Induction of IR injury in isolated and in vivo hearts Induction of experimental diabetes

1. 1
Heart models
Presented by:
Dr. Khalil Pourkhalili,
Place:
Bushehr University of Medical
Sciences

2. 2
Isolated heart model
 Langendorff perfused heart
 The Langendorff heart or isolated perfused heart assay is a predominant in
vitro technique used in pharmacological and physiological research using
animals.
 It allows the examination of cardiac contractile strength and heart rate
without the complications of an intact animal
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3. 3
Method of isolation
 Anaesthetizing the animal
 Bilateral sternotomy
 Cutting the heart vessels
 Cannulation of the aorta
 Connecting the cannula to the perfusion system
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4. 4
Method of isolation
 Anaesthetizing the animal
 Isoflurane, ketamine, pentobarbital sodium,…
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Face mask IP injection

5. 5
Method of isolation
 Bilateral sternotomy
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6. 6
Method of isolation
 Cutting the heart vessels and cannulation of the
aorta
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7. 7
Aortic cannulation
 Connecting the cannula to the perfusion system
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8. 8
Aortic cannulation and heart perfusion
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Correct
Incorrect
Aortic Valve
Left Coronary
Ostia

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Aortic cannulation and heart perfusion
Video

10. 10
Retrograde perfusion
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11. 11
Perfusion solution
 Krebs-Henseleit (K-H) solution
 NaCl 118.5 mmol/L, NaHCO3 25.0 mmol/L, KCl 4.7
mmol/L, KH2PO4 1.2 mmol/L, MgSO4·7H2O 1.2
mmol/L, glucose.H2O 11.1 mmol/L, CaCl2.2H2O 1.8
mmol/L.
 Equilibrated with Carbogen gas (95% O2-5%
CO2)
 PH 7.4
 Tempreture, 37°C
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12. 12
Perfusion solution
 Krebs-Henseleit (K-H) solution
 NaCl 118.5 mmol/L, NaHCO3 25.0 mmol/L, KCl 4.7
mmol/L, KH2PO4 1.2 mmol/L, MgSO4·7H2O 1.2
mmol/L, glucose.H2O 11.1 mmol/L, CaCl2.2H2O 1.8
mmol/L.
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Salt 1 lit (gr)
NaCl 6.93
Kcl 0.35
NaHco3 2.1
KH2Po4 0.163
Mgso4. 7 H2O 0.294
Glucose 1.98
CaCl2 0.166

13. 13
Models of retrograde perfusion
 Constant flow
 Constant pressure
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14. 14
Advantages of the ex vivo heart perfusion
 Quick, relatively cheap, and easy to perform technique
 High reproducibility, large number of experiments
 Broad spectrum of biochemical, physiological,
morphological and pharmacological studies
 Suitable for investigating cardiac-specific effects
 Controlled environment
 Ischemia/reperfusion
 Allows those experiments to be continued which would
lead to termination of an in vivo experiment (e.g.
infarction-induced loss of pump function, cardiac arrest or
arrhythmias)
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15. 15
Protocols of ischemia-reperfusion injuries
 Heart isolation and perfusion
 Stabilization period (15-20 min)
 Ischemia (20-40 min)
 Reperfusion (60-180 min)
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15 30 120
Time (min)
Ischemia Reperfusion
0-15 30 150
Stabi

16. 16
Data acquisition
 Mechanical activity of the heart
 Rate (bpm)
 Systolic pressure (mmHg)
 Diastolic pressure (mmHg)
 Left ventricular developped pressure (LVDP)
 Rate pressure product (mmHg/min)
 dp/dt
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17. Flexible Balloon Catheter
#170423 The balloon catheter is
for ventricular insertion. It is a
simple, reliable way to measure
left ventricular isovolumetric
pressure.
Data acquisition (LVDP)
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LVP Max Developed Pressure and Preload
(Balloon Method, Langendorff Only)

