**3. Models of diabetic retinopathy**

Animal models of DR can be broadly classified into (1) diabetic models by pharmacological induction, diet induction or genetic manipulation and (2) non-diabetic models of proliferative retinopathy and angiogenesis. To date, no diabetic models fully develop end-stage retinopathy, arguably due to the short lifespan of animals and differing anatomical structure from humans. Non-diabetic models are thus used to mimic the pathophysiology of end-stage DR, specifically the proliferative pathogenesis and neovascularization in the retinal vasculature. These models, however, are not DR-specific, and display phenotypes common to other conditions with retinal neovascularization. While animal models are useful for drug testing and furthering our understanding of the molecular and cellular pathological processes involved in DR, no single model can holistically reproduce the pathological features of human DR. BRB breakdown, for example, is exhibited in numerous animal models. Yet macular edema resulting from the increase in permeability of retinal capillaries is seldom observed. Judicious evaluation and selection of models according to research objectives is critical to avoid inappropriate translation of experimental findings to the clinical situation. An overview of existing models used to study DR is summarized in **Table 2**. The cellular, molecular and morphological features of existing animal models of DR are described in Section 4 of this chapter and Section 1 of the following chapter (Animal Models of Diabetic Retinopathy Part 2).

#### **3.1. Diabetic models**

#### *3.1.1. Pharmacological induction of diabetes*

Pharmacological induction of diabetes is most commonly performed using streptozotocin (STZ), a naturally occurring antibiotic in *Streptomyces acromogenes*, or alloxan, a pyrimidine derivative. Both chemicals destroy the β-cells of the pancreatic islets. STZ is preferentially used over alloxan due to its greater stability and more preferable chemical properties [31]. T1D or T2D can be induced by varying the dosage and/or number of doses administered, or by combination administration with other treatments (e.g. STZ injection with nicotinamide administration or high fat diet feeding). The use of this model to induce T1D is more common due to the inability of the two chemicals to directly induce insulin resistance. Low doses of

**Model** Diabetic

Pharmacological

• STZ-induced

• Alloxan-induced

models

induction

Genetically diabetic

• Mice


Type 1

• Consistent phenotype

• High success rate of

hyperglycemia induction

• No further manipulation

required

or 2


• Rats:



Zucker diabetic fatty (ZDF) rat, Otsuka Long-Evans Tokushima fatty (OLETF) rat, non-obese Goto-Kakizaki (GK) rat, spontaneously diabetic

Torii (SDT) rat, TetO rat

Diet-induced

Galactose-feeding

Type 2

• Longer lifespan of animals

• Longer time required to develop

DR features

• Allows for analysis of

retinal features in animals

beyond 1 year of age

• Isolated elevation of hexose

levels without metabolic

abnormalities of diabetes

Animal Models of Diabetic Retinopathy (Part 1) http://dx.doi.org/10.5772/intechopen.70238 17

**Diabetes**

Type 1

• Quick induction

• Lower cost

(or 2)

**Advantages**

**Limitations**

• Individual animals may

demonstrate resistance to STZhyperglycemia induction • Requires exogenous injections

• Short lifespan of animals

• Toxicity of drugs

• Higher cost

• Breeding time required


potential (OPs), particularly OP1, precede retinopathy development [23, 24]. The OPs are generated by inner retinal neurons and are often considered to be reflections of feedback circuits between amacrine and bipolar cells and/or circuits between amacrine and ganglion cells. Eyes with NPDR display a reduction in OP amplitudes [24, 25] and an increase in OP peak latencies [25]. There is some discrepancy regarding the onset of changes in b-wave responses, which are largely generated by depolarizing bipolar cells with some contribution from Müller cells. B-wave implicit times appear to be increased even in early stages of DR [26] while reductions in b-wave amplitudes have been suggested to be predominantly found in eyes with PDR [25, 27, 28]. Changes in OP amplitude and implicit times have also been suggested to be a reflec-

Animal models of DR can be broadly classified into (1) diabetic models by pharmacological induction, diet induction or genetic manipulation and (2) non-diabetic models of proliferative retinopathy and angiogenesis. To date, no diabetic models fully develop end-stage retinopathy, arguably due to the short lifespan of animals and differing anatomical structure from humans. Non-diabetic models are thus used to mimic the pathophysiology of end-stage DR, specifically the proliferative pathogenesis and neovascularization in the retinal vasculature. These models, however, are not DR-specific, and display phenotypes common to other conditions with retinal neovascularization. While animal models are useful for drug testing and furthering our understanding of the molecular and cellular pathological processes involved in DR, no single model can holistically reproduce the pathological features of human DR. BRB breakdown, for example, is exhibited in numerous animal models. Yet macular edema resulting from the increase in permeability of retinal capillaries is seldom observed. Judicious evaluation and selection of models according to research objectives is critical to avoid inappropriate translation of experimental findings to the clinical situation. An overview of existing models used to study DR is summarized in **Table 2**. The cellular, molecular and morphological features of existing animal models of DR are described in Section 4 of this chapter and Section 1

