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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_896_Библиотеки_им_академика_М_И_Перельмана
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M. Contreras and E. Wang
Humanized Skin-Graft STZ Mouse Model
For studying conditions of diabetic skin, such as diabetic
foot ulcerations, the humanized skin graft mouse model can
be a good option. The humanized skin graft mouse model
describes STZ-induced diabetic immunocompromised mice
with human skin grafts.
To replicate this model, researchers start with a mouse of
the background Foxn1nu: lack of innate immune response is
needed to forgo host rejection response. A human skin graft
is obtained from the discarded tissues from cosmetic surgeries. This skin is taken with a dermatome at split thickness
(8 mm)—split-thickness skin is preferred to full-thickness
skin as the former leads to better integration. The mouse is
then grafted with human skin along the dorsum and left to
heal for several weeks, with sharp debridement as necessary
to remove scabs and nonintegrated graft sections. After the
skin has integrated with the body of the mouse, diabetes is
then induced into the mice through the multiple-dose STZ
administration method at 4weeks after grafting. One week
later, diabetic state is conrmed in these mice through blood
glucose measurement [26].
The full process of creating this model is illustrated in
Fig.19.3. Of note, this model involves complex and delicate
processes over 5 weeks. Handling immunodecient mice
requires sterile environments, but this model requires in addition sterilized human skin, sterile surgical supplies, and for all
other tools to be pathogen-free. In the 4weeks after grafting,
the mice must be monitored for skin integration state, with
debridement when necessary. After diabetic induction, these
immunodecient mice tend to have more extreme diabetic
complications. Overall, it is important to carefully monitor
these animals during their procedures due to their immunodeciency, skin graft integration state, and diabetic state.
The resultant animal model supports living human skin
while also having a diabetic circulatory and immune system.
The combination of these factors is ideal, as this model replicates invivo diabetic human skin and opens new possibilities for studying diabetic skin morphology and new
experimental treatments. Researchers have already used this
model to study diabetic foot ulcerations and to test novel
therapies, as its modeling of human skin architecture with
diabetic system has greater likeness to diabetic human skin
and increased preclinical signicance [27].
Type 2 Diabetes STZ Rat Models
The previous methods describe induction of T1D states into
rodent models. However, only 9% of the global diabetic
human population has type 1 diabetes. The other 91% of diabetics have type 2 diabetes, which is characterized by insulin
deciency and insulin resistance from an underperforming
pancreas. The following T2DM models are created through a
few modications to the protocol for a single-dose STZ
induction in rodents. Typically, these methods are used with
Sprague-Dawley or Wistar rats, but the procedures can also
be used with C7BL5/6 mice with appropriate adjustments in
STZ dosage.
Nicotinamide STZ Model
In this model, rodents are given nicotinamide, a protective
agent of beta cells, 15min prior to STZ injection. By administering both chemicals, the nicotinamide will alleviate the
effect of streptozotocin on the pancreatic islet cells and
allows for partial function of the pancreas and lowered levels
of insulin production [25]. The insulin deciency caused by
this induction method allows for the animal to replicate
aspects of type 2 diabetes.
Fig. 19.3 Schematic representation of human skin grafting and subsequent streptozotocin induction
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Researchers should dissolve nicotinamide in 0.9% sodium
chloride, which can be injected intraperitoneally to the rodent
at a dosage of 1mL/kg body weight. Following a 15-min waiting period following the nicotinamide injection, the rodent can
then be injected with STZ following the previously described
method for single-dose induction. Of note, protocols in literature for the nicotinamide STZ rat model vary in glycemic outcomes and reagent dosages—nicotinamide dosages range from
60 to 230mg, and STZ dosages ranges from 32.5 to 60mg.
Research teams may need to optimize their nicotinamide and
STZ ratios prior to starting experimental treatments [25].
High-Fat Diet STZ Model
The rats of the high-fat diet (HFD) STZ model are placed on
a high-fat diet for 3weeks prior to a low-dose STZ injection.
The HFD should have around 60% of all calories from fat
and cause hyperglycemia and insulin resistance. The STZ
acts to create insulin deciency in addition to the metabolic
abnormalities caused by HFD.The dosage of STZ is 1.0mL/
kg bodyweight of 40mg STZ dissolved in 50mM sodium
citrate buffer [ 25]. This model better describes T2DM compared to the nicotinamide STZ rat model, as it confers both
insulin deciency of diabetes and the insulin resistance and
obesity that accompany type 2 diabetics.
Conrmation ofSTZ-Induced Diabetes
To conrm diabetes induction, a blood glucose test can be
performed on day 10 after STZ administration. If the glucose
concentration is higher than 150mg/dL, the animal is considered diabetic. These animals can be used for studies
examining early-stage diabetes. If the animal is not diabetic,
a retest of the blood glucose concentration can be performed
at day 21 after STZ administration. For mild hyperglycemia,
the glucose concentration should be >150mg/dL.For rodents
injected with STZ following the single-dose or multipledose method, the blood glucose concentration will range
between 300 and 600mg/dL.
Spontaneous Autoimmune Diabetes Models
Models of spontaneous autoimmune diabetes are useful for
studying mechanisms of type 1 diabetes. These animals
share similar pathways of diabetes onset: generally, there is
an injury to the pancreas within their rst month of age,
which leads to autoimmune cascade. In this immune inltration, also referred to as insulitis, T-cells begin to attack the
pancreas and cause the pancreas to completely lose insulin
production [15, 22]. In this section, the nonobese diabetic
mouse, AKITA mouse, biobreeding, and LED IDDM diabetic rat model will be discussed.
