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M. A. Niewczas and H. Shah
32. Kobayashi H, Looker HC, Satake E, etal. Neuroblastoma suppressor of tumorigenicity 1 is a circulating protein associated with
progression to end-stage kidney disease in diabetes. Sci Transl
Med. 2022;14(657):eabj2109. (In eng). https://doi.org/10.1126/
scitranslmed.abj2109.
33. Ziegler D, Strom A, Bonhof GJ, etal. Decits in systemic biomarkers of neuroinammation and growth factors promoting
nerve regeneration in patients with type 2 diabetes and polyneuropathy. BMJ Open Diabetes Res Care. 2019;7(1):e000752.
https://doi.org/10.1136/bmjdrc- 2019- 000752.
34. Herder C, Kannenberg JM, Carstensen-Kirberg M, etal. A systemic inammatory signature reecting cross talk between innate
and adaptive immunity is associated with incident polyneuropathy: KORA F4/FF4 study. Diabetes. 2018;67(11):2434–42.
https://doi.org/10.2337/db18- 0060.
35. Willenborg S, Sanin DE, Jais A, etal. Mitochondrial metabolism
coordinates stage-specic repair processes in macrophages during
wound healing. Cell Metab. 2021;33(12):2398–2414.e9. (In eng).
https://doi.org/10.1016/j.cmet.2021.10.004.
36. Go YM, Jones DP.Cysteine/cystine redox signaling in cardiovascular disease. Free Radic Biol Med. 2011;50(4):495–509. (In
eng). https://doi.org/10.1016/j.freeradbiomed.2010.11.029.
37. Swaney MH, Kalan LR.Living in your skin: microbes, molecules,
and mechanisms. Infect Immun. 2021;89(4):e00695-20. (In eng).
https://doi.org/10.1128/iai.00695- 20.
38. Chen YE, Fischbach MA, Belkaid Y.Skin microbiota-host interactions. Nature. 2018;553(7689):427–36. (In eng). https://doi.
org/10.1038/nature25177.
39. Schmidt BM, Erb-Downward J, Ranjan P, Dickson
R.Metagenomics to identify pathogens in diabetic foot ulcers and
the potential impact for clinical care. Curr Diab Rep. 2021;21(8):26.
(In eng). https://doi.org/10.1007/s11892- 021- 01391- 7.
40. Naik S, Bouladoux N, Linehan JL, etal. Commensal-dendriticcell interaction species a unique protective skin immune signature. Nature. 2015;520(7545):104–8. (In eng). https://doi.
org/10.1038/nature14052.
41. Schmidt BM. Emerging diabetic foot ulcer microbiome analysis using cutting edge technologies. J Diabetes
Sci Technol. 2022;16(2):353–63. (In eng). https://doi.
org/10.1177/1932296821990097.
42. Dangwal S, Stratmann B, Bang C, etal. Impairment of wound
healing in patients with type 2 diabetes mellitus inuences
circulating microRNA patterns via inammatory cytokines.
Arterioscler Thromb Vasc Biol. 2015;35(6):1480–8. (In eng).
https://doi.org/10.1161/atvbaha.114.305048.
43. Liang L, Stone RC, Stojadinovic O, etal. Integrative analysis of
miRNA and mRNA paired expression proling of primary broblast derived from diabetic foot ulcers reveals multiple impaired
cellular functions. Wound Repair Regen. 2016;24(6):943–53. (In
eng). https://doi.org/10.1111/wrr.12470.
44. Marjanovic J, Ramirez HA, Jozic I, et al. Dichotomous role
of miR193b-3p in diabetic foot ulcers maintains inhibition of
healing and suppression of tumor formation. Sci Transl Med.
2022;14(644):eabg8397. (In eng). https://doi.org/10.1126/sci-
translmed.abg8397.
45. Petkovic M, Sørensen AE, Leal EC, Carvalho E, Dalgaard
LT.Mechanistic actions of microRNAs in diabetic wound healing. Cells. 2020;9(10):2228. (In eng). https://doi.org/10.3390/
cells9102228.
46. Ramirez HA, Pastar I, Jozic I, etal. Staphylococcus aureus triggers
induction of miR-15B-5P to diminish DNA repair and deregulate
inammatory response in diabetic foot ulcers. J Invest Dermatol.
2018;138(5):1187–96. (In eng). https://doi.org/10.1016/j.
jid.2017.11.038.
47. Chung WK, Erion K, Florez JC, etal. Precision medicine in diabetes: a consensus report from the American Diabetes Association
(ADA) and the European Association for the Study of Diabetes
(EASD). Diabetes Care. 2020;43(7):1617–35. (In eng). https://
doi.org/10.2337/dci20- 0022.
48. Collins FS, Varmus H. A new initiative on precision medicine.
N Engl J Med. 2015;372(9):793–5. https://doi.org/10.1056/
NEJMp1500523.
49. Tahir UA, Gerszten RE.Omics and cardiometabolic disease risk
prediction. Annu Rev Med. 2020;71:163–75. (In eng). https://doi.
org/10.1146/annurev- med- 042418- 010924.
50. Komorowsky CV, Brosius FC III, Pennathur S, Kretzler
M.Perspectives on systems biology applications in diabetic kidney disease. J Cardiovasc Transl Res. 2012;5(4):491–508. (In
eng). https://doi.org/10.1007/s12265- 012- 9382- 7.
51. Hirohama D, Abedini A, Moon S, etal. Unbiased human kidney
tissue proteomics identies matrix metalloproteinase 7 as a kidney
disease biomarker. J Am Soc Nephrol. 2023;34(7):1279–91. (In
eng). https://doi.org/10.1681/asn.0000000000000141.
52. Fadini GP, Albiero M, Millioni R, etal. The molecular signature of impaired diabetic wound healing identies serpinB3 as a
healing biomarker. Diabetologia. 2014;57(9):1947–56. (In eng).
https://doi.org/10.1007/s00125- 014- 3300- 2.
