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\begin{document}

\title{Animal models of diabetes-associated vascular diseases: An update on
available models and experimental analysis}



\author[1]{Judy Choi}%
\author[1]{Judy de Haan}%
\author[1]{Arpeeta Sharma}%
\affil[1]{Baker Heart and Diabetes Institute}%


\vspace{-1em}



  
  \date{May 17, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Diabetes is a chronic metabolic disorder associated with the accelerated
development of macrovascular (atherosclerosis, coronary artery disease)
and microvascular complications (nephropathy, retinopathy and
neuropathy), which remain the principal cause of mortality and morbidity
in this population. Current understanding of cellular and molecular
pathways of diabetes-driven vascular complications as well as
therapeutic interventions have arisen from studying disease pathogenesis
in animal models. Diabetes-associated vascular complications are
multi-faceted, involving the interaction between various cellular and
molecular pathways. Thus, the choice of an appropriate animal model to
study vascular pathogenesis is important in our quest to identify
innovative and mechanism-based targeted therapies to reduce the burden
of diabetic complications. Herein, we provide up-to-date information on
available mouse models of both Type 1 and Type 2 diabetic vascular
complications as well as experimental analysis and research outputs.%
\end{abstract}%



\sloppy


\textbf{Animal models of diabetes-associated vascular diseases:}

\textbf{An update on available models and experimental analysis}

Judy S.Y. Choi\textsuperscript{1}, and Judy B. de
Haan\textsuperscript{1,3,4,5*} and Arpeeta Sharma\textsuperscript{1,2*}

\textsuperscript{1}Baker Heart and Diabetes Institute, Melbourne,
Victoria, Australia

\textsuperscript{2}Department of Diabetes, Monash University, Central
Clinical School, Melbourne, Victoria, Australia

\textsuperscript{3}Dept Immunology and Pathology, Central Clinical
School, Monash University, Melbourne, Vic, Australia

\textsuperscript{4}Department of Physiology, Anatomy and Microbiology,
La Trobe University, Melbourne, Australia

\textsuperscript{5}Faculty of Science, Engineering and Technology,
Swinburne University, Melbourne Australia.

*Joint Senior authors

\emph{Addresses for correspondence}\textbf{:}

\textbf{Dr. Arpeeta Sharma}

Group Leader (Oxidative Stress Laboratory)

Diabetic Complications Division

Baker Heart and Diabetes Institute

75 Commercial Road

Melbourne, Victoria 3004, Australia

Phone : +61(3) 8532 1179

Fax : +61(3) 8532 1100

e-mail : Arpeeta.sharma@baker.edu.au

\textbf{DECLARATIONS}

\textbf{Availability of data and materials}

Non-applicable

\textbf{Funding}

AS is supported by an early career fellowship from the National Health
and Medical Research Council (NHMRC) and a Diabetes Australia Research
Project (DARP) General Grant. JBDH is supported by a Baker Fellowship.
This work was also supported by operational infrastructure grants
through the Australian Government Independent Research Institute
Infrastructure Support Scheme (9000220) and the Victorian State
Government Operational Infrastructure Support, Australia.

\textbf{Competing interests}

The authors declare that they have no competing interests.

\textbf{Ethics}

Ethics approval was not required for the current submission

\textbf{Author Contributions}

AS and JBDH conceived and designed the review. JC, JBDH and AS provided
intellectual input, wrote and edited the review. JC and AS prepared the
figures. JC, JBDH and AS approved final version of the manuscript.

A/Prof Judy de Haan and Dr Arpeeta Sharma take full responsibility for
the work as a whole, and the decision to submit and publish the review.

\textbf{Abstract}

Diabetes is a chronic metabolic disorder associated with the accelerated
development of macrovascular (atherosclerosis, coronary artery disease)
and microvascular complications (nephropathy, retinopathy and
neuropathy), which remain the principal cause of mortality and morbidity
in this population. Current understanding of cellular and molecular
pathways of diabetes-driven vascular complications as well as
therapeutic interventions have arisen from studying disease pathogenesis
in animal models. Diabetes-associated vascular complications are
multi-faceted, involving the interaction between various cellular and
molecular pathways. Thus, the choice of an appropriate animal model to
study vascular pathogenesis is important in our quest to identify
innovative and mechanism-based targeted therapies to reduce the burden
of diabetic complications. Herein, we provide up-to-date information on
available mouse models of both Type 1 and Type 2 diabetic vascular
complications as well as experimental analysis and research outputs.

\textbf{Introduction}

Diabetes Mellitus (DM), either Type1 (insulin-deficient) or Type2 (adult
onset, insulin resistance), is a chronic metabolic disorder that has
serious implications on cardiovascular health. Indeed, macro- and
microvascular complications associated with diabetes account for
approximately 65\% of deaths in the diabetic population{[}1, 2{]}.
Macrovascular complications associated with diabetes are mainly
represented by atherosclerosis and/or hypertension and their sequelae,
which include heart attacks and stroke, whilst microvascular
complications include chronic kidney disease, retinopathy and
neuropathy. The increased risk of cardiovascular complications in
diabetes is attributed to the elevated blood glucose levels and the
interplay between different cellular mechanisms in the disease setting
as a consequence of hyperglycemia{[}3{]}. These include, but are not
limited to, genetic, epigenetic, and cellular signalling pathways,
particularly the interplay between oxidative stress and inflammation,
which then result in imbalances in cellular metabolism contributing to
the development of cardiovascular complications.

Current standard of care for diabetic patients focusses on
glucose-lowering, lipid lowering and blood pressure control, however
these treatment options have yielded suboptimal outcomes on
cardiovascular end points, with the development of tolerance and
resistance to these therapies, particularly with respect to blood
pressure lowing medications limiting their long-term use{[}4, 5{]}.
Newer, more innovative CV-specific drug treatments are urgently needed
to prevent diabetes-mediated vascular diseases. In this regard, animal
models have proven to be an invaluable tool in exploring and
characterising novel pathophysiological pathways, identifying
therapeutic targets and evaluating their \emph{in vivo} potential.
However, it is critical to choose appropriate animal models that most
closely mimic the human pathophysiology of diabetes and its sequalae
such as the cardiovascular complications, to investigate potential
treatments and therapeutic strategies. To date, there is no perfect
animal model that encompasses all the pathophysiology and
characteristics of human disease but with advances in genetic
engineering and chemical manipulation, rodents have proven to be the
most reproducible, effective and preferred animal model for studies of
diabetic vascular diseases, particularly with respect to emulating
specific conditions that occur in humans. In this review, we provide a
comprehensive and up-to-date analysis on proven animal models of
diabetes-associated vascular diseases, with a focus on the analysis and
experimental outputs that can be achieved with these models.

\textbf{Animal models of diabetes-associated macrovascular diseases}

\emph{Vascular pathophysiology: key features that need to be represented
in animal models}

Atherosclerosis is the principal cause of death and disability in the
diabetic population, with the incidence of an atherosclerotic event
occurring approximately 14.6 years earlier in this patient cohort. The
pathophysiological events that lead to disease progression in the
presence of diabetes have been extensively reviewed{[}1, 6, 7{]},
however, key factors that are important to consider in choosing a
relevant animal model of diabetes--associated atherosclerosis will be
discussed. A main clinical feature that presents in uncontrolled or
poorly controlled Type1 and Type2 diabetic patients is hyperglycemia,
the key driver of vascular injury. Elevated blood glucose, primarily via
advanced glycation end products (AGEs) and their interaction with
receptor for advanced glycated end products (RAGE) drives
pro-inflammatory/pro-oxidant pathways to elicit molecular, cellular and
vascular injury{[}8, 9{]}. A further key feature of diabetes is
dyslipidemia manifested through elevated triglycerides, decreased high
density lipoproteins (HDL) and changes in low density lipoprotein (LDL)
structure to a more atherogenic profile{[}10{]}, which results from the
ineffective clearance of post-prandial lipoprotein rather than increased
circulating levels of fasting lipoproteins{[}11{]}. In addition, the
heightened state of oxidative stress and inflammation perturbs cellular
metabolism particularly within endothelial cells which leads to
endothelial dysfunction. Together with a build-up of oxidised LDL
particles, endothelial dysfunction promotes increased adhesion and
infiltration of leukocytes into the vessel wall and the formation of
foam cells, thereby leading to the acceleration of atherosclerotic
processes{[}1{]}.

