BRM is a successful biopharmaceutical company formed in 1996 by Dennis Guberski and Dr. Arthur Like of the University of Massachusetts Medical School (UMass). Over the course of 20 years the founders developed proprietary diabetes research models under the sponsorship of the National Institute of Diabetes, Digestive and Kidney Diseases (NIDDK). BRM licensed this intellectual property portfolio from UMass in 1998 and since that time has used these proprietary tools to become one of the leading sources of customized preclinical contract research specializing in type 1 and type 2 diabetes.
16. Severe atherosclerosis:
10 months ~8 months duration of diabetes
Complete occlusion of renal artery, x100 (Left);
Arrows identify plaque, x400 (Both panels).
17. 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)
18. Neointima Thickness in Lean and Obese Male
BBZDR Rats One Week Post Balloon Injury
BBZDR Lean Rats Type 2 Diabetic Rats
19. Neointima Thickness in Lean and Obese Male
BBZDR Rats 4 Weeks Post Balloon Injury
BBZDR Lean Rats Type 2 Diabetes Rats
30. RAGE in BBZDR Rat Kidneys
Obese T2 Diabetic Lean Control
31. 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)
33. 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.
35. 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
36. Progression of Axo-glial Dysjunction
Axo-glialdysjunction(%)
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-glialdysjunction(%)
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
43. 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.
44. 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)
Diameter(µM)
45. 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
%Myogenictone
-10 -9 -8 -7 -6 -5 -4 -3
0
25
50
75
100
125
log[Histamine] M
%Myogenictone
-10 -9 -8 -7 -6 -5 -4 -3
0
25
50
75
100
125
log[Histamine] M
%Myogenictone
-10 -9 -8 -7 -6 -5 -4 -3
0
25
50
75
100
125
log[Histamine] M
%Myogenictone
Pre-diabetic age 3-4 Weeks of Diabetes
5 Months of Diabetes 10 Months of Diabetes
55. 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.
56. 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 ¯ ¯
57. 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)
Diameter(µM)
66. 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
%Myogenictone
-10 -9 -8 -7 -6 -5 -4 -3
0
25
50
75
100
125
log[Histamine] M
%Myogenictone
-10 -9 -8 -7 -6 -5 -4 -3
0
25
50
75
100
125
log[Histamine] M
%Myogenictone
-10 -9 -8 -7 -6 -5 -4 -3
0
25
50
75
100
125
log[Histamine] M
%Myogenictone
Pre-diabetic age 3-4 Weeks of Diabetes
5 Months of Diabetes 10 Months of Diabetes
67. 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
%KClConstriction
-12 -11 -10 -9 -8 -7 -6 -5
0
25
50
75
100
log[5-Hydroxy tryptamine] M
%KClConstriction
-12 -11 -10 -9 -8 -7 -6 -5
0
25
50
75
100
log[5-Hydroxy tryptamine] M
%KClConstriction
-12 -11 -10 -9 -8 -7 -6 -5
0
25
50
75
100
log[5-Hydroxy tryptamine] M
%KClConstriction
68. 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
%Myogenictone
-9 -8 -7 -6 -5 -4
0
25
50
75
100
125
log[U-73122] M
%Myogenictone
-9 -8 -7 -6 -5 -4
0
25
50
75
100
125
log[U-73122] M
%Myogenictone
-9 -8 -7 -6 -5 -4
0
25
50
75
100
125
log[U-73122] M
%Myogenictone
Arterial smooth muscle hyper-reactivity PLC activation
69. Autoregulation in rat (Sprague Dawley)
ophthalmic artery
0 30 60 90 120 150 180 210
100
150
200
250
300
Diameter with tone
Passive diameter
Myogenic tone
0
25
50
Intraluminal Pressure (mm Hg)
Lumendiameter(microns)
Myogenictone(%)
70. -15 -14 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3
0
25
50
75
100
125
Serotonin
Phenylephrine
Vasopressin
UK-14304
U-46619
log M [Vasoconstrictor]
%KCl(60mM)response
-15 -14 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3
0
50
100
150
log M [Endothelin-1]
%KCl(60mM)response
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
71. -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]
Myogenictone(%)
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
72. -9 -8 -7 -6 -5 -4 -3
20
60
100
140
180
L-NAME
Indomethacin
log M [Inhibitor]
Myogenictone(%)
Effect of the inhibition of NOS
and prostaglandin synthesis on myogenic tone
in rat ophthalmic artery
73. 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
78. 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