53. Krisp C, Jacobsen F, McKay MJ, Molloy MP, Steinstraesser L,
Wolters DA. Proteome analysis reveals antiangiogenic environments in chronic wounds of diabetes mellitus type 2 patients.
Proteomics. 2013;13(17):2670–81. (In eng). https://doi.
org/10.1002/pmic.201200502.
54. Doupis J, Lyons TE, Wu S, Gnardellis C, Dinh T, Veves
A.Microvascular reactivity and inammatory cytokines in painful and painless peripheral diabetic neuropathy. J Clin Endocrinol
Metab. 2009;94(6):2157–63. (In eng). https://doi.org/10.1210/
jc.2008- 2385.
55. Liu C, Debnath N, Mosoyan G, etal. Systematic review and metaanalysis of plasma and urine biomarkers for CKD outcomes.
J Am Soc Nephrol. 2022;33(9):1657–72. (In eng). https://doi.
org/10.1681/asn.2022010098.
56. Niewczas MA, Gohda T, Skupien J, etal. Circulating TNF receptors 1 and 2 predict ESRD in type 2 diabetes. J Am Soc Nephrol.
2012;23(3):507–15. https://doi.org/10.1681/ASN.2011060627.
ASN.2011060627 [pii].
57. Pavkov ME, Nelson RG, Knowler WC, Cheng Y, Krolewski AS,
Niewczas MA.Elevation of circulating TNF receptors 1 and 2
increases the risk of end-stage renal disease in American Indians
with type 2 diabetes. Kidney Int. 2015;87(4):812–9. https://doi.
org/10.1038/ki.2014.330.
58. Dayon L, Cominetti O, Affolter M.Proteomics of human biological uids for biomarker discoveries: technical advances and recent
applications. Expert Rev Proteomics. 2022;19(2):131–51. https://
doi.org/10.1080/14789450.2022.2070477.
59. Smith JG, Gerszten RE.Emerging afnity-based proteomic technologies for large-scale plasma proling in cardiovascular disease. Circulation. 2017;135(17):1651–64. (In eng). https://doi.
org/10.1161/circulationaha.116.025446.
60. Uhlen M, Fagerberg L, Hallstrom BM, etal. Proteomics. Tissue- based
map of the human proteome. Science. 2015;347(6220):1260419.
https://doi.org/10.1126/science.1260419.
61. Uhlen M, Karlsson MJ, Hober A, etal. The human secretome. Sci
Signal. 2019;12(609):eaaz0274. https://doi.org/10.1126/scisignal.
aaz0274.
62. Herder C, Maalmi H, Strassburger K, etal. Differences in biomarkers of inammation between novel subgroups of recent-onset
diabetes. Diabetes. 2021; https://doi.org/10.2337/db20- 1054.
63. Hohendorff J, Drozdz A, Borys S, etal. Effects of negative pressure wound therapy on levels of angiopoetin-2 and other selected
circulating signaling molecules in patients with diabetic foot
ulcer. J Diabetes Res. 2019;2019:1756798. (In eng). https://doi.
org/10.1155/2019/1756798.

18 Biomarkers ofDiabetic Foot Ulcers andIts Healing Progress
https://t.me/med1917
337
64. Schlesinger S, Herder C, Kannenberg JM, et al. General and
abdominal obesity and incident distal sensorimotor polyneuropathy: insights into inammatory biomarkers as potential mediators
in the KORA F4/FF4 cohort. Diabetes Care. 2019;42(2):240–7.
(In eng). https://doi.org/10.2337/dc18- 1842.
65. Md Dom ZI, Satake E, Skupien J, etal. Circulating proteins protect
against renal decline and progression to end-stage renal disease in
patients with diabetes. Sci Transl Med. 2021;13(600):eabd2699.
(In eng). https://doi.org/10.1126/scitranslmed.abd2699.
66. Csosz E, Toth N, Deak E, Csutak A, Tozser J. Wound-healing
markers revealed by proximity extension assay in tears of patients
following glaucoma surgery. Int J Mol Sci. 2018;19(12):4096. (In
eng). https://doi.org/10.3390/ijms19124096.
67. Donatti A, Canto AM, Godoi AB, da Rosa DC, Lopes-Cendes
I. Circulating metabolites as potential biomarkers for neurological disorders—metabolites in neurological disorders.
Metabolites. 2020;10(10):389. (In eng). https://doi.org/10.3390/
metabo10100389.
68. Guijas C, Montenegro-Burke JR, Warth B, Spilker ME, Siuzdak
G. Metabolomics activity screening for identifying metabolites
that modulate phenotype. Nat Biotechnol. 2018;36(4):316–20. (In
eng). https://doi.org/10.1038/nbt.4101.
69. Pang Z, Zhou G, Ewald J, et al. Using MetaboAnalyst 5.0
for LC-HRMS spectra processing, multi-omics integration
and covariate adjustment of global metabolomics data. Nat
Protoc. 2022;17(8):1735–61. (In eng). https://doi.org/10.1038/
s41596- 022- 00710- w.
70. Álvarez R II, Castaño-Tostado E, García-Gutiérrez DG, et al.
Non-targeted metabolomic analysis reveals serum phospholipid
alterations in patients with early stages of diabetic foot ulcer.
Biomark Insights. 2020;15:1177271920954828. (In eng). https://
doi.org/10.1177/1177271920954828.
71. Hung SY, Tsai JS, Yeh JT, etal. Amino acids and wound healing in people with limb-threatening diabetic foot ulcers. J
Diabetes Complicat. 2019;33(10):107403. (In eng). https://doi.
org/10.1016/j.jdiacomp.2019.06.008.
72. Mathew AV, Jaiswal M, Ang L, Michailidis G, Pennathur S,
Pop- Busui R. Impaired amino acid and TCA metabolism and
cardiovascular autonomic neuropathy progression in type 1 diabetes. Diabetes. 2019;68(10):2035–44. (In eng). https://doi.
org/10.2337/db19- 0145.