Diabetes-associated atherosclerosis is most commonly studied in mice
despite the fact that humans and mice differ in certain characteristics
that affect the development of disease progression. Firstly, mice are
generally protected from atherogenesis due to differences in their
lipoprotein profile, with mice exhibiting elevated HDL levels due to the
absence of cholesteryl ester transfer protein (CETP){[}12, 13{]}.
Therefore, atherosclerotic plaque generation requires genetic
manipulation such as the deletion of the apolipoprotein E (ApoE) gene or
the LDL receptor (LDLR) gene together with dietary interventions.
Additionally, humans and mice have differences in cardiovascular anatomy
and haemodynamic physiological forces that influence the sites of
atherosclerosis{[}14{]}. Lesions predominantly occur in the coronary
arteries, carotid and peripheral vessels in humans whilst in mice,
lesions are commonly studied in the aortic sinus and the aortic
arch{[}14{]}. A summary of the models of diabetes-associated
atherosclerosis is shown in Figure 1.

\emph{Rodent models of diabetes-associated atherosclerosis2.2.1. Models
of Type1 diabetes-associated atherosclerosis}

To replicate human disease, most mouse models of Type1 diabetes are
insulin deficient, either by chemical induction or genetic manipulation.
The most extensively studied Type1 model of diabetes-associated
atherosclerosis is the streptozotocin (STZ) induction model in the ApoE
knockout (\textsuperscript{-/-}) mouse, initially characterised by Park
et al{[}15{]}. A major advantage of the STZ-induced
ApoE\textsuperscript{-/-} mouse model is the vast amount of published
literature available utilising this model allowing researchers to
compare and contrast data, cellular mechanisms and therapeutic
interventions. STZ, a pancreatic \selectlanguage{greek}β-\selectlanguage{english}islet cell toxin, enters the cell via
Glut-2 transporters to mediate its cytotoxic effect, thereby limiting
insulin production, leading to hyperglycaemia{[}16{]}. Generally, STZ is
administered as five consecutive low doses (55mg/kg/day){[}17-19{]} but
certain groups have demonstrated more consistent elevations in blood
glucose by using two consecutive high doses (100mg/kg/day){[}20-24{]}.
This becomes particularly important for longer term studies where blood
glucose levels may decline due to recovery of \selectlanguage{greek}β-\selectlanguage{english}islet cells. Despite the
benefits of a robust and consistent increase in hyperglycemia of around
25-30mmol/L, there are limitations that may preclude the use of STZ
under certain circumstances. For example, STZ at high doses has been
shown to affect the function of other cells expressing the Glut-2
transporter, such as hepatocytes and epithelial cells of the renal
tubules, which may be exposed to the toxic action of STZ leading to
unwanted side effects in the liver and kidneys{[}25{]}. In addition, a
further limitation of using STZ as the chemical inducer of diabetes is
that females, particularly mice, have been shown to be resistant to STZ,
often requiring higher doses of STZ to obtain equivalent levels of
hyperglycemia compared to male counterparts{[}26{]}. Thus, female mice
are commonly omitting in pre-clinical research for diabetic
complications when STZ is the preferred method of Type1 diabetes
induction. STZ also exhibits broad spectrum antibacterial properties
which may alter the gut microbiota{[}27{]}.

Despite the noted limitations, the STZ-induced diabetic
ApoE\textsuperscript{-/-} mouse model is the preferred model for
studying atherosclerosis, since these mice exhibit hyperglycemia as well
as dyslipidemia, as indicated by the elevated blood glucose levels,
total cholesterol, LDL and triglyceride levels. Plaque formation is
increased 3-6 fold in this model over a 10-20 week period, with early
discrete lesions observed within the aortic sinus and the aortic arch;
however, over a longer course of diabetes, lesions additionally develop
over the thoracic and abdominal aortic regions. This model has been
associated with endothelial dysfunction, increased inflammation and
oxidative stress as well as macrophage foam cell formation, all of which
are hallmarks of atherosclerotic pathogenesis. More importantly, the
STZ-induced ApoE\textsuperscript{-/-} mouse model has been particularly
useful in investigating potential therapies, such as but not limited to
soluble RAGE (sRAGE), peroxisome proliferator-activator gamma agonists,
anti-oxidants (ebselen, dimethyl furate), SGLT2 inhibitors and ApoA1
mimetics, in their ability to attenuate diabetes-associated
atherosclerosis, independent of blood glucose lowering effects {[}21,
22, 28-32{]}. As the ApoE\textsuperscript{-/-} mouse model is well
established and easy to breed, cross-breeding
ApoE\textsuperscript{-/-}mice with mice harbouring genetically altered
proteins such as transcription factors, receptors and/or enzymes enables
the elucidation of their role in diabetic atherosclerotic mechanisms. On
the contrary, STZ-induced diabetic LDLR\textsuperscript{-/-} mice do not
exhibit extensive atherosclerotic lesions despite having hyperglycemia
and elevated LDL/vLDL levels and often require additional diet
supplementation{[}33{]} making them a more ideal model of Type2
diabetes.

A less well-known model that is used in Type1 diabetic atherosclerotic
research, particularly if the side-effects of STZ are of concern (as
detailed above), is the spontaneous genetic Akita mouse, cross bred with
either the ApoE\textsuperscript{-/-} or LDLR\textsuperscript{-/-} mouse.
The Akita mouse, first selected in Japan, carries an autosomal dominant
missense mutation in the insulin2 gene, resulting in impaired proinsulin
processing and aggregation of misfolded insulin protein, rendering it
insulin deficient. Akita mice develop typical Type1 diabetes
characteristics, including hyperglycemia, polyuria and polydipsia, as
well as impaired vascular tone as early as 3-4 weeks of age, making it
an ideal model to study the effects of diabetes on endothelial
dysfunction and macrovascular complications{[}34{]}. When cross-bred to
the ApoE\textsuperscript{-/-} or LDLR\textsuperscript{-/-} mouse, this
model exhibits dyslipidemia, mainly manisfested as increases in
triglycleride and non-HDL cholesterol levels, and an approximately 2 to
3-fold increase in atherosclerotic lesions{[}35-37{]}. This is
accompanied by increased infiltration of immune cells into the plaque,
in particular macrophages and T-cells, as well as augmentation of
vascular inflammation as demonstrated by increased levels of TNF-\selectlanguage{greek}α,
\selectlanguage{english}IL-1\selectlanguage{greek}β \selectlanguage{english}and MCP-1, all of which are key mediators involved in plaque
progression{[}36, 37{]}. Interestingly, the differences in plaque size
are not as pronounced in female Akita/LDLR\textsuperscript{-/-} mice as
they are in male mice, suggesting gender differences could play a part
in diabetic atherosclerosis in this model.

The models described thus far are promising to investigate the
mechanisms of endothelial dysfunction, vascular inflammation and plaque
formation in a diabetic setting, however, it is difficult to discern
whether these arise as a direct effect of the hyperglycemia or as a
consequence of other confounding atherogenic factors such as the
dyslipidemia under these conditions. To circumvent this problem,
Vikramadithyan et al have developed a mouse model to study the effects
of hyperglycemia in accelerating atherogenesis by manipulating Aldose
reductase, an enzyme involved in catalysing the reduction of glucose to
sorbitol, in combination with STZ{[}38{]}. Aldose reductase has been
shown to accelerate diabetic vascular complications through the
increased production of reactive oxygen species (ROS). Indeed,
overexpression of human aldose reductase in the diabetic
LDLR\textsuperscript{-/-} mouse resulted in a 2-fold increase in lesion
size as compared to diabetic LDLR\textsuperscript{-/-} mice alone,
driven by defects in glucose-driven ROS signalling{[}38{]}. Another
model developed to closely mimic the autoimmune destruction of
pancreatic \selectlanguage{greek}β \selectlanguage{english}cells as observed in human Type1 diabetes is the induction
of lymphocytic choriomeningitis virus (LCMV) infection {[}39, 40{]}. The
LCMV glycoprotein (GP) transgene is controlled by the insulin promoter
in \selectlanguage{greek}β \selectlanguage{english}cells, which is rapidly destroyed upon infection. These
LDLR\textsuperscript{-/-} mouse injected with the LCMV GP developed
severe hyperglycemia that was associated with accelerated lesion
formation in the absence of hypertriglyceridemia{[}39, 40{]}. Moreover,
atherosclerotic lesions were morphologically similar to human lesions
particularly with increased arterial macrophage accumulation and
intralesional haemorrhage{[}39{]}. Nonetheless, further investigation is
required to delineate the exact role of altered lipoprotein profiles and
hyperglycemia in preclinical animal models of Type1 diabetes-induced
atherosclerosis.