Drawbacks to these models include greater expense of
breeding and the need for careful insulin monitoring from an
early age as these mice are diabetic earlier than other models.
However, benets to this model include the ability for
researchers to study the early onset of type 1 diabetes and the
immunological causes and the incidence of diabetes in both
male and female animals. In addition, there is no added procedure for diabetes induction that must be performed by
researchers.
Nonobese Diabetic Mouse Model
Nonobese diabetic (NOD) mouse is one of the most regularly utilized models for investigations of type 1 diabetes,
among a variety of other autoimmune diseases. This species
develops insulitis by its rst month and has pancreatic islets
inltrated by immune cells, primarily CD4+ and CD8+
T-cells. Type 1 diabetes complications begin at 10–14weeks,
with the animal losing weight rapidly. The genetic and
immunological traits of this model resemble the human condition, including MHC II proteins of the NOD that do not
properly modulate immune response. Due to the similarity of
immunological causes of proles, the NOD mouse is well
suited for studying the pathophysiology of type 1 diabetes.
However, drawbacks include its autoimmune condition,
which requires sterile conditions while handling and comorbidities to the mouse. Autoimmune disorders present in the
NOD mouse include thyroiditis, which is present in human
T1D conditions, and other conditions such as Sjögren’s syndrome and thyroiditis, which are not [15, 22].
Biobreeding Diabetes-Prone Rat (BB Rat)
The BB rat is a diabetes-prone outbred branch of the Wistar
rat. These rats develop spontaneous autoimmune diabetes
early on, with insulitis and pancreatic abnormalities, including
overexpression of interferon alpha and MHC class 1 proteins,
and insulitis in the next 2months. Because of the early onset,
these rats must be closely monitored with insulin throughout
their life, and they develop extreme diabetic conditions. This
model is popular for studies involving islet transplantation,
diabetic neuropathy, and interventional studies. In addition,
BB rats develop a Th1-type lymphocyte- driven insulitis,
which is distinct from NOD mice and more similar morphologically to T1D in humans [25]. However, one drawback is
that these rats have a mutation in the GTPase Ian5, which
causes the diabetes in this animal and limits genetic studies of
T1D causes [28]. This mutation also causes lymphopenia in
the rats, with reduced levels of CD8+, CD4+, and ART2+
T-cells, which makes these animals immune- decient and
may limit the potential of immunological studies.
Lewis-Insulin-Dependent Diabetes Mellitus
(LEW IDDM) Rat Model
These rats originate from the LEW.1AR1 strain and develop
diabetes in 2–3 months. Diabetic rats have an autosomal
recessive mutation which causes their phenotype, with a
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M. Contreras and E. Wang
60% incomplete penetrance in mutants [28]. Both sexes
develop the diabetic syndrome equally, with 20–60% incidence. The beta cells of the LEW IDDM undergo apoptosis
as a result of proinammatory cytokines, causing diabetes.
The prediabetic onset period of diabetes is dened and lasts
for 1week, which can be useful for researchers studying islet
inltration. In addition, these rats have a high survival rate
after diabetes onset, making them suitable for studying longterm diabetic complications.
Genetically Induced Models
Rodents with impaired leptin pathways are commonly used
as models for type 2 diabetes. Leptin is a protein that is
responsible for inducing satiation feelings and limiting
hunger in the brain. Without proper leptin signaling, animals have hyperphagia which leads to obesity and the
development of insulin resistance that is characteristic of
type 2 diabetes. These models are sometimes referred to as
monogenic models, as their phenotype stems from a single
mutation in a single gene on one or both chromosomes.
There is some criticism of monogenic models, as obesity
and T2DM have multifaceted causes. Current research on
genetically induced diabetic models focus on creating polygenic models instead, which may be better to replicate conditions behind obesity and type 2 diabetes. These polygenic
models, such as the NONcNZO10, show promising results
especially in similarities to human diabetic wound healing
processes; however, more testing needs to be done to validate these new models [29].
These monogenic models include Lep db/db mice and
Zucker diabetic fatty rats, which are decient in the leptin receptor, and Lep ob/ob mice, which are decient in functional leptin.
In addition, the AKITA mouse, a model for type 1 diabetes with
mutations in insulin and proinsulin, will be discussed.
Genetically induced diabetes models are more expensive
peranimal compared to other models, and they have lower life
expectancy due to their long-term diabetic state. However, these
models share similar metabolic phenotypes to human type 2
diabetes, including insulin resistance, hyperglycemia, and obesity, as well as their diabetic complications. In particular, the
Lep db/db mouse, Zucker diabetic fatty rats, and the AKITA
mouse are the models of choice in scientic literature.
Lepr db/db Mice
These mice are homozygous for a recessive spontaneous
mutation in the db gene, leading to mutations in their leptin
receptors. The nonfunctioning receptor leads to oversecretion of insulin, which in turn causes obesity and then hyperglycemia, beta-cell dysfunction, insulin resistance, and
dyslipidemia. This model is relevant to wound healing, as it
is the most commonly used type 2 diabetes model that dis-
plays impaired wound healing in not only cuts but open
wounds. Of other commonly used diabetic mouse models
such as the AKITA mouse or STZ-induced mouse, the Lepr
db/db mouse model shows greater impairment of wound
healing for both cuts and open wounds [31].