73. Niewczas MA, Mathew AV, Croall S, etal. Circulating modied
metabolites and a risk of ESRD in patients with type 1 diabetes
and chronic kidney disease. Diabetes Care. 2017;40(3):383–90.
(In eng). https://doi.org/10.2337/dc16- 0173.
74. Niewczas MA, Sirich TL, Mathew AV, etal. Uremic solutes and
risk of end-stage renal disease in type 2 diabetes: metabolomic
study. Kidney Int. 2014;85(5):1214–24. (In eng). https://doi.
org/10.1038/ki.2013.497.
75. Moon S, Tsay JJ, Lampert H, etal. Circulating short and medium
chain fatty acids are associated with normoalbuminuria in type
1 diabetes of long duration. Sci Rep. 2021;11(1):8592. (In eng).
https://doi.org/10.1038/s41598- 021- 87585- 1.
76. Shah HS, Moreno LO, Morieri ML, etal. Serum orotidine: a novel
biomarker of increased CVD risk in type 2 diabetes discovered
through metabolomics studies. Diabetes Care. 2022;45(8):1882–
92. (In eng). https://doi.org/10.2337/dc21- 1789.
77. Sawaya AP, Stone RC, Brooks SR, etal. Deregulated immune
cell recruitment orchestrated by FOXM1 impairs human diabetic
wound healing. Nat Commun. 2020;11(1):4678. (In eng). https://
doi.org/10.1038/s41467- 020- 18276- 0.
78. Theocharidis G, Thomas BE, Sarkar D, et al. Single cell
transcriptomic landscape of diabetic foot ulcers. Nat
Commun. 2022;13(1):181. (In eng). https://doi.org/10.1038/
s41467- 021- 27801- 8.
79. Schmidt BM, Holmes CM, Najarian K, et al. On diabetic foot
ulcer knowledge gaps, innovation, evaluation, prediction markers,
and clinical needs. J Diabetes Complicat. 2022;36(11):108317.
(In eng). https://doi.org/10.1016/j.jdiacomp.2022.108317.
80. Sumpio BJ, Li Z, Wang E, Mezghani I, Theocharidis G, Veves
A.Future directions in research in transcriptomics in the healing
of diabetic foot ulcers. Adv Ther. 2023;40(1):67–75. (In eng).
https://doi.org/10.1007/s12325- 022- 02348- 2.
81. Kato M, Castro NE, Natarajan R.MicroRNAs: potential mediators and biomarkers of diabetic complications. Free Radic
Biol Med. 2013;64:85–94. (In eng). https://doi.org/10.1016/j.
freeradbiomed.2013.06.009.
82. Natarajan R, Putta S, Kato M. MicroRNAs and diabetic complications. J Cardiovasc Transl Res. 2012;5(4):413–22. (In eng).
https://doi.org/10.1007/s12265- 012- 9368- 5.
83. Kato M, Natarajan R.Diabetic nephropathy—emerging epigenetic mechanisms. Nat Rev Nephrol. 2014;10(9):517–30. (In eng).
https://doi.org/10.1038/nrneph.2014.116.
84. Friedman RC, Farh KK, Burge CB, Bartel DP. Most mammalian mRNAs are conserved targets of microRNAs. Genome
Res. 2009;19(1):92–105. (In eng). https://doi.org/10.1101/
gr.082701.108.
85. Backes C, Meese E, Keller A.Specic miRNA disease biomarkers in blood, serum and plasma: challenges and prospects. Mol
Diagn Ther. 2016;20(6):509–18. (In eng). https://doi.org/10.1007/
s40291- 016- 0221- 4.
86. Banerjee J, Sen CK. microRNA and wound healing. Adv
Exp Med Biol. 2015;888:291–305. (In eng). https://doi.
org/10.1007/978- 3- 319- 22671- 2_15.
87. Ramirez HA, Liang L, Pastar I, et al. Comparative genomic,
microRNA, and tissue analyses reveal subtle differences
between non-diabetic and diabetic foot skin. PLoS One.
2015;10(8):e0137133. (In eng). https://doi.org/10.1371/journal.
pone.0137133.
88. Kalan L, Loesche M, Hodkinson BP, etal. Redening the chronicwound microbiome: fungal communities are prevalent, dynamic,
and associated with delayed healing. mBio. 2016;7(5):e01058-16.
(In eng). https://doi.org/10.1128/mBio.01058- 16.
89. Kalan LR, Meisel JS, Loesche MA, etal. Strain- and species-level
variation in the microbiome of diabetic wounds is associated with
clinical outcomes and therapeutic efcacy. Cell Host Microbe.
2019;25(5):641–655.e5. (In eng). https://doi.org/10.1016/j.
chom.2019.03.006.
90. Sloan TJ, Turton JC, Tyson J, etal. Examining diabetic heel ulcers
through an ecological lens: microbial community dynamics associated with healing and infection. J Med Microbiol. 2019;68(2):230–
40. (In eng). https://doi.org/10.1099/jmm.0.000907.
91. McShane LM, Cavenagh MM, Lively TG, et al. Criteria for
the use of omics-based predictors in clinical trials. Nature.
2013;502(7471):317–20. https://doi.org/10.1038/nature12564.
92. Hirata S, Dirven L, Shen Y, etal. A multi-biomarker score measures rheumatoid arthritis disease activity in the BeSt study.
Rheumatology (Oxford). 2013;52(7):1202–7. https://doi.
org/10.1093/rheumatology/kes362.
93. Kobayashi H, Looker HC, Satake E, etal. Results of untargeted
analysis using the SOMAscan proteomics platform indicates
novel associations of circulating proteins with risk of progression
to kidney failure in diabetes. Kidney Int. 2022;102(2):370–81.
https://doi.org/10.1016/j.kint.2022.04.022.
