Biomedical Research Models, Inc Contract Discovery Research

BBZDR/Wor Rat • Obese (dyslipidemic)

• Hyperleptinemic

• Insulin Resistant (hyperinsulinemic)

• Hyperglycemic

• Hypertensive

BBZDR Rats Clinical Characteristics

Genetic Predisposition (fa/fa homozygous)

Obesity (Hyperleptinemia)

Insulin Resistance (Hyperinsulinemia)

Impaired Glucose Tolerance (IGT)

Type 2 Diabetes (Hyperglycemia)

Macrovascular Diabetic Complications: Hypertension, Accelerated Stenosis (Inducible), Atherosclerosis (Duration Dependent

Microvascular Diabetic Complications: Retinopathy, Neuropathy, and Nephropathy

Features of BBZDR/Wor Rats

OBESE LEAN MALES FEMALES MALES FEMALES

Incidence of Diabetes

98% (N=225)

3% (N=217)

0% (N=729)

0% (N=783)

Age at Onset 66.8± 2.7 72.1 ±4.6 N/A N/A

Hypertension + ++ – – Insulin

Resistance + + – –

GLUT-2 – + + +

Normal Islets Lean Rat

Insulin

Glucagon

Islets from an Obese Rat with IGT Insulin Glucagon

Glut 2 Receptor

0

100

200

300

400

500

600

0 10 20 30 40 50

Time (min)

OBESE > 16

LEAN > 6

3.4 mmol/kg glucose i.v.

1.7 mmol/kg arginine i.v.

Blo

od G

luco

se (

mg/

dl)

Blood Glucose: Fasted BBZDR Females

Insulin Release: Fasted Female BBZDR Rats

0

20

40

60

80

100

0 10 20 30 40 50

Time (min)

OBESE > 16

LEAN > 6

3.4 mmol/kg glucose i.v.

1.7 mmol/kg arginine i.v.

Insu

lin (

ng/m

l)

BBZDR/Wor Obese Rat Islet: Glut-2 Receptor(400x)

Islets from Type 2 Diabetic Rat Glucagon Insulin

Glut-2 Receptor Glut-2 Control

Blood Glucose: Fasted Male BBZDR rats

0

100

200

300

400

500

600

700

0 10 20 30 40 50

Time (min)

OBESE LEAN

3.4 mmol/kg glucose i.v.

1.7 mmol/kg arginine i.v.

Blo

od G

luco

se (

mg/

dl)

Insulin Release: Fasted BBZDR Males

0

20

40

60

80

100

0 10 20 30 40 50

Time (min)

Obese

Lean

3.4 mmol/kg glucose i.v.

1.7 mmol/kg arginine i.v.

Insu

lin (

ng/m

l)

Atherosclerosis in BBZDR rats

Blood Chemistry Profile of BBZDR Rats 4 Months of age

0

100

200

300

400

500

600

700

800

Glucose Cholesterol Triglyceride HDL

mg/

dl Lean

Male Obese Female

*

*

*

*

*

* P<0.05 *

*

*

*

Atherosclerosis in Renal Artery BBZDR Diabetic Rat (4mo duration)

Severe atherosclerosis: 10 months ~8 months duration of diabetes

Complete occlusion of renal artery, x100 (Left); Arrows identify plaque, x400 (Both panels).

Measurement and Calculation of Neointima Thickness

The % stenosis was calculated as the Internal Elastic Area (IEA) minus the Residual Area (RA) divided by the IEA ( x 100)

Neointima Thickness in Lean and Obese Male BBZDR Rats One Week Post Balloon Injury

BBZDR Lean Rats Type 2 Diabetic Rats

Neointima Thickness in Lean and Obese Male BBZDR Rats 4 Weeks Post Balloon Injury

BBZDR Lean Rats Type 2 Diabetes Rats

Atherosclerosis Plaque in BBZDR/Wor Obese Female Rats

Fatty Deposits and Inflammatory Cells in Occluded Obese Female Carotid Artery

% of Stenosis In Balloon Injured Rat Carotid Artery

05

101520253035404550

7 14 21 28

Days Post Balloon Injury

% S

teno

sis Lean Rat

Obese FemaleObese Male

*

*

**

Nephropathy in the BBZDR Rat

Clinical Phases

Microalbuminuria Clinical proteinuria

Progression to ESRD

LEAN

BBZDR Kidney at 5 Months of age

Obese Female

Obese Diabetic

Lean rat: 5 months old

Normal Glomeruli: x100 (left); insert x400 (right).

