceps, (L) Metzenbaum Needle Holder - RS-7900.

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  Figure 2. Inflatable balloon occluder

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  Figure 3. Induction of type I diabetes in rabbit (A) Cannulation of the marginal ear vein with a catheter. (B) Securing the cannulation by taping to the earlobe using surgical tape. (C) Infusion of alloxan monohydride with adjusted flow rate through the pump (Harvard Apparatus, Model ‘22’ Syringe Pump MA1 55–2222 -constant pressure dispensing system).

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  Figure 4. Pre-surgical preparation of rabbit for implantation of balloon occlude (A) Rabbits were intubated with an endotracheal tube to mechanically ventilated with oxygen enriched air. (B) Betadine was applied to the surgical area.

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  Figure 5. Sequential images of implantation of balloon occluder (A–C) Surgical incision in the chest. (D) Thoracotomy. (E) Exposure of heart after thoracotomy. (F–I) Opening of the pericardial sac to access the LAD (left anterior descending artery). (J and K) Placing suture around LAD. (L–N) Implantation of Balloon Occluder. (O and P) brief inflation of the balloon occluder to confirm the blanching of the distal myocardium. (Q and R) Suturing of inner muscle. (S) Completion of muscle suture. (T–W) Tunneling balloon occluder under the skin layer between shoulder blades. (X) Suturing outer skin layer. (Y) Application of antiseptic in the incision area.

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  Figure 6. Recovery Unit with automated temperature control

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  Figure 7. Myocardial ischemia in conscious diabetic rabbit (A) Picture of conscious diabetic rabbit during myocardial ischemia by inflating the hydraulic balloon occluder. (B) A transducer sensor probe was secured to arterial line of the ear connected to Blood Pressure Analyzer during conscious ischemia/reperfusion

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  Figure 8. Cardiac function was assessed with echocardiography (A) Chest hair was removed in rabbit while anesthetized with isoflurane. (B) Cardiac function was measured by placing the MS201 probe on the parasternal view. (C) Cardiac left ventricle (LV) function was measured by two-dimensional ultrasound B-Mode (Short Axis) as well as M-Mode.

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  Figure 9. Infarct size measurement (A) Perfusion of heart with Krebs-Henseleit buffer to wash out most of the blood and infusion of Phthalo blue dye for demarcation of risk and non-risk area. (B) Image of heart section to measure infarct size.

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  Figure 10. Cardiac apoptosis assessment by TUNEL staining was monitored under a fluorescence microscope (Confocal microscope Nikon D-Eclipse C1)

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  Figure 11. Blood glucose levels after 4 weeks of alloxan treatment in rabbit Animals with consistent blood glucose level of 220 mg/dL (Blue dashed line) or above were considered diabetic. Red line denoted the average blood glucose level in 57 rabbits.

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  Figure 12. Myocardial infarct size after I/R injury (A) Representative images of myocardial sections of diabetic rabbits following I/R injury. Each grid in the scale indicates 1 mm. (B and C) (B) Infarct size and (C) risk area. Statistical analyses were performed using GraphPad Prism 8 (GraphPad Software Inc. La Jolla, CA). The data are presented as mean±SE for each treatment group. p values <0.05 were considered statistically significant. The difference between two groups was analyzed via Student’s t-test.