94. Foster DS, Januszyk M, Yost KE, et al. Integrated spatial multiomics reveals broblast fate during tissue repair. Proc Natl
Acad Sci USA. 2021;118(41):e2110025118. (In eng). https://doi.
org/10.1073/pnas.2110025118.
95. Satake E, Pezzolesi MG, Md Dom ZI, Smiles AM, Niewczas
MA, Krolewski AS. Circulating miRNA proles associated

338
https://t.me/med1917
M. A. Niewczas and H. Shah
with hyperglycemia in patients with type 1 diabetes. Diabetes.
2018;67(5):1013–23. (In eng). https://doi.org/10.2337/db17- 1207.
96. Stojadinovic O, Landon JN, Gordon KA, etal. Quality assessment of tissue specimens for studies of diabetic foot ulcers. Exp
Dermatol. 2013;22(3):216–8. (In eng). https://doi.org/10.1111/
exd.12104.
97. Kounas K, Dinh T, Riemer K, Rosenblum BI, Veves A, Giurini
JM.Use of hyperspectral imaging to predict healing of diabetic
foot ulceration. Wound Repair Regen. 2023;31(2):199–204. (In
eng). https://doi.org/10.1111/wrr.13071.
98. Kim RB, Gryak J, Mishra A, etal. Utilization of smartphone and
tablet camera photographs to predict healing of diabetes-related
foot ulcers. Comput Biol Med. 2020;126:104042. (In eng). https://
doi.org/10.1016/j.compbiomed.2020.104042.
99. Brunner PM, Suarez-Farinas M, He H, etal. The atopic dermatitis
blood signature is characterized by increases in inammatory and
cardiovascular risk proteins. Sci Rep. 2017;7(1):8707. https://doi.
org/10.1038/s41598- 017- 09207- z.
100. Pavel AB, Zhou L, Diaz A, etal. The proteomic skin prole of
moderate-to-severe atopic dermatitis patients shows an inammatory signature. J Am Acad Dermatol. 2020;82(3):690–9. https://
doi.org/10.1016/j.jaad.2019.10.039.
101. Garshick MS, Baumer Y, Dey AK, et al. Characterization of
PCSK9 in the blood and skin of psoriasis. J Invest Dermatol.
2020;141:308. https://doi.org/10.1016/j.jid.2020.05.115.
102. Haslam DE, Li J, Dillon ST, etal. Stability and reproducibility of proteomic proles in epidemiological studies: comparing
the Olink and SOMAscan platforms. Proteomics. 2022;22(13–
14):e2100170. (In eng). https://doi.org/10.1002/pmic.202100170.
103. Benjamini Y, Hochberg Y. Controlling the false discovery
rate: a practical and powerful approach to multiple testing. J
R Stat Soc Ser B Methodol. 1995;57(1):289–300. https://doi.
org/10.1111/j.2517- 6161.1995.tb02031.x.
104. Austin PC, Harrell FE Jr, Steyerberg EW.Predictive performance
of machine and statistical learning methods: impact of datagenerating processes on external validity in the “large N, small p”
setting. Stat Methods Med Res. 2021;30(6):1465–83. https://doi.
org/10.1177/09622802211002867.
105. Breiman L. Statistical modeling: the two cultures. Stat Sci.
2001;16(3):199–215. http://www.jstor.org/stable/2676681.
106. Kuhn M.Building predictive models in R using the caret package. J Stat Softw. 2008;28(5):1–26. https://doi.org/10.18637/jss.
v028.i05.
107. Zou H, Hastie T. Regularization and variable selection via the
elastic net. J R Stat Soc Ser B Stat Methodol. 2005;67(2):301–20.
https://doi.org/10.1111/j.1467- 9868.2005.00503.x.
108. Gomes B, Ashley EA.Articial intelligence in molecular medicine. N Engl J Med. 2023;388(26):2456–65. (In eng). https://doi.
org/10.1056/NEJMra2204787.
109. Florez JC. Precision medicine in diabetes: is it time? Diabetes
Care. 2016;39(7):1085–8. (In eng). https://doi.org/10.2337/
dc16- 0586.

Experimental Animal Models
https://t.me/med1917
inResearch: Diabetes andImpaired
Wound Healing
MauricioContreras andEnyaWang
19
Abstract
Animal models are used to study the development and
progression of diseases, providing unique perspectives to
better understand the primary mechanisms involved in
their pathophysiology. Animal welfare laws and strict
regulations have been established to protect and ensure
the humane treatment of animals while they are being
used for research and experimentation purposes. With the
assistance of the National Institutes of Health (NIH), reliable animal models of diabetes have been established,
through the Animal Models of Diabetic Complications
Consortium (AMDCC), to study the disease, as well as
the development and testing of effective therapies and
preventative strategies for diabetic complications. Thus,
the following chapter will review the diabetic animal
models that are available to date, with a particular emphasis on models that are best suited for the study of impaired
diabetic wound healing. Based on our own research experience, we will describe in detail the models that we currently use: the mouse, rabbit, and pig models.
Abbreviations
AAALAC Association for Assessment and Accreditation of
Laboratory Animal Care
ALX Alloxan
AWA Animal Welfare Act
AZT Streptozotocin
BGL Blood glucose level
IACUC Institutional Animal Care and Use Committee
M. Contreras (*)
Beth Israel Deaconess Medical Center and Harvard Medical
School, Boston, MA, USA
e-mail: mcontrer@bidmc.harvard.edu
E. Wang
The Rongxiang Xu, MD, Center for Regenerative Therapeutics,
Beth Israel Deaconess Medical Center, Boston, MA, USA
IP Intraperitoneal
IV Intravenous
NIH National Institutes of Health
NZW New Zealand White
OLAW Ofce of Laboratory Animal Welfare
PPE Personal protective equipment
SC Subcutaneous
SURG Surgical
T2D Type 2 diabetes
USDA US Department of Agriculture
Animal Models: Background
andSignicance
Animal models have provided invaluable information in the
pursuit of medical knowledge and alleviation of human suffering. The foundations of our basic understanding of disease pathophysiology and human anatomy can largely be
attributed to preclinical investigations using various animal
models [1].