Obese female: non-diabetic, 5 month old

Evidence of mild glomeruli enlargement and mesangial expansion (arrows), x100 (Right); Insert: x400 (Right)

Aldose Reductase

Lean BBZDR Obese BBZDR 6 months 4months duration

Sorbitol Dehydrogenase in BBZDR Kidneys

Lean Control Obese T2 Diabetic

RAGE in BBZDR Rat Kidneys

Obese T2 Diabetic Lean Control

Summary • The diabetic BBZDR male rat is a good model of

diabetic nephropathy. • Supported by Clinical blood chemistry and

Histopathology – Evidence of hyperfiltration and glomeruli

hypertrophy at 3months age – Microalbumuria progressing to proteinuria from

3month through 12month – Mesangial expansion, basement membrane thicking

and interstitial fibrosis – Tubule expansion, aschemia and diffuse necrosis after

12months of age (10 months duration)

Diabetic Polyneuropathy

Neuropathy in BBZDR • Experimental rat models of types 1 and 2 diabetes differ in

sympathetic neuroaxonal dystrophy. J Neuropathol Exp Neurol. 2004 May;63(5):450-60

Schmidt RE, Dorsey DA, Beaudet LN, Parvin CA, Zhang W, Sima AA.

• C-peptide corrects endoneurial blood flow but not oxidative stress in type 1 BB/Wor rats. Am J Physiol Endocrinol Metab. 2004 Sep;287(3):E497-505.

Stevens MJ, Zhang W, Li F, Sima AA.

• A comparison of diabetic polyneuropathy in type II diabetic BBZDR/Wor rats and in type I diabetic BB/Wor rats. Diabetologia, 2000 Jun;43(6):786-93

Sima AA, Zhang W, Xu G, Sugimoto K, Guberski D, Yorek MA.

Type 1 and 2 Diabetes Type 1 Diabetes

↑ Hyperglycemia Insulin / C-peptide ↓

↑ Polyol Pathway ↓ NO

↓ Na+/K+- ATPase

↑ Oxidative Stress

↑ Nonenzymatic Glycation

↓ Neurotrophism

Apoptosis ?

Axonal Degeneration /Loss

Impaired Regeneration Nodal / Paranodal Degeneration

Acute Reversible NCV-Slowing

Chronic Irreversible NCV-Slowing

Glucose Sorbitol Fructose

NADPH NADP

Arginine Citrullin + NO ↓ GSSG GSH ↓

Pathogenetic Scheme of DPN

Sima, Cell Mol Life Sci 2003.

Diabetic Polyneuropathy (DPN) Type 2 vs Type 1

Type 2 Type 1 • 35% /25 years • Slow progression • Mild axonal

degeneration • No nodal changes

• Close to 100% /25years • More rapid progression • Severe axonal degeneration

and loss • Progressive nodal/paranodal • degeneration

Progression of Axo-glial Dysjunction A

xo-g

lial d

ysju

ncti

on (

%)

A. BB/Wor – BBZDR/Wor- rats

Duration Of Diabetes

Baseline 4mo 6mo 8mo 12mo 14mo 0

10

20

30

40

50 Control BB/Wor BBZDR/ Wor

Axo

-glia

l dys

junc

tion

(%

)

20

40

60

20 30 40 50 60 70 Age (years)