In particular, this model addresses many aws that other
mouse models contain—including mechanism of wound closure. In wild-type mice, the primary response to injury is to
heal through contraction of the skin, through the muscle panniculus carnosus, which humans do not have. The db/db mouse
heals wounds primarily through reepithelialization, which is
the natural wound response in humans. This is especially true
when wounds are inicted on the dorsum of these mice. The
wound environment of this model additionally allows for distinct aspects of ulceration healing to be observed including collagen deposition and granulation formation [30–32].
Lep ob/ob Mice
These mice are homozygous for a recessive mutation in the
leptin gene, leading to nonfunctional leptin. This model is
obese at 1month and has hyperphagia, hyperglycemia, and
hyperinsulinemia. Of note, the Lep ob/ob genotype in the
C57BL/KS strain has high mortality rates.
Although the Lepr db/db mice and the Lep ob/ob mice
both target the leptin pathway, they show metabolic differences. Glucose tolerance is impaired more for db/db mice
with lower blood insulin levels, while ob/ob mice have
higher blood insulin levels indicating higher insulin resistance. Lep ob/ob mice are more severely obese than Lepr db/
db mice. The ob/ob mice have more adipose fat, while db/db
mice show impaired adipocyte differentiation. These are
some differences, among others, that must be considered
while choosing between the two models [32].
Zucker Diabetic Fatty (ZDF) Rats
These rats are another popular model for type 2 diabetes studies. These rats have a homozygous mutation in the leptin
receptor (fa/fa), which leads to hyperphagia, hyperinsulinemia,
high levels of triglycerides and cholesterol, high blood pressure, insulin resistance, and impaired glucose tolerance. These
conditions begin to develop from the rst month and male rats
become overtly diabetic at 2months. Female ZDF rats, in contrast, do not become overtly diabetic. Male ZDF rats also
develop infertility at high rates, which is an obstacle for breeding the species, but can be an area of interest for researchers
studying testosterone agonists [33].
Overall, the ZDF rat is widely used in studies of type 2
diabetes due to the complications of lipoapoptosis, mild
hypertension, insulin resistance, and impaired glucose tolerance. In addition, the ZDF rat shares similar pancreatic morphology to humans, with disrupted islet architecture, and
similar immunology with increased B-cell degranulation and
death [28, 33, 34].
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Akita Mouse
The Akita mouse originates from the C57BL/6NSlc strain
in Akita, Japan. These mice have a spontaneous mutation
in Ins2 which leads to misfolded insulin. These misfolded
insulins create improper proinsulin processing and ER
stress in the pancreatic beta cells, which leads to their
apoptosis. This causes severe type 1 diabetes conditions in
the mice at 1month of age. The Akita mouse has validated
diabetic sympathetic autonomic neuropathy, although it
has not been validated by antineuropathic drugs [28].
However, this model has many benets over the STZinduced mice, including a lack of beta-cell mass which
makes this model more suitable for islet cell transplantation. In addition, this model is suitable for studying diabetic nephropathy [33].
Other Small Animal Models ofDiabetes
The models listed above are the most commonly used rodent
models of diabetes. However, in this section, we will give a
quick overview of several less commonly used models for
diabetes.
Surgical removal of part of the pancreas can be used to
create diabetic animal models in any species. It is not commonly used in mice and rats as this procedure is more effortintensive, invasive for the animal, and less effective in
inducing hyperglycemia and blood insulin changes compared to other procedures or breeds. However, this method
may be useful for investigating pancreas regeneration or
used in combination with administration of another diabetogenic chemical [35].
Viruses can also be used to induce diabetes in animals.
Examples include the Kilham rat virus, the Coxsackie B
virus, and the encephalomyocarditis virus. These models can
be used to study the role of viruses in the development of
type 1 diabetes [15].
Small animals other than mice and rats can also be used
for diabetic investigations. Both African hamsters and
Chinese hamsters have hereditary diabetes mellitus. Tucotucos, spiny mice, and the sand rat are also considered diabetic or diabetes-prone.
In addition, nonmammalian species that share similar
pathways to the human may be used for metabolic studies.
Benets to the use of these species include less rigorous
IACUC regulations and larger experimental numbers. These
include the silkworm, Bombyx mori, which shares glucose
regulation pathways with humans. Zebrash are another diabetic animal model, and they feature shared lipid metabolic
pathways, as well as diet-fueled insulin resistance and glucose intolerance [22].
Table 19.2
shows a comprehensive summary of nonUSDA covered species models of diabetic induction and
their advantages and limitations.
Table 19.2 Summary of non-USDA covered species
Induction method Animal model Description Advantages and limitations
Chemical ablation Alloxan Alloxan injection i.v. in any
Single-dose STZ Single high-dose STZ injection
Multiple-dose STZ Low-dose STZ injections in ve
Humanized skin graft
mouse
Nicotinamide-STZ Nicotinamide injected prior to
High-fat diet STZ High-fat diet (HFD) given for
Spontaneous
autoimmune diabetes
Nonobese diabetic
mouse
animal to destroy beta cells
through free radicals and induce
T1D state
i.p. in mice and rats to destroy
beta cells through DNA damage
to replicate T1D
consecutive days to inict
pancreatic beta-cell damage
Employs multiple-dose STZ
method with Foxn1nu mice with
human skin grafted
STZ induction for partial insulin
deciency for T2DM models
3weeks prior to low-dose STZ
injection for insulin resistance
Abnormalities in MHC class II
proteins cause autoimmune
reactions against islet cells,
leading to type 1 diabetes
Used to study T1D outcomes; however alloxan has high
toxicity to host and small windows of efcacy and is
difcult to work with due to chemical instability
Used to study T1D outcomes; STZ is more chemically
stable than alloxan, and STZ induction is easier than
alloxan induction. However, single high doses have
greater toxicity to animals compared to other methods
Benets include lowered mortality, higher conversion,
and an autoimmune response that reects human T1D
acquisition. However, more injections are more
effort-intensive
Ideal for studying diabetic human skin as an invivo
model. Benets include human skin architecture and
diabetic host. Limitations include difculty of model
creation centering around immunodeciency
Used for studies of type 2 diabetes and is less expensive
than other models of T2DM such as genetic models.