The decision to use animals in research requires critical
thought, judgment, and analysis. Using animals in research
is a privilege granted by society to the research community
with the expectation that such use will provide either signicant new knowledge or lead to improvement in human and/
or animal well-being [2, 3]. It is a trust that mandates responsible and humane care and use of these animals. Regulations
and animal welfare laws vary considerably around the world,
and the literature on the subject includes numerous publications in which the use of animals in research is discussed [4,
5]. For the purpose of this review, a owchart (Fig.19.1) has
been created to exhibit the different regulatory agencies that
are involved in the use and care of animals in medical
research.
© The Author(s), under exclusive license to Springer Nature Switzerland AG 2024
A. Veves et al. (eds.), The Diabetic Foot, Contemporary Diabetes, https://doi.org/10.1007/978-3-031-55715-6_19
339

340
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M. Contreras and E. Wang
Fig. 19.1 Flowchart: USDA
animal care and use program
National Research
Council
LAM
Veterinary &
Husbandry
USDA (PHS Policy)
APHIS
Animal Welfare Act
Research Institution
Institutional Official
Scientists
Animal Users
AAALAC / OLWA
IACUC
(Compliance)
The Animal Welfare Act
In 1966, the US Congress passed legislation pertaining the
use of animals, the Animal Welfare Act (AWA), which sets
general standards for humane care and treatment that must
be provided to animals that are bred for commercial sale and
used in biomedical research. The Congress assigned the US
Department of Agriculture (USDA) the responsibility for
enforcing the AWA.The Animal and Plant Health Inspection
Service (APHIS) is the agency within USDA responsible for
ensuring this occurs. APHIS publication, the Animal Welfare
Act and Animal Welfare Regulations, known as the “Blue
Book,” was intended to be used as a tool to improve
compliance among licensees and registrants, to enhance the
consistency of inspections by eld overseers. The Blue Book
[6] consolidates into one source the AWA and the applicable
regulations and standards. [United States Code, Title 7
(Agriculture), Chapter 54 (Transportation, Sale, and
Handling of Certain Animals), Sections 2131–2159] and
[Code of Federal Regulations, Title 9 (Animals and Animal
Products), Chapter 1 (Animal and Plant Health Inspection
Service, Department of Agriculture), Subchapter A (Animal
Welfare), Parts 1–4].
The Guide fortheCare andUse ofLaboratory
Animals
The Governing Board of the National Research Council,
whose members are drawn from the councils of the National
Academy of Sciences, the National Academy of Engineering,
and the Institute of Medicine, chosen for their special prociencies and expertise, was delegated the responsibility to
bring together recommendations and guidelines, expressed
in a publication, also known as the Guide for the Care and
Use of Laboratory Animals.
The purpose of the Guide is to assist institutions in caring
for and using animals in ways judged to be scientically,
technically, and humanely appropriate. The Guide is also
intended to assist investigators in fullling their obligation to
plan and conduct animal experiments in accordance with the
highest scientic, humane, and ethical principles.
Recommendations in the Guide are based on published data,
scientic principles, expert opinion, and experience with
methods and practices that have proved to be consistent with
both high-quality research and humane animal care and use.
These recommendations should be used as a foundation for
the development of a comprehensive animal care and use

19 Experimental Animal Models inResearch: Diabetes andImpaired Wound Healing
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program, recognizing that the concept and application of
performance standards, in accordance with goals, outcomes,
and considerations dened in the Guide, is essential to this
process. The Guide is an internationally accepted primary
reference on animal care and use, and its routine practice is
required in the United States by the Public Health Service
Policy. It was rst published in 1963, under the title Guide
for Laboratory Animal Facilities and Care, and was revised
in 1965, 1968, 1972, 1978, 1985, and 1996 [7].
National Institutes ofHealth Policy
Pertaining theUse ofAnimals inResearch
It should be noted that in 1971, the National Institutes of
Health (NIH) Policy required institutions or organizations
using warm-blooded animals in research or teaching supported by NIH grants, awards, or contracts to “assure the
NIH that they would evaluate their animal facilities in regard
to the maintenance of acceptable standards for the care, use,
and treatment of animals.” The institution could show that a
recognized professional laboratory animal accrediting body
(AAALAC) had established an animal care committee to
carry out that assurance function either accredited it. Thus,
the 1979 revision of the PHS policy required each
animal- using grantee institution to have “a committee to
maintain oversight of its animal care program” and expanded
the denition of animal to include all vertebrates. The revised
policy also required an institution to submit an assurance
statement to the Ofce for Protection from Research Risks
(OPRR), now the Ofce of Laboratory Animal Welfare
(OLAW), that it is committed to follow the Guide, the principles, and the PHS policy requirements, before receiving
PHS support for studies in which animals or animal facilities
were used. Institutions, therefore, would be required to
include in their assurance a list of committee members with
their position titles and credentials. Committees would be
composed of at least ve members including at least one veterinarian. The members had to be knowledgeable regarding
the care and use of animals used in research. Consequently,
each institution that receives PHS support for activities
involving vertebrate animals or is subject to the authority of
the Animal Welfare Act (AWA) must operate an animal care
and use program with clear lines of authority and
responsibility.
Institutional Animal Care andUse
Committee (IACUC)
The Institutional Animal Care and Use Committee (IACUC)
is the association that provides oversight and ensures appropriate review of the use of vertebrate animals in teaching,
testing, and research. The committee composition is generally designed to be broad enough to represent both scientic
and nonscientic interests. IACUCs derive their authority
from the law.