Common slope: df =39, f=13.91, p< 0.001

Control: a= 0.24 b= 0.26

NIDM: a= 3.44 b= 0.26

IDDM: a= 25.43 b= 0.26

B. Human

Sima et al., J Clin Invest 1984 Sima et al., N Engl J Med, 1986

0

20

40

60

80

100

0

5

10

15

20

25

30

35

Teased Fiber Pathology in Type 1 and Type 2 Diabetic Patients

Control Type 1 Type 2 n=19 n=11 n=17

Control Type 1 Type 2 n=19 n=11 n=17

p<0.001

p<0.005

p<0.001

p<0.001

Sima et al, JCI,1988

Structural Abnormalities In Sural Nerve Axonal Degeneration Fiber Number

Nerve Regeneration

P<0.001

P<0.001

P<0.01 P<0.001

Perc

enta

ge o

f Axo

nal

D

egen

erat

ion

(%)

0

5

10

15

20

25

C8 D1-8 D1CP D2-8

P<0.05 P=0.07

P<0.05 1000

800

600

400

200

0 Num

ber

of M

yelin

ated

Fi

bers

C8 D1-8 D1CP D2-8

P<0.001

P<0.001

P<0.05

C8 D1-8 D1CP D2-8

Fibe

rs (%

)

0 1 2 3 4 5 6 7 8

Sima et al., Diabetologia,2002

*: p<0.01, **: p<0.001 vs control.

Blood Glucose Serum Insulin Serum C-peptide

0

100

200

300

400

500

600

0

200

400

600

800

1000

0

100

200

300

400

500

600

700

C D1 D1+C D2 C D1 D1+C D2 C D1 D1+C D2

** ** ** *

** ** **

Blo

od G

luco

se (m

mol

/L)

Insu

lin (p

mol

/L)

C-p

eptid

e (p

mol

/L)

Glucose, insulin and C-peptide plasma levels in BB/Wor- and BBZDR/Wor-rats

Sima et al: Diabetologia, 2001

Na+ /K

+ - A

TPa

se A

ctiv

ity

(mm

ol/A

DP/

mg/

wet

wt./

hr)

Control Sham 10µg 100µg 500µ 1000µg hrC-peptide

0

200

400

600

800

1000

*

†

†

*: p<0.01, vs age-matched controls;

†: p<0.05 vs duration-matched sham-treated BB/W-rats.

The Effect of C-peptide on Neural Na+/K+- ATPase

Zhang et al: Exp. Diab.Res. 2001

35

40

45

50

55

60

65

70

75

Control BB/Wor BB/Wor+C BBZDR/Wor

Onset 1 Week 2 Week 3 Week 4 Week 5 Week

Hyperglycemic Component

Insulin/C-Peptide Deficiency

Component

Acute Metabolic NCV Defects

Diabetic Retinopathy (DR)

Retinopathy in BBZDR • N-3 polyunstaurated Fatty acids prevent diabetic

retinopathy by inhibition of retinal vascular damage and enhanced endothelial progenitor cell reparative function. Plos One. 2013;8(1):e55177. Epub 2013 Jan29.

Tikhonenko M, Lydic TA, Opreanu M, Li Calzi S, Bozack S, McSorley KM, Sochacki AL, Faber MS, Hazra S, Duclos S, Guberski D, Reid GE, Grant MB, Busik JV

• Diabetic retinopathy is associated with bone marrow neuropathy and a depressed peripheral clock. Journal of Experimental Medicine. 2009 Dec21;206(13):2897-906

Busik JV, Tikhonenko M, Bhatwadekar A, Opreanu M, Yakubova N, Caballero S, Player D, Nakagawa T, Afzal A, Kielczewski J, Sochacki A, Hasty S, Li Calzi S, Kim S, Duclos S, Segal MS, Guberski DL, Esselman WJ, Boulton ME, Grant MB.

• Time course of NADH oxidase,inducible nitric oxide

synthase and peroxynitrite in diabetic retinopathy in the BBZ/Wor rat. Nitric Oxide. 2002 May;6(3): 295-304.

Ellis,E.A., Guberski, D.L., Huston, B. and Grant, M.B.

