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M. A. Niewczas and H. Shah
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Experimental Animal Models
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inResearch: Diabetes andImpaired Wound Healing
MauricioContreras andEnyaWang
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 Ofce of Laboratory Animal Welfare PPE Personal protective equipment SC Subcutaneous SURG Surgical T2D Type 2 diabetes USDA US Department of Agriculture
Animal Models: Background andSignicance
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 signi­cant 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
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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 fortheCare andUse ofLaboratory 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 pro­ciencies 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 scientically, technically, and humanely appropriate. The Guide is also intended to assist investigators in fullling their obligation to plan and conduct animal experiments in accordance with the highest scientic, humane, and ethical principles. Recommendations in the Guide are based on published data, scientic 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 inResearch: Diabetes andImpaired Wound Healing
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program, recognizing that the concept and application of performance standards, in accordance with goals, outcomes, and considerations dened 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 ofHealth Policy Pertaining theUse ofAnimals inResearch
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 denition of animal to include all vertebrates. The revised policy also required an institution to submit an assurance statement to the Ofce for Protection from Research Risks (OPRR), now the Ofce 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 andUse 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 scientic and nonscientic interests. IACUCs derive their authority from the law.
There has been an evolution in the ways that IACUCs fullled 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 invitro alternatives to the production of monoclonal antibodies. The IACUC community has also gained a greater understanding of and appreciation for the role of nonafliated and nonscientic 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 signicantly. 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 andConsiderations intheSelection Process ofAnimal 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, rene­ment, 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.
Renement: Refers to modications 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 renement, 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 sci­entic 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.
Renement 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 signicant 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 justication 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 toHelp Determine Pain andDistress 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 scientically justi­ed 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 scientically justied point at which pain or distress in an experimental animal can be prevented, terminated, or relieved while meeting the sci­entic aims and objectives of the research study. By law, the institution must annually report all category E procedures to the USDA and include a scientic justication supporting the IACUC’s decision to approve them. It is important for the information on category E procedures to be complete and accurate.
Recommendations andStrategies inChoosing aParticular 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 efcacy 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 denition, 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 immunodecient 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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Animal Models ofDiabetes: Background andSignicance
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 inuence 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 beneted 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 tissue­specic 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 Ofce of Laboratory Animal Welfare. The following sections will discuss specic 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 andStreptozotocin 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 spe­cically 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 [2325]. 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 230mg nicotinamide 15min
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
4weeks after skin graft
before STZ induction. High variability in effective reagent dosages
3weeks 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 specic 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 50mg/kg and for injection into rats, the dosage of alloxan is 70mg/kg [24].
Some studies report lower mortality rates when animals are given a 50mg/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 10days, with glucose concentrations >150mg 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 specics of induction method protocols are summarized in Table19.1 [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 200mg/ kg at a 20 mg/mL concentration in 50 mM sodium citrate buffer (pH4.5), and the dosage for rats is 65 mg/kg at a
32.5mg/mL concentration in 50 mM sodium citrate buffer. These animals should be fasted before the procedure (4 h without food for mice, 8h 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].