There has been an evolution in the ways that IACUCs
fullled their mandate. This has been in part due to
increased experience implementing the PHS policy and
AWRs. Other factors contributing to this evolution have
originated from the research community itself, such as the
development of transgenic animals and invitro alternatives
to the production of monoclonal antibodies. The IACUC
community has also gained a greater understanding of and
appreciation for the role of nonafliated and nonscientic
IACUC members. Humane endpoints in research and innovative ways to address environmental enrichment of primates are other areas that grew in sophistication during the
1990s. Training of IACUC members and animal users has
received greater attention and the number of training programs and modules has increased signicantly. Finally,
IACUCs derive their authority from the law. They are mandated by the Health Research Extension Act (HREA) of
1985, and the AWA, OLAW, USDA, and AAALAC
International have all placed an increased focus on IACUC
functions.
The original OPRR/ARENA IACUC Guidebook was published in 1992 and has served as a useful resource to the animal research community. The revised edition in 2008
continues to support the fundamental principle on which the
animal care and use program is based: self-regulation with
oversight. It clearly demonstrates the increased role of the
IACUC in ensuring the ethical and sensitive care and use of
animals in research, teaching, and testing [8].
General Guidelines andConsiderations
intheSelection Process ofAnimal Models
Over the years, the three Rs [9] have become an internationally accepted approach for researchers to apply when deciding to use animals in research and in designing humane
animal research studies. The three Rs, replacement, renement, and reduction, represent a practical method for implementation of the principles described earlier.
Replacement: Refers to methods that avoid using animals.
The term includes absolute replacements (i.e., replacing animals with inanimate systems such as computer programs
now supported by AI: mechanistic modeling, software engineering and science data optimization) as well as relative
replacements (i.e., replacing animals such as vertebrates
with animals that are lower on the phylogenetic scale). It
should be noted that there are those who believe that animals
should not be used for experimental purposes. Nevertheless,
it is possible to minimize distress and have humane end-

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Three R’s
B: No Research use
Animal Protocol
Fig. 19.2 Flowchart: experimental animal protocol owchart
USDA Category
Model Type
C: Teaching No Research
D: Anesthesia & Analgesia
E: Humane Endpoints
1: Disease induction
I: Replacement
II: Refinement
III: Reduction
2: Xenograft
3: Inbred Strain
4: Tr ansgenic
Pre-Review
Vaterinary
&
Res. Adm.
M. Contreras and E. Wang
IACUC Review
Corrections
&
Recommendations
Final Approval
points with well-designed studies. Research involving animals should be a balance between knowledge gained and
potential harm to animals.
Renement: Refers to modications of husbandry or
experimental procedures to enhance animal well-being and
minimize or eliminate pain and distress. While institutions
and investigators should take all reasonable measures to
eliminate pain and distress through renement, IACUCs
should understand that with some types of studies, there
might be either unforeseen or intended experimental outcomes that produce pain. These outcomes may or may not be
eliminated based on the goals of the study.
Reduction: Involves strategies for obtaining comparable
levels of information from the use of fewer animals or for
maximizing the information obtained from a given number
of animals (without increasing pain or distress) so that in the
long run, fewer animals are needed to acquire the same scientic information. This approach relies on an analysis of
experimental design, applications of newer technologies,
the use of appropriate statistical methods, and control of
environmentally related variability in animal housing and
study areas.
Renement and reduction goals should be balanced on a
case-by-case basis. Principal investigators are strongly discouraged from advocating animal reuse as a reduction strategy, and reduction should not be a rationale for reusing an
animal or animals that have already undergone experimental
procedures especially if the well-being of the animals would
be compromised. Studies that may result in severe or chronic
pain or signicant alterations in the animals’ ability to maintain normal physiology, or adequately respond to stressors,
should include descriptions of appropriate humane endpoints
or provide science-based justication for not using a particu-
lar, commonly accepted humane endpoint. Veterinary consultation must occur when pain or distress is beyond the level
anticipated in the protocol description or when interventional
control is not possible (Fig.19.2).
USDA Directives toHelp Determine
Pain andDistress Categories
The USDA mandates that research animals subject to experimentation be placed by species into one of four USDA pain/
distress categories [7]:
Category B: Animals that are being “bred,” conditioned,
or held for use in teaching, testing, experiments, research, or
surgery but not yet used for such purposes.” These animals
have not been used for any research procedure, however
minor.
Category C: Animals that are not subjected to procedures
that involve pain or distress or would require the use of painrelieving drugs. Routine procedures such as injections and
blood sampling from veins that produce only mild, transient
pain or discomfort. Procedures such as an observational
study of animal behavior or animals that are euthanized
before tissue collection or other manipulations are also commonly placed in this category, if no other procedures are performed that put them in a higher pain/distress category.
Category D: Animals subjected to potentially painful procedures for which anesthetics, analgesics, or tranquilizers
will be used. The important concept is that animals are given
appropriate anesthesia and/or pain relief to limit their pain
and distress as much as possible. Examples under this category are surgery conducted with appropriate anesthesia and
postoperative analgesia, rodent retro-orbital eye bleeding

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performed under anesthesia, removal of small tumors under
local or general anesthesia, use of analgesia after an animal’s
skin is exposed to ultraviolet light to cause a “sunburn,” and
terminal exsanguinations (euthanasia by removal of blood)
under anesthesia.
Category E: Animals that are subjected to painful or
stressful procedures without the use of anesthetics, analgesics, or tranquilizers. Withholding of anesthetics, analgesics,
or tranquilizers can only be allowed if it is scientically justied in writing and approved by the IACUC.Examples under
this category are induction of illness without intervention to
alleviate pain or distress, pain studies that would not be possible if pain-relieving agents were administered, and psychological conditioning experiments that involve noxious/
painful stimuli that cannot immediately be avoided by an
animal. Under this category, humane endpoints (HEP) have
to be followed. A HEP is the earliest scientically justied
point at which pain or distress in an experimental animal can
be prevented, terminated, or relieved while meeting the scientic aims and objectives of the research study. By law, the
institution must annually report all category E procedures to
the USDA and include a scientic justication supporting
the IACUC’s decision to approve them. It is important for the
information on category E procedures to be complete and
accurate.