Small Pressurized Artery Myography: As close as it gets to true organ

perfusion or function

0 50 100 150 200 250

50

75

100

125

150

175

Diameter with tone Passive diameter

WKY

Pressure (mmHg)

Dia

met

er (µ

M)

Endothelial Dysfunction in BBZDR Rats

Obese-Diabetic; O Lean control (age-matched)

-10 -9 -8 -7 -6 -5 -4 -3

0

25

50

75

100

125

log[Histamine] M

% M

yoge

nic

tone

-10 -9 -8 -7 -6 -5 -4 -3

0

25

50

75

100

125

log[Histamine] M

% M

yoge

nic

tone

-10 -9 -8 -7 -6 -5 -4 -3

0

25

50

75

100

125

log[Histamine] M

% M

yoge

nic

tone

-10 -9 -8 -7 -6 -5 -4 -3

0

25

50

75

100

125

log[Histamine] M

% M

yoge

nic

tone

Pre-diabetic age 3-4 Weeks of Diabetes

5 Months of Diabetes 10 Months of Diabetes

Levels of Blood Sugars and Glycosylated Hemoglobin

Blood Sugars (mg/dl)

Total Glycosylated

Hemoglobin (%)

Diabetic (BBZ/Wor) 496 ± 15 13.0 ± 0.4

Non-Diabetic (BB DR /Wor) 123 ± 3 5.3 ± 0.5

VEGF and H2O2 Correlation

H 2 O 2 % VEGF CORRELATION COEFFICIENT

DIABETIC 78.7 ± 4.84 24.67 ± 0.33 0.82*

NON-DIABETIC 39.0 ± 4.47 21.52 ± 0.43 0.83**

* p = 0.001 ** p = 0.0001

Percentage of Blood Vessels Positive for Peroxide

Central Retina

Peripheral Retina

Diabetic (BBZ/Wor) 80.00 ± 10.52 44.24 ± 12.79

Non-Diabetic (BB DR /Wor) 38.28 ± 9.68 26.47 ± 8.62

Basement Membrane Thickness (nm) in Type 2 BBZDR Rats

Diabetic (BBZ/Wor) 96.0 ± 13.32nm

Non-Diabetic (BB DR /Wor) 60.4 ± 5.62nm

Immunocytochemical Localization of Fibronectin

Colloidal Gold Particles/um 2

Diabetic (BBZ/Wor) 4.45 ± 1.09

Non-Diabetic (BB DR /Wor) 1.56 ± 0.79

Animal Characteristics for iNOS and Nitrotyrosine Study

ANIMAL GROUP

AGE LENGTH OF DIABETES

BLOOD GLUCOSE

PRE-DIABETIC (BBZ/Wor)

1.5 – 2 months ND 142 ± 29 md/dl

NEW ONSET DIABETES (BBZ/Wor)

2 months 2 – 6 days 383 ± 79 mg/dl

CHRONIC DIABETES (BBZ/Wor)

7 – 20 months 4.8 – 18 months 387 ± 96 mg/dl

NON-DIABETIC CONTROLS (BB DR /Wor)

7 – 20 months ND 130 ± 28 mg/dl

Colloidal gold localization of nitrotyrosine (arrows) in retina of BBZ/Wor rat with diabetes for two days

eNOS and iNOS Immunoreactivity in Retina

eNOS iNOS

BBZ/Wor 13.9 ± 2.3 part/50 µm 2 33.9 ± 10.0 part/50 µm 2

BB DR /Wor 78.8 ± 6.0 part/50 µm 2 3.5 ± 2.8 part/50 µm 2

iNOS and Nitrotyrosine Immunoreactivity

ANIMAL GROUP iNOS NITROTYROSINE

PRE-DIABETIC (BBZ/Wor) 3.7 ± 0.81 % C 8.2 ± 1.70 part/50 µm 2 C

NEW ONSET DIABETES (BBZ/Wor) 69.6 ± 5.88 % A 60.8 ± 16.91 part./50 µm 2 A

CHRONIC DIABETES (BBZ/Wor) 49.9 ± 9.75 % B 29.5 ± 4.31 part./50 µm 2 B

NON-DIABETIC CONTROLS (BB DR /Wor) 8.7 ± 4.66 % C 9.0 ± 1.87 part./50 µm 2 C

A: p = 0.0001 B: p = 0.0001 Means with the same letter (A, B, C) are not significantly different.