\emph{2.2.2. Models of Type2 induced diabetes-associated
atherosclerosis}

The prevalence of Type2 diabetes driven by insulin resistance and
obesity is on an exponential rise worldwide with 700 million individuals
predicted to have Type 2 diabetes by 2045{[}41{]}. Metabolic features of
Type2 diabetes include hyperglycemia, hyperinsulinemia, obesity and
dyslipidemia encompassing an increase in LDL, vLDL and a decrease in HDL
levels. Thus, it is pertinent to choose an animal model that at best
encompasses all of these features in order to study the mechanisms and
explore therapeutic approaches to limit macrovascular disease induced by
Type2 diabetes. The majority of the animal models available to study
Type2 diabetes-associated atherosclerosis are either genetically
manipulated or diet induced.

Two of the most common genetic models are generated by crossbreeding
genetic models of Type2 diabetes, such as the leptin (ob/ob) and leptin
receptor (db/db) deficient mice, with atherosclerotic mice such as the
ApoE\textsuperscript{-/-} and LDLR\textsuperscript{-/-} mice. Leptin,
the satiety hormone that is produced by adipose cells and enterocytes of
the small intestine, regulates appetite via receptors in the arcuate
nucleus of the hypothalamus. Ob/ob mice and db/db mice carry mutations
in the leptin gene and the leptin receptor-coding gene respectively,
causing the induction of hyperphagia leading to obesity and Type 2
diabetes{[}42, 43{]}. The db/db;ApoE\textsuperscript{-/-} mouse is well
established and exhibits typical features of Type2 diabetes by 20 weeks,
such as increased body weight, hyperglycemia, hyperinsulinemia, elevated
cholesterol levels (triglycerides, LDL and vLDL) with a 3-4 fold
increase in atherosclerotic plaque burden{[}42, 43{]}. From a more
diabetic perspective, these mice display higher expression of RAGE,
inflammatory adhesion molecules (such as VCAM-1) and matrix
metalloproteinase (MMP-9) activity{[}43{]}. Pharmacological
interventions, such as soluble RAGE and PPAR-agonists have proven to be
effective in their pre-clinical evaluation with this model{[}43{]}.
Similar atherosclerotic observations were noted in the
ob/ob;ApoE\textsuperscript{-/-} and ob/ob;LDLR\textsuperscript{-/-}mice,
however, in these models either hyperglycemia was not evident (ob/ob;
ApoE\textsuperscript{-/-} mice) or hyperlipidemia was too pronounced
masking the diabetic effects (ob/ob;LDLR\textsuperscript{-/-}mice). A
modification of the ob/ob;ApoE\textsuperscript{-/-} and
ob/ob;LDLR\textsuperscript{-/-} mice to more closely align with the
human lipoprotein profile is to cross these mice to
ApoB\textsuperscript{100/100} producing mice which do not make ApoB-48
in their liver{[}44, 45{]}. Cholesterol in mice is mostly carried in
ApoB-48 containing vLDL particles and chylomicron remnants whereas
humans do not produce ApoB-48 in the liver{[}44, 45{]}.
Ob/ob;ApoE\textsuperscript{-/-};ApoB\textsuperscript{100/100} and
ob/ob;LDLR\textsuperscript{-/-};ApoB\textsuperscript{100/100} mice
exhibit a metabolic phenotype which includes obesity, insulin resistance
as well as atherosclerosis and hypertension{[}44{]}. Due to their
specificity in lipoprotein profiles, these models are useful for
studying cholesterol-lowering therapies from a Type2 diabetic/metabolic
syndrome perspective{[}44{]}.

For research that requires less influence of lipid levels and consistent
features of Type2 diabetes, there are other models with a more
clinically relevant lipoprotein profile that can be considered. The
LDLR\textsuperscript{-/-} mouse that is able to synthesise only
ApoB\textsuperscript{100/100} protein represents a model more closely
aligned to the human lipoprotein profile. This model exhibits
accelerated atherogenesis{[}45{]}. Furthermore, Heinonen et al crossbred
the LDLR\textsuperscript{-/-};ApoB\textsuperscript{100/100} mouse with
transgenic mice overexpressing insulin-like growth factor-II in
pancreatic \selectlanguage{greek}β \selectlanguage{english}cells which led to the development of Type2 diabetes
without major hyperlipidemia{[}46{]}. These mice demonstrated aggravated
atherosclerotic lesions with increased complications such as lesion
calcification which coincided with an increase in the expression of a
variety of genes involved in calcification (OPN, ALP-2 and BMP-2) and
inflammation (MCP-1){[}46{]}. Moreover, the complexity of the lesion and
increased calcification was more prominent in aged mice, which is in
line with clinical studies that demonstrate calcification in peripheral
and coronary arteries, particularly in older patients with a longer
duration of diabetes, and is considered a strong independent risk factor
for cardiovascular events{[}46-49{]}. Another useful genetic model is
the ApoE\textsuperscript{-/-} mouse that has a homozygous or
heterozygous deletion of the insulin receptor substrate (IRS1 and IRS2)
gene. ApoE\textsuperscript{-/-} mice that are IRS1\textsuperscript{+/-},
IRS2\textsuperscript{+/-} and IRS2\textsuperscript{-/-} mice display a
Type2 diabetes/metabolic syndrome phenotype that includes, insulin
resistance, hyperglycemia, hyperinsulinemia, and impaired glucose
tolerance as compared to IRS2\textsuperscript{+/+} mice, however lipid
levels were only modestly altered{[}50-52{]}. This is consistent with
clinical findings in Type2 diabetic patients that have reduced
expression of IRS2 in their pancreatic \selectlanguage{greek}β \selectlanguage{english}cells and a highly polymorphic
IRS1 gene{[}53{]}. These mice have augmented atherosclerotic lesions and
vascular inflammation that is attributed to impaired IRS2 signalling of
the AKT and ERK pathways that lead to the upregulation of
pro-atherogenic MCP-1{[}54{]}. Lastly, another note-worthy genetic model
of T2D-induced atherosclerosis is the heterozygous glucokinase (GK)
knockout mouse on the ApoE\textsuperscript{-/-} background. GK is the
rate-limiting enzyme of glucose-stimulated insulin secretion, thus is it
not surprising that
GK\textsuperscript{+/-}/ApoE\textsuperscript{-/-}mice on a western diet
show significant glucose intolerance and impaired glucose-stimulated
insulin secretion, a phenomenon that was consistent over time{[}55{]}.
Despite plasma lipid levels being comparable to the
ApoE\textsuperscript{-/-} mouse,
GK\textsuperscript{+/-}/ApoE\textsuperscript{-/-} mice have
significantly accelerated and highly developed atherosclerotic lesions,
strongly suggesting that lesions are driven by hyperglycemia and insulin
resistance{[}55{]}.

Diet-induced insulin resistance is extensively studied in models of
Type2 diabetes associated atherosclerosis. In particular, a high-fat
western diet (comprising cholesterol (0.2\% total cholesterol) + total
fat (21\% by weight; 42\% kcal from fat) + high saturated fatty acids
(\textgreater{}60\% of total fatty acids) + high sucrose (34\% by
weight)) in ApoE\textsuperscript{-/-} or LDLR\textsuperscript{-/-} mice
increases lesions throughout the aortic tree and exhibits hyperglycemia,
hyperinsulinemia and hypertriglyceridemia. Interestingly, in mice fed a
diabetogenic diet where 35\% of the energy is sourced from fats,
atherosclerotic lesions were inconsistent with the
LDLR\textsuperscript{-/-} mice being more susceptible to diabetes{[}56,
57{]}. LDLR\textsuperscript{-/-} mice on a diabetogenic diet had severe
dyslipidemia and increased atherosclerotic lesions, albeit with modest
increases in glucose and insulin levels. On the contrary, diabetogenic
diet-fed ApoE\textsuperscript{-/-} mice displayed no significant
differences between in lipids, glucose and atherosclerotic lesion sizes
as compared to chow-fed ApoE\textsuperscript{-/-} mice{[}56, 57{]}. In
comparing neointimal formation, ApoE\textsuperscript{-/-} mice fed a
western diet exhibited larger lesions, accompanied by higher glucose and
insulin levels, as compared to mice on a diabetogenic diet that
maintained euglycemia but developed insulin resistance{[}58{]}.
Treatment of these mice with Rosiglitazone, a thiazolidinedione class of
PPAR agonist, led to a marked reduction in macrophage content and
neointimal formation{[}58{]}.