18. Left Ventricular End Diastolic Pressure
Left
Ventricular
Pressure
Frank Starling Curve
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19. Following calibration and insertion of the balloon you will want to optimize the pre-load to obtain accurate max developed pressure
measurements. This is a combination of both the resting pressure (or systole) and max developed pressure diastole.
You will see a distinct pressure wave as you begin to increase pre-load to the balloon as seen in the RED trace. Gradually
increasing pre-load will increase end developed pressure, as shown in the GREEN trace.
The BLUE trace indicates the approximate pre-load and max developed pressure for a 250-300gm adult rat.
The ORGANGE wave indicates that pre-load has increased too far. Depicted in the trace as an acceptable max developed
pressure but an abnormally systolic or pre-load pressure. This would also be an indication of a balloon size being too small for the
donor heart.
Left
Ventricular
Pressure
Left Ventricular End Diastolic Pressure
Frank Starling Curve
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20. 20
Data acquisition
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21. 21
Data acquisition
 Electrical activity of the heart
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22. 22
Data acquisition
 Electrical activity of the heart
 ECG (PVC, Bigemini, Salvos, VT and VF)
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ScoreArrhythmias
1Single PVBs
2Bigeminy/Salvos
3VT
4Transient VF
5Sustained VF
Normal
Bigemini
Sinle VEB
Salvos
VT VF

23. 23
Data acquisition
 Coronary flow (ml/min)
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Video

24. 24
Infarct size after IR injury
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Video

25. 25
Infarct size after IR injury
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26. 26
Infarct size after IR injury
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27. 27
Apoptosis in the heart tissue (TUNEL)
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Terminal deoxynucleotidyl transferase dUTP nick end labeling

28. 28
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29. Diabetes mellitus (DM)
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Polyuria
Polydipsia
Polyphagia

30. Diabetes mellitus (DM)
 Diabetes mellitus (DM), commonly referred to as diabetes, is a group
of metabolic diseases in which there are high blood sugar levels over
a prolonged period
 Symptoms of high blood sugar
 Frequent urination
 Increased thirst
 Increased hunger
 Diabetes is due to either:
 Pancreas not producing enough insulin
 Cells of the body not responding properly to the existing insulin
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31. Diabetes mellitus (DM)
 Diabetes is due to either:
 Pancreas not producing enough insulin
 Cells of the body not responding properly to the existing insulin
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32. Diabetes mellitus (DM)
 Type 1 DM
 Results from the pancreas's failure to produce enough insulin. "insulin-
dependent diabetes mellitus" (IDDM) or "juvenile diabetes".

 Type 2 DM
 Begins with insulin resistance, a condition in which cells fail to respond to
insulin properly. As the disease progresses a lack of insulin may also develop.
non insulin-dependent diabetes mellitus" (NIDDM) or "adult-onset diabetes".
 Gestational diabetes
 Ocurs when pregnant women without a previous history of diabetes develop
high blood-sugar levels.
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33. Importance of DM for research studies
 DM is an incurable metabolic disorder affecting about
2.8% of the global population.
 Up to 2010, around 285 million people suffering from Type
2 diabetes making up about 90% of the cases.
 According to statistics, by 2030, this number is estimated
to almost double.
 Experimental animal models are one of the best strategies
for the understanding of pathophysiology of DM in order to
design and develop the drugs for its treatment
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Models of diabetes
induction

35. A. Surgical induction of DM
 Pancreatectomy (Remove the pancreas, either partially or
totally)
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36. B. Chemical induction of DM
1. Streptozotocin (STZ)-induced model of DM (69%, 1996-
2006)
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37. 2. Alloxan-induced DM (31%, 1996-2006)
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38. C. Genetically induction of DM
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Type I

39. C. Genetically induction of DM
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Type II

40. Streptozotocin (STZ)
 Is an antibiotic (glucosamine derivative of nitrosourea).
 Is a diabetogenic agent (Rakieten first demonstrated the diabetogenic
property of STZ in dogs and rats in 1963).
 Is an anticancer chemotherapy drug (used for pancreas cancer)
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41. Mechanism of action of STZ
 Streptozotocin prevents DNA synthesis in mammalian and bacterial cells.
 STZ enters the pancreatic cell via a glucose transporter-GLUT2 and causes
alkylation DNA.
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42. Important points!
 Due to their similarity in structure to glucose, glucose can compete with
alloxan and STZ, and thus, fasting animals tend to be more susceptible.
 Both alloxan and STZ are relatively unstable, and the solutions should ideally
be made immediately prior to injection.
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43. Mechanism of action of STZ
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44. Mechanism of action of STZ
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45. Alterations in insulin & glucose concentrations
 Two hours after STZ injection, the hyperglycemia is observed with a
concomitant drop in blood insulin.
 About six hours later, hypoglycemia occurs with high levels of blood insulin.
Finally, hyperglycemia develops and blood insulin levels decrease
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2 h 4 hours
STZ
Hyperglycemia
Hypoinsulinemia
Hypoglycemia
Hyperinsulinemia
finally Hyperglycemia &
Hypoinsulinemia