Time Course of Vascular Changes in BBZDR/Wor Rat With Duration of Hyperglycemia

Length of hyperglycemia 1 week 4-6 months 18-24 months

NADH oxidase - - - Free Radicals - - - iNOS - - - Nitrotyrosine - - - eNOS ¯ ¯ ¯ NOS cofactors - ¯ ¯ Vessel Diameter - ¯ ¯

Small Pressurized Artery Myography: As close as it gets to true organ

perfusion or function

0 50 100 150 200 250

50

75

100

125

150

175

Diameter with tone Passive diameter

WKY

Pressure (mmHg) D

iam

eter

(µM

)

Isolated Intact Pressurized Resistance Arteries: The heart of microvascular (patho) physiology

• Cerebral

• Coronary

• Renal

• Retinal, opthalmic

Inflow

T=37.4

outflow

The Arteriograph System: Total control over the micro vascular environment

Pressure / flow control

Open / close

micromanipulator

The Pressure Arteriograph System: A controlled two cell system in native interaction

CONTROL 116 MICRONS

U73122 185 MICRONS

U73122+KCL 77 MICRONS

1.305 1.058 1.595

1.6

0.7

Quantitative Calcium Imaging (FURA-2)

10 mmHg 122 MICRONS

70 mmHg 108 MICRONS

130 mmHg 112 MICRONS

0.2

3.0

1.095 1.408 1.564

Quantitative Membrane Potential Imaging (Di-8-ANNEPS)

0 0

-40 -47-39

-40

-60

V

-60

-20 -20Arterial membrane potential, mV:

Arterial diameter, µm:

videoedge

detection

Arterial wall [Ca ], nM:2+

60 mm Hg

105

189 245 68

205103

100 mm Hg+

10 nMNisoldipine

100 mm Hg

8 1416 2012 18Time (min) Time (min)V

Simultaneous Electrophysiology, diameter and calcium Imaging

Long-term artery culture: a physiological genomics platform

• Viral transduction

• Protein transduction

• Ribozyme, siRNA

• To VSM and/or ENDO

3-4 Weeks of Diabetes Pre-diabetic age

5 Months of Diabetes 10 Months of Diabetes

0 25 50 75 100 125 150 175 200 225

0

20

40

60

Intraluminal pressure (mm Hg)

Myo

geni

c to

ne (%

)

0 25 50 75 100 125 150 175 200 225

0

20

40

60

Intraluminal pressure (mm Hg)

Myo

geni

c to

ne (%

)

0 25 50 75 100 125 150 175 200 225

0

20

40

60

Intraluminal pressure (mmHg)

Myo

geni

c to

ne (%

) Obese-Diabetic; O Lean control (age-matched)

0 25 50 75 100 125 150 175 200 225

0

20

40

60

Intraluminal Pressure (mmHg)

Myo

geni

c to

ne (%

)

Characterization of Cerebral-arterial Dysfunction in BBZ/Wor Rats

Endothelial Dysfunction

Obese-Diabetic; O Lean control (age-matched)

-10 -9 -8 -7 -6 -5 -4 -3

0

25

50

75

100

125

log[Histamine] M

% M

yoge

nic

tone

-10 -9 -8 -7 -6 -5 -4 -3

0

25

50

75

100

125

log[Histamine] M

% M

yoge

nic

tone

-10 -9 -8 -7 -6 -5 -4 -3

0

25

50

75

100

125

log[Histamine] M

% M

yoge

nic

tone

-10 -9 -8 -7 -6 -5 -4 -3

0

25

50

75

100

125

log[Histamine] M

% M

yoge

nic

tone

Pre-diabetic age 3-4 Weeks of Diabetes

5 Months of Diabetes 10 Months of Diabetes

Arterial smooth muscle hyper-reactivity

Pre-diabetic age 3-4 Weeks of Diabetes

5 Months of Diabetes 10 Months of Diabetes

Obese-Diabetic; O Lean control (age-matched)