Pharmacological induction of diabetes is most commonly performed using streptozotocin (STZ), a naturally occurring antibiotic in *Streptomyces acromogenes*, or alloxan, a pyrimidine derivative. Both chemicals destroy the β-cells of the pancreatic islets. STZ is preferentially used over alloxan due to its greater stability and more preferable chemical properties [31]. T1D or T2D can be induced by varying the dosage and/or number of doses administered, or by combination administration with other treatments (e.g. STZ injection with nicotinamide administration or high fat diet feeding). The use of this model to induce T1D is more common due to the inability of the two chemicals to directly induce insulin resistance. Low doses of

tion of the severity and prospective progression of DR [24, 25, 27].

16 Experimental Animal Models of Human Diseases - An Effective Therapeutic Strategy

of the following chapter (Animal Models of Diabetic Retinopathy Part 2).

**3. Models of diabetic retinopathy**

**3.1. Diabetic models**

*3.1.1. Pharmacological induction of diabetes*

levels without metabolic

abnormalities of diabetes


insulin are required for maintenance of STZ or alloxan-induced diabetic animals. It is important to note that failure of hyperglycemia induction may occur in individual animals due to STZ resistance. Blood glucose monitoring is hence essential for confirmation of hyperglycemia development. A review by Lai and Lo [32] comprehensively details existing regimens for

Animal Models of Diabetic Retinopathy (Part 1) http://dx.doi.org/10.5772/intechopen.70238 19

Spontaneous hyperglycemia can occur in animals carrying endogenous mutations. Inbreeding of mutated animals with wild-type animals generates reliable hyperglycemic models with consistent phenotype expression. However, the establishment of large colonies may be timeconsuming. The target genes for genetic manipulation in specific animal models (e.g. insulin 2 gene mutation in the *Ins2Akita* mouse; leptin receptor gene mutation in the *db/db* mouse) are

Experimental galactosemia via feeding with 30–50% galactose can also be used to induce diabetic retinopathy. Galactose feeding causes the isolated elevation of blood aldohexose levels. Other metabolic abnormalities (e.g. alterations in insulin, glucose, fatty acids, amino acid levels) characteristic of diabetes are absent in this model [33]. Despite the long feeding time required for the onset of DR-like lesions, these animals have a longer lifespan than other diabetic models. The model may hence be able to reflect the retinal complications arising from a

Originally developed as a model for retinopathy of prematurity, the oxygen-induced retinopathy (OIR) model has also been used to investigate angiogenesis in other retinal diseases, including proliferative DR. The OIR model is mostly used in small rodents such as mice and rats. In brief, neonatal rodents are exposed to hyperoxia to induce vaso-obliteration. Upon removal from hyperoxia, hypoxia develops in the retina. This triggers a compensatory revascularization response, resulting in neovascularization [34]. This model differs from DR in that OIR-induced neovascularization occurs in incompletely differentiated retinae, while neovascularization in DR results from progressive retinal ischemia and capillary obliteration in fully

The OIR mouse model involves exposing postnatal 7-day-old (P7) mice to 75% oxygen for 5 days before placing them back in normoxia at P12. Upon return to room air, vessel regrowth occurs at P12–P17, with neovascularization beginning at P14. Neovascularization peaks at P17

and complete spontaneous resolution is subsequently achieved by P25 [35, 36].

detailed in Section 4 of this chapter and Section 1 of the following chapter.

prolonged period of isolated elevated hexose levels.

*3.2.1. Oxygen-induced retinopathy (OIR) model*

induction of diabetes using STZ.

*3.1.2. Genetically diabetic animals*

*3.1.3. Diet induced*

**3.2. Angiogenesis models**

differentiated retinae.

*3.2.1.1. OIR mouse model*

insulin are required for maintenance of STZ or alloxan-induced diabetic animals. It is important to note that failure of hyperglycemia induction may occur in individual animals due to STZ resistance. Blood glucose monitoring is hence essential for confirmation of hyperglycemia development. A review by Lai and Lo [32] comprehensively details existing regimens for induction of diabetes using STZ.

#### *3.1.2. Genetically diabetic animals*

Spontaneous hyperglycemia can occur in animals carrying endogenous mutations. Inbreeding of mutated animals with wild-type animals generates reliable hyperglycemic models with consistent phenotype expression. However, the establishment of large colonies may be timeconsuming. The target genes for genetic manipulation in specific animal models (e.g. insulin 2 gene mutation in the *Ins2Akita* mouse; leptin receptor gene mutation in the *db/db* mouse) are detailed in Section 4 of this chapter and Section 1 of the following chapter.

#### *3.1.3. Diet induced*

Experimental galactosemia via feeding with 30–50% galactose can also be used to induce diabetic retinopathy. Galactose feeding causes the isolated elevation of blood aldohexose levels. Other metabolic abnormalities (e.g. alterations in insulin, glucose, fatty acids, amino acid levels) characteristic of diabetes are absent in this model [33]. Despite the long feeding time required for the onset of DR-like lesions, these animals have a longer lifespan than other diabetic models. The model may hence be able to reflect the retinal complications arising from a prolonged period of isolated elevated hexose levels.