Due to ongoing discrepancies in lesion size between diet-induced and
genetically manipulated mouse models, research groups have continually
strived to modify diet composition to more closely reflect the human
Type2 diabetic cardio-metabolic risk profile. Indeed, a diabetogenic
diet supplemented with 0.15\% cholesterol in the
LDLR\textsuperscript{-/-} mice further enhances obesity, insulin
resistance, systemic inflammation and atherosclerosis with increased
macrophage accumulation{[}59, 60{]}. Additionally, Neuhofer et al found
that by altering complex carbohydrates with a~high-fat, high-sucrose
diet and the inclusion of 0.15\% cholesterol, they were able to further
augment atherosclerotic lesion size and increase adipose tissue
inflammation, hallmarks often seen in human obesity and insulin
resistance, thus creating a more robust model of metabolic syndrome with
diet-induced obesity{[}61{]}. Furthermore, supplementing rodent diets
with high fructose to emulate consumption of sugary drinks in humans and
the impact on atherosclerotic lesions has been investigated by several
groups. ApoE\textsuperscript{-/-} mice fed a high fructose diet
exhibited a pro-atherogenic state with insulin resistance, independent
of hypercholesterolemia{[}62{]}. This cohort of mice exhibited increased
vascular inflammation (VCAM-1), overproduction of NADPH-oxidase induced
ROS and significant outward vascular remodelling{[}62{]}.
Supplementation of liquid fructose to western-diet fed
LDLR\textsuperscript{-/-} mice demonstrated similar results with the
mice developing increased atheromatous plaque driven by increased
monocyte/macrophage infiltration and local inflammation. Importantly,
atherosclerotic plaque size strongly correlated with plasma lipid
levels{[}63{]}. Another method that has been described to model the
metabolic syndrome associated with diabetes is the incorporation of
low-dose STZ along with high fat feeding. Although this model has not
been validated in terms of atherosclerotic lesions, it has been shown to
develop cardiac inflammation and dysfunction, which combines both early
and late stages of the disease{[}64, 65{]}.

\emph{2.2.3. Models of atherosclerotic plaque complications}

In advanced stages of lesion development, the atherosclerotic plaque is
more vulnerable, unstable and prone to rupture. Indeed, plaque rupture
frequently leads to luminal~thrombosis which is the direct cause of
acute ischaemia responsible for cardiovascular deaths and this
phenomenon is accelerated in the diabetic setting{[}66, 67{]}. However,
plaque rupture is rarely detected in mouse models of atherosclerosis, a
limitation with respect to replication of human pathology to
recapitulate the human disease. Nonetheless, several experimental models
have proven advantageous in analysing post-rupture thrombotic events. In
general, to induce unstable plaques, an invasive approach such as the
placement of a perivascular carotid cuff or ligation of the common
carotid artery under western diet-fed conditions in the
ApoE\textsuperscript{-/-} mouse can achieve an environment of altered
haemodynamic flow to replicate the advanced stages of atherosclerotic
plaque development with evidence of an occlusive thrombus{[}68{]}.
Another method commonly used to develop the unstable plaque phenotype is
through the elevation of Angiotensin II, either endogenously via
surgical clipping of the renal vasculature, or via Angiotensin II
infusion through the placement of osmotic mini pumps. High angiotensin
II levels in the western-diet fed ApoE\textsuperscript{-/-} mice exhibit
accelerated lesions with characteristic features of a thinner fibrous
cap, larger lipid necrotic core, and increased macrophage content, all
of which contribute to plaque destabilisation and vulnerability{[}69,
70{]}. Another interesting model that displays features of advanced
unstable atherosclerotic plaque is a model harbouring a heterozygous
mutation in the fibrillin-1 gene (C1039\textsuperscript{+/-}) which
leads to the fragmentation of elastic fibres in the vessel wall,
increased arterial wall stiffness and highly sporadic plaque rupture
when crossed to the ApoE\textsuperscript{-/-}{[}71, 72{]}. When fed a
western diet for 35 weeks,
ApoE\textsuperscript{-/-}Fbn1\textsuperscript{C1039G+/-} mice exhibit a
3-fold increase in necrotic core, which was associated with increased
T-cell infiltration, augmented neovascularization and intraplaque
haemorrhage{[}72{]}. Furthermore, spontaneous plaque rupture was
observed in the ascending aorta and brachiocephalic arteries in more
than 50\% of
ApoE\textsuperscript{-/-}Fbn1\textsuperscript{C1039G+/-}mice{[}72{]}. In
line with human complications, these
ApoE\textsuperscript{-/-}Fbn1\textsuperscript{C1039G+/-} mice showed
coronary plaques and myocardial infarction as well as sudden
death{[}72{]}. Thus, this reproducible model of advanced atherosclerosis
and plaque rupture with added complications of myocardial infarction is
clinically relevant.

While these models are useful in studying the pathways involved in
plaque rupture, they are not entirely ideal as they are generated by an
artificial environment often driven by genetic manipulation or altered
biomechanical stress. Therefore, there is a need to develop preclinical
models that represent the spontaneous plaque rupture that occurs in
humans. A more innovative approach has been developed by Chen et al who
showcase a unique mouse model of atherosclerotic plaque instability that
is proving to be useful for mechanistic insights and advantageous for
therapeutic testing{[}73{]}. With the hypothesis that low shear stress
and high tensile stress contribute to plaque instability in patients,
computational haemodynamic modelling was used to develop a unique
tandem stenosis mouse model of plaque instability/rupture. The tandem
stenosis model involves applying two sutures to the right common carotid
artery in the ApoE\textsuperscript{-/-} mouse fed a high-fat western
diet, resulting in an unstable plaque reflecting the characteristics
seen in humans. These features include ruptured fibrous caps,
intraplaque haemorrhage, large necrotic cores, plaque inflammation,
intravascular occlusive thrombus formation and near-infrared
autofluorescence. Importantly, this model of plaque instability and
rupture is suitable for testing the responsiveness of pharmacological
interventions including plaque-stabilising drugs and other inflammatory
mediators, however, its role in the diabetic setting is yet to be
determined.

Experimental analysis of diabetes-associated atherosclerosis

In most preclinical animal models, atherosclerotic lesion size is the
first quantitative measure performed to determine if genetic loss or
overexpression as well as pharmacological intervention has an impact on
diabetes-associated atherosclerosis. Generally, lesions are observed in
1) aortic root/sinus and ascending aorta, 2) aortic tree which spans the
arch, thoracic and abdominal regions and 3) the brachiocephalic
(innominate) artery. Quantification of lesion area in the aortic sinus
and ascending aorta is described in detail by Paigen et al{[}74{]} and
Daughtery and Whitman{[}75{]}. This involves sectioning the whole frozen
aortic root spanning approximately 300\selectlanguage{greek}µ\selectlanguage{english}m and the ascending aorta
(400\selectlanguage{greek}µ\selectlanguage{english}m). Acquaintance with the architecture of the sinus region and the
three valve leaflets is essential for the accurate sectioning of this
region of tissue requiring considerable skill. Staining of selected
sections 30\selectlanguage{greek}µ\selectlanguage{english}m apart is then performed using Oil Red O, a fat soluble dye
for the staining of triglycerides and lipids in atherosclerotic plaque.
The advantage of obtaining serial sections through the sinus region is
that cellular composition of the plaque (collagen, macrophages, smooth
muscle cells and necrotic core) can be determined by histological (H\&E,
picosirius red, masson's trichrome) and immunohistochemical staining
(CD68 antibody to detect macrophages). Additionally, inflammation and
oxidative stress markers can be quantified by performing
immunohistochemistry and immunofluorescence using specific antibodies
{[}31, 76-79{]}.

A relatively quick and simple method to quantify atherosclerotic lesions
on the intimal surface of the aortic tree is by an \emph{en face}method,
whereby the formalin-fixed aorta is stained with Sudan IV, a lipid
soluble dye, after which the aorta is cut open, pinned flat and imaged.
This mode of analysis is normally one dimensional with lesion area being
the main assessment, however, the aorta can then be embedded in paraffin
for immunohistochemical analysis to determine inflammatory and oxidative
stress markers. Furthermore, using a separate cohort of mice, the aorta
can be snap frozen for RNA and protein extraction to determine
atherogenic proteins and pathways by qRT-PCR and Western Blotting
respectively{[}20, 76, 80, 81{]}. Similarly, the innominate artery which
is connected to the aortic arch can be dissected out and sectioned to
assess lesion area using histological stains. Lesions in the innominate
artery develop the complexities of late stage plaque, characterized by
an atrophic media, perivascular inflammation, and thinning of the
fibrous cap which in some instances can lead to plaque rupture and
intraplaque haemorrhage.