However, HFD-STZ is more representative
More representative of T2DM with insulin resistance
and insulin deciency. However, this model requires
3weeks of high-fat diet prior to experimental use
Widely used model for T1D, as the MHC II
susceptibilities and the immune cell inltration closely
resemble human pathology. However, mice are
immunodecient with unrelated comorbidities
(continued)
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Table 19.2 (continued)
Induction method Animal model Description Advantages and limitations
Biobreeding rat Diabetes-prone branch of Wistar
Lew IDDM rat Autosomal recessive mutation
Genetically induced Lepr db/db Recessive mutation in the leptin
Lep ob/ob Recessive mutation in leptin
Zucker diabetic fatty
rat
AKITA mouse Mutation in insulin causes ER
Surgical Pancreatectomy in
wild-type animals
Viral Wild-type Viruses used to induce diabetes in
Hereditary African hamsters,
Chinese hamsters,
tuco-tucos, spiny
mice, sand rat
High-fat diet Bombyx mori The silkworm can be fed a
Zebrash Zebrash fed a high-fat or
rats with early-onset insulitis from
overexpression of IFNy and MHC
I leading to T1D
leading to diabetes mellitus in
LEW.1AR1 rats
receptor leading to obesity,
hyperglycemia, and T2D
leading to hyperphagia, obesity,
and hyperglycemia
Mutation in leptin receptors leads
to obesity, insulin resistance,
dyslipidemia, and T2D
stress leading to beta-cell
apoptosis and severe T1D
Removal of a small part of the
pancreas to induce insulin
deciency
animals, including Kilham rat,
Cox B, and encephalomyocarditis
The hamsters have hereditary
diabetes mellitus, while the other
species are diabetes-prone
high-fat or high-sucrose diet to
alter their glucose regulation
high-sugar diet can lead to insulin
resistance and glucose intolerance
Popular model for T1D, well suited for studying
insulitis, islet transplantation, diabetic neuropathy, and
interventions. Genetic and immunological studies are
limited due to mutations and lymphopenia
Benets include high survival rate, allowing for diabetic
complication studies, as well as a dened prediabetic
state, allowing for diabetic onset studies. Limitations
include incomplete penetrance of diabetic state and
lower diabetic incidence
Widely used spontaneous T2D model which is suited
for wound healing studies. Db/db mouse skin is similar
to humans, showing impaired healing,
re-epithelialization process, and collagen deposition
and granulation. Limitations include high costs
peranimal
Another spontaneous model for T2D, Lep ob/ob has
similar metabolic phenotypes to humans; however, the
mouse does not have beta-cell dysfunction
Spontaneous T2D model that shares T2D metabolic
phenotypes, as well as similar pancreatic morphology.
ZDF rats can also be studied for dyslipidemia as well as
infertility. Limitations include sex differences
Spontaneous T1D model with severe T1D conditions at
an early age. Suitable for neuropathy, ER stress, islet
cell transplantation, and diabetic nephropathy studies
Suitable for pancreatic regeneration studies and can be
used in addition to a diabetogenic chemical. Alone,
pancreatectomy is less effective in inducing
hyperglycemia and insulin changes and is not the
preferred procedure for rodent models of diabetes
Can be used to convert animals quickly and is less
expensive than genetically diabetic animals. Can also
be used to study the role of viruses in T1D acquisition
Advantages to these models include spontaneous
diabetes development. Limitations include limited use
in literature, more difcult acquisition, and less
validated phenotypes and genetic background
Silkworms can be used to study glucose regulation, as
they share pathways with humans. They are also not
rigorously regulated by animal use committees,
allowing for larger animal numbers. However, the other
pathways are not similar to human diabetic conditions
Zebrash are a commonly used model organism, with a
well-researched genetic background. These sh share
similar lipid metabolic pathways to humans, as well as
insulin signaling and glucose regulations pathways.
However, the physiology of zebrash differs more from
humans compared to rodent models
M. Contreras and E. Wang
USDA Covered Species
USDA covered species is a term that refers to animals whose
care is governed by the Animal Welfare Act. In addition to
dogs, cats, and nonhuman primates, USDA covered species
currently include guinea pigs, hamsters, rabbits, and any other
warm-blooded animal with the exception of mice of the genus
Mus, rats of the genus Rattus, and birds. This also excludes
“cold-blooded” animals such as sh, reptiles, and amphibians.
Although there are several USDA covered species models
of type 2 diabetes which are commonly used, such as (a)
swine, (b) rabbit, (c) nonhuman primate, and (d) canine
models, we will describe the models we use and are experienced with. Nonhuman primate and canine models pose
increased regulatory burden and restrictions that require specialized animal research facilities that could provide the care
and oversight these species need, in order to fulll USDA
regulations.