46. Alterations in insulin & glucose level after STZ and alloxan
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47. Routes of administration
 IV injection
 IP injection
 SC injection
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48. Streptozotocin dosage for induction of diabetes
 Type I (IDDM)
 Single dose
 Rats: 40-60 mg/kg dose of STZ (IV) or higher are used to induce insulin
dependent diabetes , but higher doses are also used especially after IP
administration.
 Mice: 100-200 mg/kg
 Multiple doses: doses below 40 mg/kg in adult mice, STZ given in
multiple low doses (15 mg/kg, i.v. for 5 days) induces an insulin dependent
diabetes that is quite similar to the autoimmune forms (islet inflammation and
cell death) of Type 1 diabetes.
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49. Important point!
 Regeneration of the pancreatic islets can occur after
STZ treatment; and thus sufficient controls should be in
place to determine that any improvement in glycaemia is
not due to spontaneous regeneration of endogenous
beta cells (Grossman et al., 2010).
 STZ should be dissolved in 0.1 M citrate buffer (pH 4.5)
to induce diabetes.
49
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50. Streptozotocin dosage for induction of diabetes
 Type II (NIDDM)
1. Combination of STZ and NAD administration (rats).
 NAD (230 mg/kg, ip) 15 min before STZ (65 mg/kg,iv) has been shown to
develop moderate and stable non-fasting hyperglycaemia without any
significant change in plasma insulin level.
 As NAD is an antioxidant which exerts protective effect on the cytotoxic action
of STZ by scavenging free radicals and causes only minor damage to
pancreatic beta cell mass producing Type 2 diabetes. Therefore, this model is
found to be an advantageous tool for investigation of insulinotropic agents in
the treatment of Type 2 diabetes.
2. Multiple doses
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51. DM-type I or DM-type II
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52. Effect of Varying Dose and Administration of streptozotocin on Blood Sugar in
Male CD1 Mice (Ventura-Sobrevilla et al 2011)
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53. Alloxan
 The chemical name of alloxan is 2,4,5,6 tetraoxypyrimidine, which is
an oxygenated pyrimidine derivative which is present as alloxan
hydrate in aqueous solution.
 Alloxan is a toxic glucose analogue, which selectively destroys beta
cells.
 When administered to rodents and many other animal species. This
causes an insulin-dependent diabetes mellitus, with characteristics
similar to type 1 diabetes in humans.
 Alloxan is selectively toxic to insulin-producing pancreatic beta cells
because it preferentially accumulates in beta cells through uptake via
the GLUT2 glucose transporter.
 Alloxan, in the presence of intracellular thiols, generates reactive
oxygen species (ROS) in a cyclic reaction with its reduction product,
dialuric acid. The beta cell toxic action of alloxan is initiated by free
radicals formed in this redox reaction.
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54. Mechanism of action
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55. Dosage of alloxan
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 Doses
 Mice: 50 to 200 mg/kg
 Rats: 40 to 200 mg/kg
 Depending on the strain and the route of administration with i.p and s.c.
administration requiring up to three times as high a dose as the i.v. route
(Szkudelski, 2001).
 A dose of 100 mg/kg has been used to create a long-term diabetes models in
rabbits (Wang et al., 2010). It should be noted that alloxan has a narrow
diabetogenic dose, and even light overdosing can cause general toxicity,
especially to the kidney (Szkudelski, 2001).

56. Routes of administration
 IV injection
 IP injection
 SC injection
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57. Important points
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 Alloxan could not induce diabetes in human and guinea
pig.

58. References
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 The use of animal models in diabetes research, Aileen JF King et al,
2012.
 Animal models of diabetes mellitus, D. A. Rees et al 2004.
 Experimental Models on Diabetes: A Comprehensive Review, Radha
Sharma et al 2013.
 Alloxan Induced Diabetes: Mechanisms and Effects, Ankur Rohilla et
al 2012.
،‫زاده‬ ‫حسين‬ ‫حسيين‬ ،‫شهيدي‬ ‫ايمن‬ ‫محسن‬ ،‫ديابت‬ ‫ايجاد‬ ‫حيواني‬ ‫هاي‬ ‫مدل‬1381.
‫هدايت‬ ‫مهدي‬ ‫دکتر‬ ،‫فرد‬ ‫معينی‬ ‫مرضيه‬ ،‫ديابت‬ ‫پژوهش‬ ‫ابزار‬ ،‫استرپتوزوتوسين‬ ‫و‬ ‫آلوکسان‬،‫ی‬1393.

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‫ممنوع‬ ‫امتحان‬ ‫در‬ ‫تقلب‬!!