A major limitation of these methods is that the analysis does not
provide volumetric three-dimensional assessment of the plaque, nor the
degree of vascular occlusion. Often tissue integrity is compromised
during preparation and processing for histology. To overcome these
issues, over the past decade, there have been improvements in imaging
technologies to provide a more in-depth view of plaque complexity. For
example, an \emph{ex vivo} scanning method, known as microscopic
computed tomography (microCT) has been adapted for the visualisation of
atherosclerotic plaque{[}82{]}. Excised hearts and vessels from diabetic
ApoE\textsuperscript{-/-} mice, can be stained \emph{en bloc} with metal
solutions and scanned using microCT. Then, an image analysis software is
able to recreate a virtual histological section to view multi-focal
images in both 2-dimensional and 3-dimensional formats, allowing
volumetric assessments of the atherosclerotic lesion{[}82{]}. The tissue
is intact and can further be processed for traditional histology and
immunohistochemistry{[}82{]}. In addition, \emph{ex vivo}high-resolution
three dimensional MRI is able to sensitively and volumetrically
quantitate atherosclerotic lesions in the mouse aortic root and
innominate artery with specific insight into microanatomy of the
lipid-rich necrotic core and fibrous cap components which can be
verified by histology, allowing for tracking of lesion progression over
time{[}83{]}. Using fluorescence emission computed technology, Htun et
al were able to detect autofluorescence in the near-infrared range that
correlated with plaque instability and vulnerability, establishing this
technique as a vital imaging tool for the detection of high risk plaques
in patients{[}84{]}.

The small size of mouse aortae can limit the availability of tissue for
analysis, often requiring separate cohorts of mice to be run for
extensive histological analysis as well as gene expression and protein
quantification, making these studies time, labour and cost intensive. To
circumvent this, a new emerging technology, known as Laser Capture
Microdissection (LCM) is gaining significant interest. This technique
can be performed on frozen or paraffin-embedded sectioned tissue. LCM
enables subpopulations of tissue cells to be collected under the
microscope using a low-power infrared laser, which harvests cells of
interest directly by first removing unwanted cells to give
histologically pure enriched cell populations{[}85, 86{]}. Furthermore,
this technology preserves the integrity of the tissue and does not
damage RNA, DNA and protein, therefore, enabling a number of downstream
molecular analyses to be performed. The precision with which tissues are
dissected enables the extraction of lesion-specific areas allowing
researchers to explore cell populations and differentially expressed
genes that make up the different plaque regions. Indeed, LCM of the
atherosclerotic lesion showed a significant enrichment of foam-cell
specific RNA transcripts{[}87{]}. In addition, this cell-specific
approach enables analysis of genetic and pharmacological manipulations
of atherosclerotic lesions.

Over the last decade, research has focused on examining cell-specific
populations and markers that are responsible for~disease progression. As
such, flow cytometry has proven useful to sort, identify and quantify
cell subpopulations within the atherosclerotic aorta. In particular,
flow cytometry has been used to track leukocyte infiltration,
composition and subtypes in aortae, which is significantly heightened in
disease settings{[}88{]}. A major advantage of flow-cytometry is the
ability to simultaneously stain for surface and intracellular markers
using a range of antibodies to identify different cell types and their
functional stage{[}88, 89{]}. This enables both quantitative and
qualitative results from one sample. Using this technique, Nagareddy et
al elegantly demonstrated significant increases in pro-inflammatory
circulating monocytes (Ly6-C\textsuperscript{hi} subset) and neutrophils
but not lymphocytes, which were significantly lessened after glucose
lowering, suggesting a role for leukocytosis in promoting
diabetes-associated atherosclerosis{[}90{]}. Recently, Brennan et al
cultured diseased carotid arteries \emph{ex vivo} from diabetic and
non-diabetic patients who underwent carotid endarterectomies and
analysed tissue cytokine responses in response to anti-inflammatory
treatment with a pro-resolving mediator known as LipoxinA4{[}91{]}.
Additionally, other groups have utilised this method to perform flow
cytometry of immune cell subsets{[}92{]}. This reproducible technique
allows the investigation of important aspects of atherogenesis,
particularly the function of immune cell and cell-cell interactions
within the tissue from a human disease perspective{[}91, 92{]}.

In line with flow cytometry data, more recently, single-cell RNA
sequencing (scRNAseq) technologies allow the evaluation of transcriptome
information from a single cell to reveal cell population differences at
the RNA level. Moreover, a key feature is the ability to detect
heterogeneity among individual cells to define the cellular landscape
via cell maps{[}93{]}. Since its inception a few years ago, several
research groups have employed scRNAseq technology to uncover the
transcriptome-based cellular landscape of human and murine
atherosclerotic plaque, with a particular focus on unrecognized immune
cell populations and their role in atherogenesis{[}94-97{]}. Indeed,
current work is underway to profile the transcriptional landscape in
diabetic atherosclerotic aortae using scRNAseq.

Lastly, as atherosclerosis is a dynamic process involving the
interaction between different cell types, it is pertinent to explore the
use of real-time functional assays. As an initial step in atherogenesis,
vascular inflammation triggers the cell adhesion cascade, contributing
to the recruitment and infiltration of leukocytes to the endothelium.
Intravital microscopy is now widely used to visualise and record
leukocyte rolling and adhesion using whole blood perfusion in
real-time{[}98{]}. As this method is performed on vessels \emph{ex vivo}
, it does not require invasive surgery in animals. Additionally, this
technique has been validated in diabetes-induced vasculopathies enabling
localised inhibitor treatment to evaluate the role of therapeutics in
modulating leukocyte-endothelial interactions{[}20, 98, 99{]}. Vessel
myography \emph{ex vivo} is another important technique to assess
endothelial viability and function, which are critical early markers for
the development of diabetes-associated atherosclerosis. This technique
allows researchers to evaluate various parameters including the
assessment of vascular tone, endothelium-dependent and independent
function and nitric oxide bioavailability, which can be determined in
the presence or absence of pharmacological interventions/genetic
manipulations{[}20, 100{]}. A summary of experimental and research
outputs to evaluate diabetes-associated atherosclerosis is shown in
Table 1.

\textbf{Animal models of diabetes associated microvascular diseases}

In diabetic patients, it is apparent that microvascular and
macrovascular complications often develop simultaneously. Microvascular
complications are characterised by functional and structural organ
damage arising from changes affecting capillaries and arterioles. Three
major manifestations of diabetic microvascular disease are nephropathy,
retinopathy and neuropathy. While animal models that describe diabetic
microvascular diseases have been extensively reviewed{[}101-103{]}, we
aim to highlight the most promising murine models that most closely
align with three debilitating human microvascular pathologies, diabetic
nephropathy, retinopathy and neuropathy.

\emph{Diabetic nephropathy}

\emph{3.1.1. Kidney pathophysiology: key features that need to be
represented in animal models}

Diabetic nephropathy is a progressive complication of DM, a leading
cause of end-stage renal failure globally. A main clinical feature that
presents in both Type1 and Type2 diabetic patients is albuminuria where
the first manifestation typically occurs as microalbuminuria, defined as
albumin excretion of 30-299mg in 24 hours. Over time, microalbuminuria
progresses to proteinuria, defined as albumin excretion of over 500mg in
24 hours leading to overt diabetic nephropathy{[}104{]}. Hyperglycaemia
acts as the major driving force of the progression to diabetic
nephropathy with the generation of ROS, formation of AGEs, and the
activation of polyol and PKC pathways all contributing to glomerular
damage. The downstream consequences include inflammation, thickening of
the glomerular basement membrane and microaneurysm formation{[}105{]}.
In addition, accumulation of extracellular matrix proteins such as
collagens and fibronectin replaces healthy renal architecture, leading
to mesangial expansion and reduction of filtration surface area, which
in turn is further disrupted by the thickening of glomerular basement
membranes{[}106{]}.