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Rabbit Diabetes Model
Although rabbits and hares have long been classied as close
relatives of rodents (mice, rats, squirrels), a new study has
concluded that they are more closely related to primates [36],
and under USDA animal classication, rabbits are considered covered species.
The alloxan diabetes induction rabbit model has been
used for a very long time, in spite of its challenges (high
associated mortality). Therefore, it is critically important to
compensate for a potentially lethal hypoglycemic phase that
inevitably follows alloxan administration in order to avoid
any animal mortality. Monitoring rabbit behavior and blood
glucose level (BGL) testing for up to 36h post induction,
followed by a preventive glucose administration based on
rabbits’ individual needs, should be mandatory, as it seems
to be an effective way to keep animal mortality rates to a
minimum [37]. In this model, there seems to be a small number of islet cells in the pancreas, a change similar to that seen
clinically in patients with type 1 diabetes [38]. Furthermore,
alloxan-induced diabetes has shown to accelerate the development of atherosclerotic lesions in the rabbit [39], even
though there are undeniable differences in the atherosclerotic
process in humans when compared to rabbits. Previous studies suggests that diabetes rapidly creates a relatively hypoxic
oxygen microenvironment within the arterial wall. This rapid
induction of global arterial wall hypoxia may contribute to
the diffuse, severe, and accelerated form of atherosclerotic
vascular occlusive disease seen with diabetes [40].
The major blood vessels in the rabbit ear lie in a thin
skin sheet and can be easily viewed. The study of this feature has been devoted to the growth and differentiation of
blood vessels during wound healing and vascular physiology [41]. Although a rabbit ear ischemic wound model has
been described in the past, [42, 43] this model resulted in a
much longer ischemic time and has not been adopted
widely, due to the requirement of a surgical microscope,
electrocautery, and extensive skin interruption. The wide
skin incision also makes the ear vulnerable to infection.
However, a minimally invasive technique to create an ischemic ear model for wound healing studies in animals with
weakened tolerance such as aging or diabetes has also been
described by Chen [44]. The operation in this model is simple, without the use of a surgical microscope or electrocautery, the skin continuity is preserved, and a longer ischemic
time is achieved. Thus, the hyperglycemic rabbit ear wound
provides a suitable preclinical animal model to evaluate
new therapeutic modalities to improve healing and promote
wound closure. This is a model which more closely resembles the human condition in comparison to other rodent
models. Furthermore, there are extensive studies using this
model with histological analysis providing insight into the
neovasculature in healing wounds [45–47]. This investiga-
tive approach is relevant as a central pathological process in
nonhealing diabetic ulceration where vascular supply is
impaired [48].
Diabetic Rabbit Ear Model ofImpaired
Wound Healing
We have designed the following protocol for alloxan diabetes induction in New Zealand White (NZW) rabbits,
with a high degree of success of diabetic conversion and
eliminating the high mortality rate associated with this
model. Furthermore, our diabetic rabbit ear model incorporates the components of neuropathy and blood vessel
disease that manifest in chronic nonhealing ulcers.
Therefore, the rabbit ear can serve as an appropriate
model for studying diabetic wound healing caused by
nerve and blood vessel disease.
Animal Procurement: It is critical to acquire animals from
a Class A vendor, with a strong reputation of providing animals that are in the best health and that are evaluated using a
comprehensive health monitoring program, which includes
bacteriology, parasitology, and pathology, as well as serol-
ogy. The rabbit colonies should also be screened quarterly
for Helicobacter using PCR and tested annually for infec-
tious agents. Thus, the NZW rabbit strain should be main-
tained under a VAF/Plus® health status that indicates that the
colony has been tested for, and is free of, an extensive list of
viruses and other pathogens.
Acclimation Period: NZW rabbits, weighing 3–4 kg,
4 months old, and both male and female, are allowed to
acclimate for 7 days. Although the USDA requirement is
48h of acclimation, we like to provide additional time for
animal to distress from shipping and get used to their new
environment and being handled by new ARF staff.
Baseline Measurements: After acclimatization, rabbits are
initially fasted overnight (nothing by mouth/NPO for
10–12h). The following morning, initial baseline measurements are made.
1. Medical hyperspectral imaging (MHSI): Rabbits are
weighed and sedated with acepromazine (0.75 mg/kg
i.m.). Rabbit ears are shaved and depilatory cream is used
to remove any excess hair.
2. Blood draw: First blood draw (5cc) through ear marginal
vein is done right after MHSI while the animals are still
sedated.
3. Blood glucose (BG) check: Using commercially avail-
able glucometer, BG is measured using 1μL of blood
(generally blood drop left from blood draw).
4. Hemoglobin A1C (HbA1C): Using commercially avail-
able HbA1C monitor, HbA1C is measured using 5μL of
blood (generally blood drop left from blood draw).
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Following these initial baseline measurements, rabbits are
allowed to recover from sedation and will receive Gatorade
instead of water for 72-h period. This is to ensure that they get
used to the taste of Gatorade, which they will have to drink postalloxan injection. It is extremely important that they receive
high glucose content following alloxan because alloxan administration leads to severe hypoglycemia within the rst 24–48h
and their stress response post-alloxan injection is minimized.