-12 -11 -10 -9 -8 -7 -6 -5

0

25

50

75

100

log[5-Hydroxy tryptamine] M

% K

Cl C

onst

rict

ion

-12 -11 -10 -9 -8 -7 -6 -5

0

25

50

75

100

log[5-Hydroxy tryptamine] M

% K

Cl C

onst

rictio

n

-12 -11 -10 -9 -8 -7 -6 -5

0

25

50

75

100

log[5-Hydroxy tryptamine] M

% K

Cl C

onst

rict

ion

-12 -11 -10 -9 -8 -7 -6 -5

0

25

50

75

100

log[5-Hydroxy tryptamine] M

% K

Cl C

onst

rictio

n

Pre-diabetic age 3-4 Weeks of Diabetes

5 Months of Diabetes 10 Months of Diabetes

Obese-Diabetic; O Lean control (age-matched)

-9 -8 -7 -6 -5 -4

0

25

50

75

100

125

log[U-73122] M

% M

yoge

nic

tone

-9 -8 -7 -6 -5 -4

0

25

50

75

100

125

log[U-73122] M

% M

yoge

nic

tone

-9 -8 -7 -6 -5 -4

0

25

50

75

100

125

log[U-73122] M

% M

yoge

nic

tone

-9 -8 -7 -6 -5 -4

0

25

50

75

100

125

log[U-73122] M

% M

yoge

nic

tone

Arterial smooth muscle hyper-reactivity PLC activation

Autoregulation in rat (Sprague Dawley) ophthalmic artery

0 30 60 90 120 150 180 210

100

150

200

250

300

Diameter with tonePassive diameter

Myogenic tone

0

25

50

Intraluminal Pressure (mm Hg)

Lum

en d

iam

eter

(mic

rons

)M

yogenic tone (%)

-15 -14 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3

0

25

50

75

100

125

SerotoninPhenylephrine

Vasopressin

UK-14304

U-46619

log M [Vasoconstrictor]

% K

Cl (

60 m

M) r

esp

on

se

-15 -14 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3

0

50

100

150

log M [Endothelin-1]

% K

Cl (

60

mM

) re

sp

on

se

Effect of different vasoconstrictors in ophthalmic artery

Order of efficacy: Endothelin-1 > Vasopressin = Serotonin = Phenylephrine > U-46619 > UK 14304 Order of potency: Endothelin-1 > Vasopressin = Serotonin > U-46619 > UK 14304 > Phenylephrine

-14 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3

0

25

50

75

100

125

Histamine

Carbachol

Adenosine

CGRP

Isoprenaline

VIP

log M [Vasodilator]

Myo

geni

c to

ne (%

)

Order of efficacy: Carbachol > Isoprenaline > Histamine = CGRP = VIP = Adenosine Order of potency: CGRP > VIP > Carbachol = Isoprenaline > Adenosine = Histamine Bradykinin, substance P and octreotide did not produce sustained dilatory responses

Effect of different vasodilators in ophthalmic artery

-9 -8 -7 -6 -5 -4 -3

20

60

100

140

180L-NAMEIndomethacin

log M [Inhibitor]

Myo

geni

c to

ne (%

)

Effect of the inhibition of NOS and prostaglandin synthesis on myogenic tone

in rat ophthalmic artery

CNS Controls Release of EPC’s • Bone marrow derived epithelial

progenitor cells (EPCs) home to sites of vascular injury and help to maintain capillary integrity or re-perfuse acellular capillaries

• The release of EPCs from bone marrow follows a circadian rhythmicity in response to local adrenergic signals (Mendez-Ferrer et al. 2008). Circadian rhythms are driven by a central clock (suprachiasmaticnucleus, SCN) and peripheral clocks (tissues)

• Clock genes induce circadian rhythm for EPC release by synchronous interplay of positive (Clock, Bmal1) and negative (Per1, Per2, Cry1, Cry2) loops of the peripheral circadian clock

J Busik et. al,. (2009) J. Exp. Med

Diabetes Decreases Circadian Release of EPCs.

qPCR mRNA Expression Analysis for Clock Genes

EPC Dysfunction in T2D

Decreased Adrenergic Function in T2D

Conclusions

• BBZ has most of the features of diabetic retinopathy as observed in humans

• Evaluation of drug therapies can be performed using – Histological studies – Immunological studies – Functional studies using small vessels