\emph{3.1.2. Models of diabetic nephropathy and research outputs}

For many years, mouse models have been the primary and most extensively
studied species to investigate kidney pathogenesis and for the
development of novel therapies to limit diabetic nephropathy. As
previously mentioned, the diabetogenic \selectlanguage{ngerman}ß-islet cell toxin, STZ, is also
by far the most widely used drug to induce diabetic nephropathy.
However, development of overt diabetic nephropathy is strain dependent.
C57BL/6J and BALB/c strains of mice are highly responsive to developing
STZ-mediated diabetes although C57BL/6J mice are relatively resistant to
developing diabetic nephropathy{[}107{]}. Hence, C57Bl/6J mice are
commonly crossbred with genetic mouse models known to have impaired
renal function. One approach is to breed eNOS\textsuperscript{-/-} mice
onto the C57BL/6 background. Deletion of eNOS diminishes NO
availability, thereby phenocopying the well-documented decreased NO
levels and its effects on the vasculature in human diabetic nephropathy.
STZ-induced diabetic eNOS\textsuperscript{-/-} mice on the C57BL/6
background develop significant impairment of renal function in which
clear pathophysiological changes are visible after 24 weeks of
diabetes{[}108{]}. On its own, a reduction in eNOS functionality is
sufficient to develop vascular dysfunction, hypertension and
albuminuria, all of which accelerate renal injury, however, when
superimposed on a diabetic background the severity of the albuminuria
appears to be enhanced, and more advanced glomerular lesions and
glomerular endothelial injury are exhibited in the STZ-induced diabetic
eNOS\textsuperscript{-/-} mouse model. However, high dose administration
of STZ should be avoided in this model since this can be highly lethal,
although the extent of albuminuria and renal injury observed is milder
than that of the eNOS\textsuperscript{-/-} db/db (Type2 diabetes) mouse
model{[}108{]}.

Genetic mouse models, such as the ob/ob and db/db mice, have been widely
studied as models of diabetic nephropathy, although the degree of
severity differs between these two models. In the db/db mice, larger
glomeruli with increased mesangial matrix are visible by 5-6month of
age, becoming more pronounced over time, along with thickening of the
glomerular basement membrane. The extent of renal disease is far less in
ob/ob mice when compared to db/db mice. When on the C57Bl/6 background,
ob/ob mice are insulin resistant but do not develop severe diabetes. The
major renal pathology in ob/ob mice primarily includes diffuse and
nodular lipo-hyaline changes in the glomerulus with a relative paucity
in mesangial expansion{[}109{]}. Both ob/ob and db/db mice have been
crossbreed with numerous genetically modified mice. Initially
established by Zhao et al. the eNOS\textsuperscript{-/-};db/db mouse
model has been identified as the most robust model of Type2
diabetes{[}110{]}. These mice display many human pathophysiological
features of diabetic nephropathy including significant albuminuria (more
than 30-fold that of controls), reduction in glomerular filtration rate
and glomerular basement membrane thickening{[}110{]}.

The importance of performing animal studies in both male and female
mice, as opposed to studies in one gender only (mostly male), is
becoming increasingly apparent to improve the translation of novel
therapies to both males and females. Recent findings showed that there
were no significant differences in renal structural and functional
damage between male and female diabetic eNOS\textsuperscript{-/-}db/db
mice {[}111{]}. Both male and female eNOS\textsuperscript{-/-}db/db mice
showed hyperglycaemia and obesity as early as 8 weeks of age. Although
male diabetic mice had a tendency towards greater body weights than
female counterparts, the difference was not significant. Both male and
female diabetic mice showed progressive albuminuria, renal hypertrophy,
and significantly elevated serum creatinine, although this was slightly
greater in female diabetic mice{[}111{]}. Notably, both male and female
mice displayed significantly greater glomerular injury compared to their
wild-type counterparts, but no differences were noted between genders.
Hence, the eNOS\textsuperscript{-/-}db/db mouse model represents a
robust model of diabetic nephropathy enabling the study of drug
treatment in both male and female mice without gender impacting
experimental outcomes. However, it should not be assumed that drug
treatments will always necessitate the same outcomes, and for this
reason both genders should be evaluated. However, historically male mice
strains have been used to demonstrate more robust hyperglycaemia and
renal injury.

Initially characterized by Clee et al., the black and tan, brachyuric
(BTBR) mouse crossed onto the ob/ob background is a widely used mouse
model to investigate Type2 diabetic nephropathy{[}112{]}. The BTBR mouse
strain has alleles promoting insulin resistance resulting in natural
hyperinsulinemia. When crossed with the ob/ob mouse, it develops a
severe Type2 diabetic phenotype, maintaining sustained
hyperglycaemia{[}112{]} and closely recapitulating the kidney lesions
observed in human diabetic nephropathy. In particular, BTBR ob/ob mice
exhibit glomerular hypertrophy, accumulation of mesangial matrix, loss
of podocytes and glomerular lesions that are similar to that seen in
human T2D kidney disease{[}113{]}. BTBR ob/ob mice display
overactivation NF-\selectlanguage{greek}κ\selectlanguage{ngerman}ß and JAK/STAT pathways, thereby promoting
inflammation and the infiltration of lymphocytes and macrophages
compared to BTBR wild type mice. Indeed, use of the BTBR ob/ob mouse
model has demonstrated the therapeutic potential of targeting the
transcription factor NF-\selectlanguage{greek}κ\selectlanguage{ngerman}ß in the prevention of diabetic
nephropathy{[}114{]}.

An additional, albeit invasive approach to develop diabetic nephropathy,
is through the use of unilateral nephrectomy (UNx) that can
significantly shorten the time required to develop the
pathophysiological signs of diabetic nephropathy. This technique
involves removing the left kidney after ligation of the renal artery,
vein and ureter. Initially performed on STZ-induced diabetic
Sprague-Dawley rats, it was later applied to mouse models to develop
enhanced renal injury. Together with STZ, it has a synergistic effect by
accelerating the development of glomerular lesions and impaired renal
function. It has been shown that the combination of STZ and UNx is an
effective way to induce diabetic nephropathy consistent with Type1
diabetes, especially in the resistant C57BL/6J mouse strain{[}115{]}.
This model includes albuminuria, thickening of the glomerular basement
membrane, mesangial matrix expansion, glomerular hypertrophy and
upregulation of pro-fibrotic markers{[}115{]}. However, the use of a
western-diet to induce Type2 diabetes was not effective in inducing
significant albuminuria and glomerular injury when combined with STZ and
UNx, despite the presence of hyperglycaemia. A possible explanation for
this protective effect may have been driven by metabolic changes in the
renal cells of the remaining kidney after UNx, where the
hyperfunctioning nephron is able to avoid hypoxia-induced injury.
Moreover, 15-weeks of a western diet may not have been sufficient to
induce renal injury due to the high cholesterol, potentially providing
protective effectives in the shorter-term (\textless{}4 months),
therefore longer HFD-feeding may be required
(\textasciitilde{}9months){[}115{]}.

More recently, the incorporation of proteomics and/or metabolomics
studies is providing novel insights into the molecular pathways of
diabetic nephropathy, as well as identifying mouse models that more
closely phenocopy human diabetic kidney disease. Metabolomics analysis
predominantly involves nuclear magnetic resonance spectroscopy or
chromatography-mass spectrometry, enabling either the detection of known
metabolites or numerous metabolites respectively, the latter technique
without any prior knowledge of the metabolites required{[}116{]}. Using
a proteomics and metabolomics approach, Rossi et al showed that a tissue
inhibitor of metalloproteinases (TIMP3) knockout mouse model alone or in
combination with STZ-mediated diabetes, led to significant alterations
in kidney lipid metabolism and peripheral acylcartinine levels, which
have been implicated in Type2 diabetes{[}117{]}. Loss of TIMP3 in the
kidney was associated with features of diabetic nephropathy including
higher levels of medium and long chain acylcarnitine in the blood. In
contrast, levels of free carnitine and short chain beta-hydroxy and
dicarboxy-acylcarnitines were lower diabetic in TIMP3 knockout mice as
compared to WT-STZ mice{[}117{]}. Similar metabolomics signatures were
found in a study by Lim et al who showed the accumulation of a number of
short acylcarnitines in the plasma of patients who developed diabetic
kidney disease over time, as well as data by Gurley et al who found
elevated levels of short acylcarnitines in the Akita mouse{[}118{]},
indicating that this model might closely resemble the human metabolic
profile. Furthermore, the incorporation of flow cytometry allows the
identification of immune cell-specific population in diabetic
nephropathy. Indeed, a significant increase of intrarenal CD3+ T cells
that was associated with augmented production of interferon-\selectlanguage{greek}γ \selectlanguage{english}and TNF-\selectlanguage{greek}α
\selectlanguage{english}in diabetic mice was revealed via flow cytometry{[}119{]}. In addition,
transcriptomic profiling using scRNA-seq is identifying factors
underlying the pathophysiology of diabetic nephropathy and is therefore
a useful tool for identifying new therapeutic avenues for research.
Using scRNA-seq, Fu et al demonstrated that the number of glomerular
cell-specific markers that were significantly higher in diabetic mice
compared with controls. These immune cells were predominantly
macrophages and greater number expressed M1 phenotype than M2. Moreover,
regulation of angiogenesis and migration pathways in endothelial cell
from diabetic mice were altered{[}120{]}. Hence, these studies, in
addition to adding valuable insights into the diseased kidney, also
highlight the potential to generate a mouse model more prone to diabetic
nephropathy.