Alloxan Administration: Day 0: 48h following baseline
measurements, animals are weighed and sedated with
acepromazine (0.75mg/kg i.m.). The alloxan solution is prepared fresh and ltered-sterilized just prior to animal administration (100mg/kg). Alloxan is dissolved in sterile normal
saline to achieve a 5% concentration (W/V) and used right
away, since it is known to be highly unstable. The syringe is
then loaded to an automated infusion pump set up to dispense the total volume over 1min, through a 22g angiocatheter previously placed into the marginal ear vein. In addition,
to decrease the risk of nephrotoxicity (hyperuricemia), an i.v.
injection of 0.9% saline (4–5mL/kg) at an infusion rate of
1mL/min is administered. To mitigate any distress or pain
from the alloxan injection, meloxicam (0.308mg/kgs.c.), a
NSAID, is administered to the rabbits.
To counteract the hypoglycemic effect of alloxan, 4 and
8 h after alloxan injection, 10 mL of 5% dextrose i.v. is
administered through the i.v. buttery catheter left in place
from the previous alloxan injection. This catheter is removed
after the 8-h dextrose i.v. injection. At 12-h post-alloxan,
10 mL of 5% dextrose is administered s.c. in their back
(intrascapular region). Rabbits continue to receive Gatorade
at lib for another 48h.
Twenty-four hours after alloxan injection, rabbits receive a
second dose of meloxicam at (0.308 mg/kg) s.c. Because
alloxan is also known to cause distress, animals should be
closely observed and monitored throughout the diabetes induction period for any signs of discomfort and distress including a
rise in temperature, absence or reduced urine and feces, ketones
in the urine, hyper-/hypoglycemia, reduced water and food
intake, and decreased activity, and the normal rectal temperature
is between 38 and 39°C (100.4–103.8 °F). Measures of relief
should be considered, such as increasing or decreasing the dose
of insulin, administering uids (lactated Ringer’s solution i.v.)
or dextrose (in case hypoglycemic), and administering meloxicam (in case temperature is >103.8°F). Additionally, Diet Gel
Criticare could be given ad lib to support nourishment.
Personal Protective Equipment (PPE): Any personnel
handling alloxan will require using PPE and following strict
rules for hazardous substance handling and administration
during and during the 72h post-alloxan administration. The
PPE includes a disposable gown, hair bonnet, and face
shield. An absorbent pad/chuck is placed under the cage so
that urine does not splash around the cage. PPE and the waste
collected from the cage are disposed of in a biohazard container for later disposal/incineration.
Day 2: Post-alloxan diabetes conrmation—Rabbits are
placed NPO by staff for 4h prior to BGL determination and
Gatorade is switched to regular water. Rabbits exhibiting a
BGL >250 are considered diabetic (this value was chosen
based on previous studies). Only rabbits that do not become
diabetic receive a second dose of alloxan (100mg/kg i.v.),
following the same procedure described above. In our experience, more than 95% of the animals turn diabetic with one
single alloxan dose.
Insulin Administration Considerations: Rabbits that have
become diabetic (BGL >250mg/dL) are weighed and receive
long-lasting insulin (starting dose: 0.5unit/kgs.c. of Lantus
insulin glargine). Rectal temperature is measured every day
for the rst week after alloxan and meloxicam is administered PRN (if temperature is >103.8 °F). Dietary supplements are given such as ad lib Diet Gel CritiCare to ensure
proper nourishment until the end of the study. Day 3, 5, 7, or
9: BGL and ketone check: BGLs are checked at the same
time in the morning and afternoon, up to day 7 or 9 using the
same glucometer. Urine ketones should be checked also in
the morning up to day 7; however, should a rabbit show any
signs of distress, then the BGLs and ketones are monitored
every day until the rabbit is stabilized. If hyperglycemia
(BG > 500 mg/dL) or hypoglycemia (BG < 250 mg/dL)
occurs, insulin dose should be adjusted (Table19.3). In addition to BGL and ketone check, rabbits are closely observed
throughout the study period for any visual signs of discomfort and distress including no or reduced urine and feces,
reduced water and food intake, and reduced activity.
Once stable hyperglycemia has been established, BGLs
are measured once a day, same time in the morning, using the
same blood glucometer. The following BGLs will help determine long-acting insulin (Lantus) to be administered to each
individual rabbit [49].
Neurovascular Surgical Injury and Skin Wound Punch
Biopsy: All surgeries are performed 30 days following
alloxan induction and the hyperglycemia state has been
instituted.
Animals are initially sedated with acepromazine (0.75mg/
kg i.m.). Rabbit ears are shaved and a depilatory cream is used
to remove any excess hair and MHSI performed. Animals are
then fully anesthetized with a combination of ketamine
(35 mg/kg i.m.) and xylazine (2.5 mg/kg i.m.). Blood draw
(5cc) is drawn from a 22g catheter previously placed in the
saphenous vein. An i.v. drip with LRs will be maintained during the surgical procedure at a rate of 6–8mL/h.
Table 19.3 Lantus insulin (long-acting) dosing chart
Blood glucose level (BGL) Lantus insulin administered
BGL 350–450mg/dL 1U/kg
BGL 450–550mg/dL 2U/kg
BGL 550–600mg/dL 3U/kg
BGL >600mg/dL 4U/kg
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353
Rabbits are E.T. intubated and isourane (1.5–2%) and
oxygen (100% O2 at 4 L/min) mixture is administered to
maintain anesthesia throughout the surgical procedure.
Rabbit ears are cleaned with betadine solution and isopropyl
alcohol (three times). A sterile eld is maintained around the
ears with sterile drapes. The rabbit ear has three main vascular pedicles—large central, medium-sized rostral, and small
caudal—which are easily visualized through the dorsal ear
skin. Once identied, they are inltrated subcutaneously
with 1% lidocaine (0.25mL/incision site) at each of the three
surgical sites, and doing so decreases vasospasm from surgical dissection, maintaining the vessels distended and patent.