Recommendations andStrategies
inChoosing aParticular Animal Model
The primary criteria for evaluating the utility of a potential
animal model are the ability to conduct research using the
model that otherwise would not be possible or feasible in the
primary system of interest (e.g., humans) and the generalizability and validity of the results obtained in the model to the
primary system of interest [10]. Therefore, animal models
are used to study the development and progression of diseases and to help determine the safety and efcacy of new
treatments before they are either administered or implemented in humans. Thus, an animal model is a nonhuman
species used in biomedical research because it can either
mimic or replicate aspects of a biological process or disease
found in humans, in which novel therapeutics can be tested
and evaluated for future clinical translation into meaningful
human applications.
Unfortunately, most investigators choose their model for
convenience of availability, or because they perceive a particular model to be favored by others in the eld or by funding agencies. In fact, the most convenient, most easily
available, or most “popular” model may not be the best
model for the research in question [11, 12]. Therefore, the
animal model selection should be conducted carefully. The
primary factor in the choice should be the research goals;
while factors such as convenience, availability, and ease of
maintenance are important, they should not be permitted to
override the requirements of good science. Therefore, whatever the source of the models, the most important aspect of
the choice is that it must be well informed. In addition, this
will facilitate IACUC’s review process and help expedite
nal animal protocol approval (Fig.19.2).
Even though transgenic and knockout practices have
transformed the manipulation of murine and other species to
better understand the pathogenesis of human illness, we
have yet to be able to develop perfect animal models of
many of the human diseases. Therefore, the challenge
remains when selecting an appropriate animal model, and
researchers should consider the following recommendations
when doing so.
There are four main categories of animal models that are
used in preclinical research [13]:
1. Disease induction models. Where an animal is used dur-
ing the research and investigation of particular human
disease, for better understanding the disease process and
for testing new pharmacological agents or therapeutic
interventions.
2. Xenograft animal models. By denition, a xenograft is a
tissue graft or organ transplant from a donor of a different
species from the recipient. Usually, they are patientderived xenograft (PDX) mouse models that are involved
in the direct transfer of fresh human tumor (or tissue)
samples into immunodecient mice following surgical
resection or other medical operations.
3. Inbred strains. They are a population of animals that
result from a process of at least 20 sequential generations
of brother-sister matings. The resultant animals are essentially clones of each other at the genetic level.
4. Transgenic models. They are mouse and rat models that
have their genomes altered to include a transgene or foreign sequence for studying gene functions. Transgenic
animals are used to study oncology, obesity, diabetes,
aging, heart disease, and more.
Although no single model will be completely suitable for
all purposes within a eld of study, an appropriate animal
model for any research should be based on the following
considerations [14]: (1) appropriateness as an equivalent, (2)
transferability of information, (3) genetic uniformity of
organisms, (4) background knowledge of biological properties, (5) availability, (6) statistical framework and reliability
of the results, (7) ease and adaptability to experimental
manipulation, and (8) ethical and societal implications.

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M. Contreras and E. Wang
Animal Models ofDiabetes: Background
andSignicance
Animal models have been used extensively in the study of
diabetes mellitus. They give researchers the opportunity to
control in vivo the genetic and environmental factors that
may inuence the development of the disease and establishment of its complications and therefore gain new information about its handling and treatment in humans [15, 16].
Without a doubt, these models have provided an invaluable insight into the pathogenesis of the human disease, and
patients have beneted from experimentation using animals.
One such example was the discovery of insulin by Dr.
F. Banting in 1921, by reporting “encouraging results with
isletin,” controlling blood sugar levels in depancreatized
dogs [17].
Although most diabetes experiments are performed in
rodents, studies on larger animals are also employed. Several
toxins, including streptozotocin and alloxan, can induce
hyperglycemia in mice and rats, as well as other species,
such as, rabbits, swine, and nonhuman primates. Selective
inbreeding has produced several strains of animals that are
considered reasonable models of type 1 diabetes, type 2 diabetes, and related phenotypes such as obesity and insulin
resistance. Apart from their use in studying the pathogenesis
of the disease and its complications, all new treatments for
diabetes, including islet cell transplantation and preventative
strategies, are initially investigated in animals. In recent
years, molecular biological techniques have produced a large
number of new animal models for the study of diabetes,
including knock-in, generalized knockout, and tissuespecic knockout mice [18].
In an attempt to standardize reliable animal models of
diabetes that mimic human disease, the National Institutes
of Health initiated the Animal Models of Diabetic
Complications Consortium (AMDCC) in 2001 to create
and characterize such models to enhance the development
and testing of effective therapies and preventative strategies for diabetic complications. Unfortunately, while the
primary goal of the consortium was to develop murine
models of diabetic micro- and macro-vascular complications that would completely replicate the human disease,
this has yet to be accomplished [19]. Nonetheless, considerable progress has been made in model development,
mouse phenotyping, strain analysis, and understanding
the pathogenesis of diabetic complications. In spite of
these limitations, just because an animal model of diabetes does not replicate all of the conditions and processes
involved in the disease, it should not be excluded from
consideration [20].
USDA Non-covered Species
USDA non-covered species is a term to describe the use of
mice, rats, and birds in the laboratory. Their use is governed
by the US Department of Health and Human Services, the
Public Health Service, and the Ofce of Laboratory Animal
Welfare. The following sections will discuss specic rodent
models of diabetes and non-rodent models in USDA noncovered species.
Rodent Models
There are several major considerations in choosing a rodent
model that will be suitable for the conditions and complications that researchers target. Overall, rodent models are less
costly and easier to house and care for compared to larger
animal species. However, the cost of each rodent model varies depending on the method of diabetes induction, and diabetic rodent models also vary in diabetic complications,
diabetes type, delity to the human condition, and power of
predictiveness into clinical success.