\emph{Diabetic retinopathy}

\emph{3.2.1. Retinal pathophysiology: key features that need to be
represented in animal models}

Diabetic retinopathy is the leading cause of visual impairment leading
to blindness impacting approximately one third of the diabetic
population{[}121{]}. Hyperglycaemia induced vascular damage involves
several metabolic pathways analogous to those affecting diabetic
nephropathy, including the polyol pathway, formation of AGEs and the PKC
pathway. These pathways accelerate the loss of pericytes, apoptosis of
endothelial cells, microaneurysms and thickening of the basement
membrane, resulting in capillary occlusions and ischaemia. These
processes trigger the upregulation of the growth factor VEGF, promoting
vascular permeability and angiogenesis. This contributes to the
progression of proliferative diabetic retinopathy and diabetic macular
oedema, ultimately leading to vision loss and blindness{[}122{]}.

\emph{3.2.2. Models of diabetic retinopathy}

Rodent models are the most commonly used animal models to study diabetic
retinopathy. Traditionally, the mouse model of retinopathy of
prematurity (ROP) has been used as a surrogate model to study the
formation of new malformed and leaky vessels, a hallmark of diabetic
retinopathy. In this model, pups with their mothers are placed in an
oxygen chamber (75\% O2 \selectlanguage{ngerman}± 5\% and 2\% CO2 for 22h/day) at postnatal day
7 until postnatal day 11, after which they are returned to room air
until day 18. The early exposure to elevated oxygen causes a central
obliteration of the vasculature, whilst the return to room air mediates
an hypoxia-driven increase in VEGF via HIF-1a resulting vascular
neogenesis. Using this model, we have shown that oxidative stress is a
key driver of neovascularisation{[}123{]}, and that interventions like
ebselen to bolster specific antioxidants such glutathione
peroxidase-1{[}124{]} leads to significant attenuation of
vaso-obliteration and neovascularisation. In addition, by targeting the
master regulator of oxidative stress, namely the transcription factor
Nrf2 via the bardoxolone-methyl mimetic dh404, we additionally
demonstrated that a global approach to bolstering antioxidant defence
improved ROP in our mouse models{[}125{]}.

To study diabetic retinopathy \emph{per se} mouse or rat models can be
used after exposure to STZ. Rat models (a single tail vein injection of
STZ at 55mg/kg with daily insulin) enable robust analysis of vascular
leakage after 10-weeks of diabetes. In most experimental models,
Sprague-Dawley rats are used for diabetic retinopathy studies. Gene and
protein expression analysis of diabetic retinae enable the study of the
molecular pathways involved, with emphasis on VEGF since this growth
factor is the main causal factor in the vascular permeability that
accompanies diabetic retinopathy. Similarly, the use of STZ has enabled
the long-term study of diabetic retinopathy in mice. As mentioned
earlier, female mice are known to be resistant to STZ. Recently, a study
by Saadane et al have reported that the resistance of female C57BL/6J
mice to STZ-induced diabetes can be overcome by injecting higher doses
of STZ{[}26{]}. Female mice administered a dose of 75mg/kg of body
weight over 5 consecutive days, developed equivalent levels of
hyperglycaemia as male counterparts administered 55mg/kg of body weight
over 5 consecutive days, which was maintained for at least 8months,
enabling a robust evaluation of diabetic retinopathy in this model.
Importantly, both male and female diabetic mice developed similar
abnormalities in retinal oxidative stress as measured by the level of
superoxide in the retinae. This was accompanied by significantly
increased expression of pro-inflammatory proteins, leucocyte mediated
cytotoxicity and similarities in the number of degenerated capillaries
in both male and female mice{[}26{]}. Thus, varying STZ dosage is an
easy adjustment to enable the inclusion of both sexes in the exploration
of therapeutic agents for complications such as diabetic retinopathy.

Inflammatory processes play a key role in driving diabetic retinopathy.
To directly assess a role for inflammatory cytokines in the progression
of diabetic retinopathy, Mugisho et al have developed a novel animal
model that incorporates both hyperglycaemia and inflammation via single
intravitreal injections of the pro-inflammatory cytokines, IL-1ß and
TNF-\selectlanguage{greek}α, \selectlanguage{english}in non-obese diabetic (NOD) mice{[}126{]}. The injection of
intravitreal cytokines evokes inflammation in the surrounding ocular
tissue and exhibits retinal oedema, increased astrogliosis and microglia
upregulation. In addition, pericyte loss observed by advanced vessel
dilation and vessel beading, endothelial cell death and blood-retina
barrier breakdown, are prominent{[}126{]}. The model may provide a new
opportunity to further study the pathology of diabetic retinopathy,
although its proneness to autoimmunity due to a genetic defect causing
cytokine dysregulation could be a limitation. However, the effectiveness
of this model was further highlighted in a follow-up study where
connexcin43, an inhibitor of inflammation and inflammasome activation,
was shown to significantly attenuate diabetic retinopathy{[}127{]}.

\emph{3.2.3 Emerging experimental analysis of diabetic retinopathy}

In most preclinical animal models, fluorescein angiography, optical
coherence tomography and electroretinography are widely utilized methods
to determine the functional changes of diabetic retinopathy.
Additionally, inflammation and oxidative stress markers can be
quantified by performing histology, real-time PCR, western blots, ELISA,
immunohistochemistry and immunofluorescence using specific antibodies.

More recently, the discovery of a novel laser speckle flowgraphy imaging
technique has emerged as a rapid and non-invasive method to determine
basal blood flow and neuronal activity-dependent alterations in ocular
diseases. Not only serving as a useful biomarker to detect and monitor
postoperative outcomes for retinal diseases, this technique enables the
detection of reduced blood flow volume and dysregulated retinal
microvasculature that can cause hypoxia and eventually retinal
neovascularization{[}128{]}. Moreover, the use of positron emission
tomography (PET) imaging using {[}\textsuperscript{18}F{]}FP-(+)-DTBZ
has been reported as an early diagnostic tool to evaluate diabetic
retinopathy in rat models. The {[}\textsuperscript{18}F{]}FP-(+)-DTBZ
PET targeting of vesicular monoamine transporter 2 (VMAT2) changes have
been shown to detect dopaminergic neuron loss in the retina, a known
characteristic symptom of early diabetic retinopathy{[}129{]}.

A major limitation of current screening processes to detect diabetic
retinopathy is that disease progression can go undetected until
irreversible damage and blindness has occurred. The development of a
biomarker for clinical screening would be a useful tool to overcome
limitations such as the need for diagnosis by a qualified specialist,
which can be costly, thereby restricting the population that can be
effectively screened. To overcome this, Tawfik et al evaluated
homocysteine (Hcy), a sulfur-containing amino acid known to induce the
death of retinal ganglion cells, as a biomarker of diabetic
retinopathy{[}130{]}. Indeed, an increase in Hcy levels was observed in
the serum, vitreous humour and retina of both experimental animal models
of diabetes and diabetic patients, while optical coherence tomography
and fluorescein angiography has confirmed that Hcy-induced retinal
changes in diabetic mice are aggravated{[}130{]}. In summary, Hcy should
be investigated further as a strong biomarker candidate for diabetic
retinopathy screening.

\emph{Diabetic neuropathy}

\emph{3.3.1. Nerve pathophysiology: key features that need to be
represented in animal models}

Diabetic neuropathy, characterised by damage to peripheral nerve
function, is a highly prevalent complication of diabetes occurring in at
least 6\%-50\% of diabetic patients depending on age and duration of
diabetes{[}131, 132{]}. Diabetic patients often experience a high degree
of morbidity, including poor balance and co-ordination leading to
frequent falls, numbness, pain and ulceration of the lower limbs that
can lead to limb amputation. Amongst the different forms of neuropathy,
distal symmetric polyneuropathy commonly affecting the hands and lower
limbs, is the most prevalent form of diabetic neuropathy. Hyperglycaemia
induces accumulation of acylcarnitines that can trigger axonal
degeneration. In addition, increased glucose levels lead to increased
ROS, inflammation and AGE production, contributing to nervous system
dysfunction{[}131{]}. Despite its high prevalence, the exact aetiology
remains unknown, with available treatment options mostly ineffective,
leaving the focus for patients mainly on improving glycaemic control or
advocating for lifestyle modifications.