A 1cm small incision down through the perichondrium will
be made 1cm distal to the base of the experimental ear, leaving the three main vascular pedicles intact. In the experimental ear, to create ischemia, the rostral and the central arteries
are divided with the preservation of the caudal artery and all
the three veins. Since the veins are maintained intact, there is
no venous congestion. By ligating the rostral and central
arteries, the entire dermal circulation to the ear will be interrupted. The central artery is ligated and transected using 5-0
silk and the rostral artery is ligated using 7-0 silk as well.
This will create an ischemic wound with local oxygen tensions below 40mmHg and no venous congestion. In addition
to arterial ligation, central and rostral nerves are transected.
Skin incisions are sutured (individual stitches) per incision
with 5-0 nylon monolament suture. Following ligation of
arteries and/or nerves and skin closure, four full-thickness
circular wounds are created using a 6mm punch biopsy disposable device. Using a scalpel blade (#15), the punch biopsies are etched out and the overlying skin is removed
(Fig.19.4). The intact cartilage maintains the wound open,
minimizing tissue contracture to less than 3%, allowing the
wound to heal by new tissue formation.
abc
def
Fig. 19.4 (a) Schematic representation of alloxan IV injection, (b)
arterial and venous supply and innervation in the rabbit ear, (c) ligation
and transection of the rostral and central arteries and nerves, (d) four
individual ear skin wound creation (6mm punch biopsy) p/ear, (e) bilateral ear wounds (treated vs nontreated), and (f) wound measurements
for healing progression
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M. Contreras and E. Wang
Surgical incisions are covered with the triple antibiotic,
bacitracin ointment (0.1–0.2 mL), and non-adhering bandages. E-collar is placed around the neck to prevent the rabbits from removing the bandages and scratching the wounds.
Either Tegaderm dressing (control), different treatment
dressings, or new therapeutic balms are to be tested.
Protective E-collars are kept for the entire period from surgery to euthanasia.
A BGL is done at the end of the surgery. If BGL is
<150 mg/dL, then 5 mL of 5% dextrose is administered
through the 22 g angiocatheter previously placed in the
saphenous vein. When rabbits recover from anesthesia and
are awake, the ET tube is removed so they can breathe on
their own. The 22g i.v. catheter is then removed. A single
dose of buprenorphine (0.01–0.05 mg/kg) is administered
s.c., and a fentanyl dermal patch (4μg/kg) is applied to a
previously shaved portion on the back, providing analgesia
for the next 72h, maintaining the animal comfortable and
without experiencing any pain. Once vital signs are stable,
animals are returned to their individual cage and continued
to be monitored for the duration of the study.
Euthanasia and Tissue Harvest: At the end of the experimental study, animals will be initially sedated with acepromazine (0.75 mg/kg i.m.) and MHSI performed. Animals are
then fully anesthetized with a combination of ketamine
(35mg/kg i.m.) and xylazine (2.5mg/kg i.m.). A nal blood
sample (5 cc) will be taken to determine BG and HbA1C
levels, from a 22 g angiocatheter placed in the saphenous
vein. A lethal dose of Fatal-Plus (pentobarbital
sodium/1mL/10lb) will be administered i.v., and once the
animal has expired, collection of the rabbit’s ears will take
place, so all individual healing wounds could be assessed for
histology, immunohistochemistry, and additional studies
(gene expression, single-cell analysis, proteomics).
Swine Diabetes Model
Swine have been used as a model for many human conditions including type 1 (insulin-decient) and type 2 (insulinresistant) DM research because of their phenotypic
similarities to humans including cardiovascular anatomy and
function, metabolism, lipoprotein prole, size, tendency to
obesity, and omnivorous habits. There is a phenotypic overlap between the two types of DM and swine models show
characteristics and complications of both. Streptozotocin
and alloxan have been used to create insulin-decient diabetes in pigs. One of the most unique and useful phenotypes is
that these insulin-decient pigs develop more severe coronary atherosclerosis than nondiabetic controls. It is not fully
understood why patients with either type 1 or type 2 DM
have increased severity and diffuseness of atherosclerosis
compared with nondiabetic patients. The current human epidemic of type 2 DM and its attendant cardiovascular compli-
cations underscore the unmet need for creating a useful,
readily available animal model of type 2 insulin-resistant
DM.The goal would be to develop a useful animal model for
mechanistic studies as well as to develop and test novel therapeutics for both type 2 DM and its cardiovascular complications [50, 51].
Furthermore, swine models have been widely considered
as one of the best wound healing models due to their anatomical, physiological, and metabolic similarities to human
skin [52, 53]. Various wound types, including full-thickness
excision, partial-thickness excision, and thermal wounds,
have been used in wound healing studies with porcine models [54–57].
Additionally, pigs fed with a high-fat high-cholesterol diet
develop coronary, aortic, iliac, and carotid atherosclerotic
lesions, anatomical locations extremely relevant to the human
condition. Most importantly, these lesions recapitulate the histopathology seen in humans: proliferative lesions consisting of
smooth muscle cells, macrophages, lymphocytes, foam cells,
calcication, brous caps, necrotic and apoptotic cells, plaque
hemorrhage, and expanded extracellular matrices [58, 59].
Results of testing medicines (e.g., statins) and devices (e.g.,
stents) in swine have been regarded as having a high positive
predictive value for subsequent translation to humans [60].
