Heart models and diabetes

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Heart modelsPresented by:

Dr. Khalil Pourkhalili, Place:

Bushehr University of Medical Sciences

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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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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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Method of isolation

Anaesthetizing the animal Isoflurane, ketamine, pentobarbital sodium,…

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Face mask IP injection

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Method of isolation

Bilateral sternotomy

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Method of isolation

Cutting the heart vessels and cannulation of the aorta

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Aortic cannulation

Connecting the cannula to the perfusion system

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Aortic cannulation and heart perfusion

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Correct

Incorrect

Aortic Valve

Left CoronaryOstia

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Aortic cannulation and heart perfusion

Video

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Retrograde perfusion

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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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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

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Models of retrograde perfusion

Constant flow

Constant pressure

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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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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

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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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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)

Left Ventricular End Diastolic Pressure

Left Ventricular Pressure

Frank Starling Curve

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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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Data acquisition

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Data acquisition

Electrical activity of the heart

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Data acquisition

Electrical activity of the heart ECG (PVC, Bigemini, Salvos, VT and VF)

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Score Arrhythmias

1 Single PVBs

2 Bigeminy/Salvos

3 VT

4 Transient VF

5 Sustained VF

Normal

Bigemini

Sinle VEB

Salvos

VT VF

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Data acquisition

Coronary flow (ml/min)

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Video

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Infarct size after IR injury

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Video

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Infarct size after IR injury

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Infarct size after IR injury

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Apoptosis in the heart tissue (TUNEL)

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Terminal deoxynucleotidyl transferase dUTP nick end labeling

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Diabetes mellitus (DM)

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Polyuria

Polydipsia

Polyphagia

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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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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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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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

A. Surgical induction of DM

Pancreatectomy (Remove the pancreas, either partially or totally)

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B. Chemical induction of DM

1. Streptozotocin (STZ)-induced model of DM (69%, 1996-2006)

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2. Alloxan-induced DM (31%, 1996-2006)

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C. Genetically induction of DM

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Type I

C. Genetically induction of DM

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Type II

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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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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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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Mechanism of action of STZ

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Mechanism of action of STZ

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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

STZHyperglycemiaHypoinsulinemia

HypoglycemiaHyperinsulinemia

finally Hyperglycemia &Hypoinsulinemia

Alterations in insulin & glucose level after STZ and alloxan

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Routes of administration

IV injection

IP injection

SC injection

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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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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.

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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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DM-type I or DM-type II

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Effect of Varying Dose and Administration of streptozotocin on Blood Sugar in Male CD1 Mice (Ventura-Sobrevilla et al 2011)

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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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Mechanism of action

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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).

Routes of administration

IV injection

IP injection

SC injection

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Important points

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Alloxan could not induce diabetes in human and guinea pig.

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 حسیين شهيدي، ايمن محسن ديابت، ايجاد حيواني هاي مدل1381 .

دکتر فرد، معینی مرضیه دیابت، پژوهش ابزار استرپتوزوتوسین، و آلوکسانهدایتی، . 1393مهدي

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