Chemically Induced Diabetes: Alloxan
andStreptozotocin Models
Diabetes can be induced through the administration of diabetogenic chemicals, most commonly alloxan and streptozotocin. Alloxan and streptozotocin (abbreviated as STZ) are
both cytotoxic glucose analogues, and they accumulate specically in pancreatic islets through glucose uptake from the
bloodstream [21]. Thus, the effectiveness of these two diabetogenic chemicals is proportional to the glucose transporter
(GLUT2) activity of the species. As these chemicals accumulate inside pancreatic cells, they cause cell destruction
within the pancreatic islets which leads to an inability of the
pancreas to respond to insulin. However, the GLUT2 protein
is also expressed in other organs, such as the kidney and
liver, which can lead to unintentional organ damage in addition to the diabetes induction [21, 22].
The background species for the injected rodents are usually C57BL/6J and Sprague-Dawley or Wistar rats, and the
dosage of drug given is proportional to the animal’s body
weight. Of note, male animals are preferred for diabetes
induction, as female animals are less sensitive to the diabetogenic properties of both chemicals [23–25]. When planning
to work with alloxan or STZ, both of which are known carcinogens, it is essential for all researchers and staff to proceed
with care and follow all animal and lab safety guidelines.

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Table 19.1 Reagent dosages for chemical induction of diabetes in rodent animal models
Concentration
Method Animal Dosage (mg/kg)
Single-dose Mouse 200 20 4
Single-dose Rat 65 32.5 8
Multiple-dose Mouse 40 4 4 Alternate i.p. injections between left and right
Humanized skin graft Mouse (Foxn1/nu) 40 4 4 Employ multiple-dose induction method at
Nicotinamide Rat 32.5 32.5 8 Inject rats with 230mg nicotinamide 15min
High-fat diet Rat 32.5 32.5 8 Rats should be placed on a high-fat diet for
(mg/mL) Fasting (h) Additional notes
sides to minimize trauma
4weeks after skin graft
before STZ induction. High variability in
effective reagent dosages
3weeks prior to induction
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Alloxan
Alloxan is a glucose analogue which targets the GLUT2
transporter and glucokinase enzyme. When taken up into the
cell through GLUT2, alloxan reacts with thiols intracellularly to produce superoxide radicals and hydroxyl radicals,
which are cytotoxic to the pancreatic islets. Alloxan also acts
on glucokinase, a glucose sensor of pancreatic beta cells,
through specic inhibition which downregulates glucoseinduced secretion of insulin. These two pathways both contribute toward the development of type 1 diabetes [23].
Alloxan induction is performed through an intravenous
injection of alloxan dissolved in 0.9% saline. For mice, the
dosage of alloxan is 50mg/kg and for injection into rats, the
dosage of alloxan is 70mg/kg [24].
Some studies report lower mortality rates when animals
are given a 50mg/kg dose of dextrose after alloxan injection
[23]. To test for diabetes conversion, a blood glucose test of
tail vein blood can be performed at 10days, with glucose
concentrations >150mg indicating mild hyperglycemia.
Some drawbacks to alloxan induction include high toxicity to the animals, leading to lower survival rates, and possible reversion. Some studies show that alloxan-induced
animals have their blood glucose concentrations revert to
normal levels within a week. In addition, alloxan degrades
rapidly when in solution into alloxanic acid, with a half-life
of 1.5 min [21]. Thus, alloxan induction is more effortintensive for researchers to perform, as the alloxan solution
must be prepared immediately before administration, and the
drug must be given through rapid intravenous injection.
Streptozotocin
Streptozotocin (STZ) is derived from the bacteria
Streptomyces achromogenes. As another glucose analogue, it
is taken into pancreatic cells through GLUT2, where it accumulates. From there, the methyl-nitrosourea moiety of the
STZ molecule alkylates the DNA inside the cell and produces cytotoxic damage [21].STZ is more commonly used
than alloxan due to its greater chemical stability, ease of
administration, and irreversible pancreatic damage. The next
page lists several of the most common methods of STZ diabetes induction with the most commonly used dosage of
STZ, although there are a range of usable concentrations in
literature. In addition, female animals may need higher STZ
doses compared to male animals, as females are less sensitive to STZ.Depending on the method, rodent models can be
created for T1D or T2DM.The background species for this
method is generally C57BL/6J mice and Sprague-Dawley or
Wistar rats, and specics of induction method protocols are
summarized in Table19.1 [21, 25].
Single-Dose STZ
Some researchers prefer using a single high dose of STZ,
injected intraperitoneally, to induce diabetes in rodents. This
is popular due to the ease of the procedure; however, the
higher dose has less success in converting the rodents into
the diabetic state and higher toxicity leading to higher mortality. For this method, the STZ dosage for mice is 200mg/
kg at a 20 mg/mL concentration in 50 mM sodium citrate
buffer (pH4.5), and the dosage for rats is 65 mg/kg at a
32.5mg/mL concentration in 50 mM sodium citrate buffer.
These animals should be fasted before the procedure (4 h
without food for mice, 8h without food for rats) [25].
Multiple-Dose STZ
Another method of STZ induction is through a multiple-dose
administration. This is performed through multiple intraperitoneal injections for ve consecutive days of lower doses of
STZ.This method has higher conversion rates and yields less
reversion back into nondiabetic states. The pathophysiological
mechanism of pancreatic islet destruction is also more faithful
to the human condition. Due to the lower but repeated doses of
STZ, the animals have a delayed onset of hyperglycemia and
decreased severity of pancreatic cell destruction. However,
due to the partial damage from STZ, an immune response is
mounted in the pancreas, which leads to total destruction of
the pancreatic islets. This pathway of pancreatic damage is
more faithful to the process of T1D acquisition in humans
compared to the single-dose induction method [24, 25].
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