Thus, pigs have great potential as a relevant animal model of
insulin-resistant type 2 DM to identify mechanisms that lead
to the development of diabetic complications and to develop
and test novel therapeutic approaches [61–65].
Diabetic Swine Model ofImpaired
Wound Healing
We have designed the following protocol for alloxan diabetes induction in minipigs (Yucatan’s), in order to more appropriately mimic the delayed healing responses seen in chronic
wounds, and chemically induced diabetes [21] with alloxan
is the gold standard and has been successfully used in previous studies [27].
Animal Procurement: It is critical to acquire animals from
a Class A vendor, with a strong reputation of providing animals that are in the best health and that are evaluated using a
comprehensive health monitoring program, which includes
bacteriology, parasitology, and pathology, as well as serol-
ogy. The swine colonies for research should be specic
pathogen-free (SPF); thus, populated with pigs that are surgically derived, probiotic ora or sterilized colostrum may
be provided. Thus, several terms have been used to characterize the health of pig herds by dened microbial or pathogen status. These include germ-free (axenic), gnotobiotic
(known microbiota), SPF, secondary SPF (SPF2), and
dened high health status, which indicates that the colony
has been tested for, and is free of, an extensive list of viruses
and other pathogens.
ab c
19 Experimental Animal Models inResearch: Diabetes andImpaired Wound Healing
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Fig. 19.5 Schematic representation of surgical procedures. (a) Orchiectomy or oophorectomy, (b) surgical jugular vein (JV) catheterization and
infusion port implantation, and (c) alloxan i.v. injection (marginal ear vein)
355
Acclimation Period: Miniature swine (Yucatan’s),
7–9 months old with an approximate weight of 30–35 kg,
and both male and female animals, are allowed to acclimate
for 7days. Although the USDA requirement is 48h of acclimation, we like to provide additional time for animal to distress from shipping and get used to their new environment
and being handled by new ARF staff.
Initial Surgical Interventions: After acclimatization, pigs
are initially fasted overnight (nothing by mouth/NPO for
10–12h). The following morning, animals are prepared for
surgery. Anesthetic induction is performed with Telazol
(3–5 mg/kg) i.m. followed by isourane via face mask at
4–5%, 100%/liter of oxygen, followed by endotracheal intubation and placement on an isourane vaporizer at 1–3%,
100%/liter of oxygen. Pig ears are shaved and a depilatory
cream is used to remove any excess hair:
clamp is used to tie off the vessels with 1-0 or 2-0 silk
sutures; each ovary is tied and then removed using a #10
scalpel blade; and then, depending on the surgical technique
used, the uterus could be either left in place or removed,
using the same clamping and tying off technique. The surgeon will then make sure that the vascular pedicels/stumps
are well tied off and hemostasis has been achieved. The uterine and ovarian attachments are then placed back in the
abdominal cavity. Lastly, the abdomen is closed in three layers with 3-0 Vicryl suture. For skin closure, a subcuticular
suturing technique is used to eliminate the need to remove
sutures/stitches later (Fig.19.5).
Jugular Vein (JV) Catheterization and Infusion Port
S.C.Implantation. While the animal remains under general
anesthesia and under sterile surgical conditions, a midline
incision is made on the anterior aspect of the neck, approximately 6–8cm. With blunt dissection, the right internal jugu-
1. Blood Draw: First blood draw (5cc) through the ear cen-
tral artery is done.
2. Blood glucose (BG) check: Using commercially avail-
able glucometer, BG is measured using 1 ul of blood
(generally blood drop left from blood draw). An i.v. catheter (20g) is inserted into the marginal ear vein to establish an i.v. line to administer lactated Ringer’s or saline
solution (500 mL and 1 gm cefazolin) throughout the
length of the surgical procedure. A single dose of s.c.
buprenorphine is administered.
lar vein (IJV) is identied and isolated from the surrounding
tissue, and elastic vessel loops are placed circumferentially
around the vessel proximal and distal to secure blood ow
control. A tunneling rod is then passed subcutaneously from
the IJV to the lateral aspect of the neck for infusion port
placement. The catheter attached to the port is then passed
through the tunneling rod to leave it in place with retention
beads, to prevent catheter movement postsurgically. The
venotomy is performed after tightening the vessel loops
proximally and distally, leaving the blood vessel lled. The
venotomy should be less than two-thirds of the vessel diam-
Oophorectomy, JV Catheterization with Infusion Port
S.C.Implantation and Baseline Measurements: Most class “A”
vendors will provide male pigs already castrated. Thus, orchiectomy will no longer be a surgical procedure that will need to
be performed; however, females will require Oophorectomy.
Surgical oophorectomy-hysterectomy (laparotomy with
ovaries and uterus removed): An incision (10–15 cm) is
made through the skin into the abdomen, below the belly button. Using a spay hook, the uterus is brought up and out of
the abdomen. Once the ovaries are visualized, a vascular
eter made with #11 blade. The lumen of the blood vessel can
be visualized by use of a vein pick, which also facilitates
passage of the catheter into the lumen. The tip of the catheter
is advanced into the blood vessel to the point of interest
(approximately 6–8cm). After placement of the catheter, it is
sutured into place between the suture beads, leaving an extra
segment of catheter to create a loop, and the coil relieves tension on the catheter during postural changes of the animal.
The port is then secured into place with three anchoring
sutures, 3-0 Vicryl. The same surgical procedure that was
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