#### **3.2. Angiogenesis models**

#### *3.2.1. Oxygen-induced retinopathy (OIR) model*

Originally developed as a model for retinopathy of prematurity, the oxygen-induced retinopathy (OIR) model has also been used to investigate angiogenesis in other retinal diseases, including proliferative DR. The OIR model is mostly used in small rodents such as mice and rats. In brief, neonatal rodents are exposed to hyperoxia to induce vaso-obliteration. Upon removal from hyperoxia, hypoxia develops in the retina. This triggers a compensatory revascularization response, resulting in neovascularization [34]. This model differs from DR in that OIR-induced neovascularization occurs in incompletely differentiated retinae, while neovascularization in DR results from progressive retinal ischemia and capillary obliteration in fully differentiated retinae.

#### *3.2.1.1. OIR mouse model*

**Model** Nondiabetic

Oxygen-induced

• Continuous hyperoxia

• Alternating cycles of hyperoxia and

hypoxia

→ normoxia

→ normoxia

/

• Consistent and reproducible

neovascularization

retinopathy (OIR)

models

**Diabetes**

**Advantages**

**Limitations**

• Phenotype not specific to DR

• Mostly for small rodents (mice,

rats)

• Only applicable to newborn

rodents

• Neovascularization in

undifferentiated retina

• Varying ocular angiogenesis

responses in differing strains of rats

• Spontaneous regression of

neovascularization features within

1 week of neovascularization

18 Experimental Animal Models of Human Diseases - An Effective Therapeutic Strategy

development

• Phenotype not specific to DR

• Acute ischemia

Retinal occlusion

Intraocular

• Vascular endothelial growth factor (VEGF)

/

injection

• (Fibroblast)

• Retinal vein occlusion

/

• Neovascularization in fully

differentiated retinae

• Quick induction of

neovascularization response

• Displays NPDR and PDR

• Phenotype not specific to DR

• Mainly applicable to large

animals (e.g. rabbits)

• Long duration of exogenous

injection of pro-angiogenic

molecules required

• Mimics proliferative

vitreoretinopathy more than

ischemic retinopathy (fibroblast

injection)

• Cost

• Some strains not commercially

• Phenotypes may not be specific

• Changes do not necessarily occur

due to prolonged hyperglycemia

to DR

available

Transgenic mice

**Table 2.**

Overview of existing models used to study DR.

mice

• Mice: Kimba mice, Akimba mice, TgIGF-I

(Akimba:

• Exhibits reproducible

neovascularization

type 1)

features (VEGF injection)

The OIR mouse model involves exposing postnatal 7-day-old (P7) mice to 75% oxygen for 5 days before placing them back in normoxia at P12. Upon return to room air, vessel regrowth occurs at P12–P17, with neovascularization beginning at P14. Neovascularization peaks at P17 and complete spontaneous resolution is subsequently achieved by P25 [35, 36].

#### *3.2.1.2. OIR rat model*

The OIR rat model involves either continuous hyperoxia or alternating cycles of hyperoxia and hypoxia. In general, the continuous hyperoxia model involves placing rats under 80% oxygen conditions for 22 hours per day until P11. Rats are then transferred to room air for 7 days (P11–P18). In the alternating hyperoxia model, newborn rat pups are exposed to sustained cycles of hyperoxia (50–80%)/hypoxia (SHH) for 14 days and subsequently returned to room air [37, 38]. OIR methods involving the use of varying oxygen concentrations have been described.

#### *3.2.2. Retinal occlusion*

Retinal vein occlusion via laser photocoagulation or photodynamic therapy has been used to induce neovascularization in fully differentiated retinae of mice, rats, pigs and monkeys [39–43]. This model induces a near immediate neovascular response with development of retinal edema within hours and the development of intravitreal vessels within days. As DR is predominantly a chronic ischemic disorder, the use of these retinal occlusion models involving periods of reperfusion following acute ischemia induction is less suitable.

#### *3.2.3. Intraocular injection of vascular endothelial growth factor (VEGF)*

In view that pro-angiogenic molecules are strongly implicated in retinal neovascularization, researchers have injected VEGF and cultured fibroblasts into monkeys and rabbits, respectively. Intravitreal injection of VEGF in monkeys successfully induced the development of many NPDR and PDR features [44]. However, the rabbit model involving intravitreal injection of fibroblasts mimicked proliferative vitreoretinopathy more than ischemic retinopathy, as the elicited neovascular response was more traumatic and inflammatory than ischemic [45, 46].

#### *3.2.4. Transgenic models*

Transgenic mouse models of neovascularization include the Kimba mouse, Akimba mouse and transgenic mouse overexpressing insulin growth factor I, as detailed in the following section.