\emph{3.3.2. Models of diabetic neuropathy}

Although animal models have been an invaluable tool in understanding the
pathophysiology of diabetic vascular complications, the onset and
progress of diabetic neuropathy in commonly used rodent models is not
completely understood. Recent advances in animal models to study
diabetic neuropathy have concentrated on models of Type2 diabetes. These
advances have been made possible by creating models that are based on
guidelines established by the Neurodiab Study Group of the European
Association for the Study of Diabetes (NSGEASD) that determined that
assessments of behaviour, motor and sensory nerve conduction velocity
(NCVs), and nerve structure are essential requirements for a murine
model of diabetic neuropathy{[}133{]}.

Gregorio et al have characterized the onset and progression of Type2
diabetic neuropathy in the diabetic db/db mouse on a C57BLKS/J
background. These mice show hyperphagia{[}134{]} as well as other signs
of diabetic neuropathy including progressive sensory loss,
electrophysiological impairments, decreases in intraepidermal nerve
fibre density, Schwann cells apoptosis and infiltration of T
lymphocyte{[}135{]}. Other studies investigating neuropathy have been
performed in \emph{ob/ob} ~leptin-based mouse models of type 2
diabetes{[}136{]}.

A growing number of animal studies in recent years have demonstrated
that low dose STZ combined with a high-fat diet can recapitulate certain
features of Type2 diabetes as seen in humans. In addition, fructose is
now recognised as a major contributor to the T2D epidemic. Barri\selectlanguage{ngerman}ère et
al have developed a high fat/high fructose (HF/HF)-STZ model of Type2
diabetes in the rat as useful model to study the long-term complications
of Type 2 diabetes, including diabetic neuropathy, nephropathy and
retinopathy{[}137{]}. Monitored over a period of 56 weeks, the HF/HF-STZ
rats exhibited an early prediabetic phase of hyperinsulinemia with
moderate dysglycaemia, developing into late stage hyperglycaemia,
normalisation of insulinemia, marked dyslipidaemia, hepatic fibrosis and
ultimately pancreatic \selectlanguage{greek}β-\selectlanguage{english}cell failure. With respect to neuropathy, the
HF/HF-STZ rats developed a gradual increase in mechanical hyperalgesia
(an intense response to pain indicative of nerve damage) as measured by
decreased mechanical nociceptive thresholds and displayed profound
tactile allodynia, where HF/HF-STZ rats experience increased pain to
relatively minor stimuli compared with controls. These phenotypic
changes were accompanied by significant increases in impaired myelinated
fibres of the sciatic nerves, increases in mitochondrial vulnerability
and functional reorganization within the spinal dorsal horn
circuitry{[}137{]}.

In order to develop a more robust model of early onset neuropathy,
O'Brien et al have characterised the development of neuropathy over time
in a diet-induced obesity (DIO)-STZ mouse model of adult-onset Type2
diabetes and compared this with a model of DIO alone, the latter
indicative of adolescent pre-diabetes. This has enabled a detailed
analysis of neurogenic changes that align with the natural progression
from obesity and impaired glucose tolerance to overt type 2 diabetes in
adolescent and adult mice respectively{[}138{]}. To establish this
model, one cohort was fed a high-fat diet starting at 5 weeks of age,
whilst a second cohort additionally received low dose STZ at 12 weeks of
age (75mg/kg body weight for the first injection and 50mg/kg body weight
for the second injection, 72 hours later) to cause mild impairment of
insulin secretion and mild hyperglycaemia. Comparisons between the 2
cohorts at 16, 24 and 36 weeks displayed similar levels of decreased
motor and sensory NCVs as early as 16 weeks followed by hypoalgesia and
cutaneous nerve fibre loss at the later timepoints, despite the two
groups having vastly different metabolic profiles{[}138{]}. Hence, this
dual animal model approach may be more useful in understanding
neurological pathophysiology of the young, enabling the development of
novel therapies to target early neurological changes that are much
needed with the growing prevalence of obesity and neurological changes
of adolescent and young adults.

A less well-known, albeit invasive approach to develop diabetic
neuropathy can be achieved by sciatic nerve transection. Focusing on the
changes occurring in sensory nerve fibres with initial degeneration and
regeneration processes resulting in pain, Pham et al developed a Type2
diabetic mouse model using sciatic nerve transection to study
pathogenesis and treatment of early diabetic neuropathy{[}139{]}. Type2
diabetes was induced by injection of STZ and nicotinamide (NA) which was
used to partially protect pancreatic \selectlanguage{greek}β \selectlanguage{english}cells from the toxic effects of
STZ. This allowed the diabetic status to be more stable over a longer
period of time, ensuring a high survival rate of the animals{[}140{]}.
Sciatic nerve transection involves exposing the right sciatic nerve and
inserting a silicone tube into the proximal and distal stumps leaving a
gap between them. This model enables the evaluation of nerve
regeneration in the same animal over several months. Of particular
importance, these animals display mechanical hyperalgesia and a decrease
in the number of intra-epidermal nerve fibres{[}140{]}. Since an
effective treatment for diabetic neuropathy involves the prevention of
progressive nerve degeneration whilst simultaneously promoting nerve
regeneration, using the nerve transection-regeneration model enables the
study of effective treatments for pre-diabetic and early stage diabetic
neuropathy.

\textbf{Conclusions and Perspectives}

Animal models of diabetic complications have been developed over a
number of years to best replicate the changes observed in humans. The
need to more closely recapitulate critical aspects of the disease led to
the establishment of the Animal Models of Diabetic Complications
Consortium (AMDCC) in 2001 by the National Institutes of Health, with a
guidelines paper published in 2007{[}141{]} specifically focused on the
evaluation of cardiovascular complications. The goals of the AMDCC were
to best characterize and validate various animal models of diabetic
cardiovascular disease for basic, developmental, or translational
research including outlining testing, prevention, early detection,
therapy, and diagnostic imaging strategies. Since then, other consortia
(e.g. NSGEASD: neuropathy; NIDDK Diab Complications Consortium
(DiaComp): nephropathy, uropathy, retinopathy, wound healing,
gastro-intestinal and liver disease, cardiovascular disease) have made
available useful resources to standardise experimental protocols
(https://www.diacomp.org). In this review, we have focussed on more
recent advances as well as modifications to older methods that are
allowing more nuanced analyses of diabetic micro and macrovascular
complications. Careful choice of rodent model, as well as the mode of
diabetes induction, duration of disease and window of study, whether
that be younger (pre-diabetic models) or older more established T2D
diabetic models, are all critical considerations that may impact the
outcomes of a study. For example, this review has emphasized the
appropriateness of using STZ in the induction of T1D with caveats around
dose, gender, off-target toxicity and alterations in gut microbiota. In
addition, this review has highlighted relevant murine models for both
micro- and macrovascular complications and the need for the inclusion of
both male and female models. Additionally, where appropriate,
limitations of the models have been noted. These considerations will
assist in effective study design to maximise results for the evaluation
of novel therapies to limit these debilitating complications.

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\textbf{Figures and Tables}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1: Summary of common mouse models of diabetes-associated
atherosclerosis at various stages of the disease.} STZ: streptozotocin;
ApoE: Apolipoprotein E; LDLR: Low density lipoprotein receptor; db/db:
leptin receptor deficient; ob/ob: leptin deficient;
ApoB\textsuperscript{100/100}: Apoliprotein B100; IRS: Insulin receptor
substrate; Insr: Insulin receptor; WD: Western Diet; GK: Glucokinase;
LCMV: Lymphocytic choriomeningitis virus; hAR: Human aldose reductase;
DD: diabetogenic diet; IGF-11: Insulin growth factor II;
Fbn\textsuperscript{C1093G+/-}: Fibrillin-1 habouring a heterozygous
C1093G. * Model of endothelial dysfunction.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

Table 1: Summary of experimental analysis and research output for models
of diabetes-associated atherosclerosis.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

Table 2: Summary of experimental analysis and research output for models
of diabetes-associated microvascular complications.

\selectlanguage{english}
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