**4. DR features of animal models**

Among all of the existing animal models of DR, mice and rats are most commonly used, possibly due to their small size, availability, genetic tractability and relatively faster development of DR lesions as compared with larger animals. **Table 3** summarizes the cellular, molecular and morphological features of mouse models of DR. Features of rat and non-rodent models are detailed in the next chapter (Animal Models of Diabetic Retinopathy Part 2).

**Mouse model**

**Type of** 

**Hyperglycemia** 

**Cellular, morphological and vascular features of human DR displayed in mouse models**

**(Age at which correlates are first reported unless otherwise specified)**

**+Time post treatment: diabetes, galactosemia, or induction of VEGF overexpression)**

**PDR features**

*• 21 days+*:

*• 4*

*wks+*: decreased OP3

and total OP amplitude

neovascularization\* [63]

*• 17*

*wks+*: increased

[60, 61]

Prolonged OP2, 3

implicit time [61]

*• 6*

*mo+*: decreased

a-wave and b-wave

amplitudes [51]

density of capillaries

suggestive of

neovascularization [110]

\*in a novel FOB\_FT strain

of mice

**Functional changes (ERG)**

**diabetes**

**onset**

**(**

**NPDR features**

STZ injection

1 (or 2)

Within 1 week

(wk)

*• 7 days+*: Müller cell gliosis\* [63]

RGC loss\* [63]

*• 2 wks+*

*• 3–4*

*• 4*

*• 8 days+*: increased vascular permeability [109] (*2 mo+*) [52]

*:* increased RGC apoptosis [50]

*wks+*: decreased total, GCL, IPL, OPL thickness [51]

*wks+*: decreased arteriolar velocity [58, 59]

Decreased venular velocity [58]

Decreased arteriolar and venular shear rates [58]

Decreased arteriolar and venular blood flow rate [58]

Decreased arteriolar and venular diameter (not

observed at 8 wks post diabetes induction) [59]

*• 21 days+*: increased acellular capillaries\* [63] (*6 mo+*) [53]

(*9 mo+*) [51, 54]

IRMAs\* [63]

Possible venous dilation or beading\* [63]

Preretinal neovascular tufts\* [63]

*wks+*: reactive gliosis and increased number of astrocytes

*• 5*

[47]

*• 6*

[50]

*• 2* *• 10*

*• 3* *• 17*

*• 6*

Alloxan

1 (or 2)

1–4

65]

days [64,

injection

*mo+*: capillary apoptosis [53]

*• 7 days+*: disorganized capillaries\* [63]

*• 3*

*wks+*: decreased

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

b-wave amplitude [65,

66]

*• 3* wave ratio [64]

Delayed OPs [64]

21

*mo+*: decreased b/a-

*• 21 days+*: microaneurysm\* [63]

IRMA-like lesions\* [63]

[63]

*• 3*

*mo+*: shortened dendrites in microglia [64]

\*in a novel FOB\_FT strain of mice

Capillary dilatation with preretinal neovascular lesions\*

*mo+*: pericyte loss [52] (*6 mo+*) [53] (*9 mo+*) [54]

Leukostasis [56, 57] (*3 mo+*) [51, 54]

*wks+*: decreased total, INL and ONL thickness [50]

*mo+*: increased number of leukocytes [54]

*wks+*: capillary basal lamina thickening [55]

*wks+*: reduced number of RGCs [48] (*7*

*wks*) [49] (*10 wks*)


*3.2.1.2. OIR rat model*

been described.

is less suitable.

than ischemic [45, 46].

*3.2.4. Transgenic models*

**4. DR features of animal models**

section.

*3.2.2. Retinal occlusion*

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

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

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

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

Among all of the existing animal models of DR, mice and rats are most commonly used, possibly due to their small size, availability, genetic tractability and relatively faster development of DR lesions as compared with larger animals. **Table 3** summarizes the cellular, molecular and morphological features of mouse models of DR. Features of rat and non-rodent models

are detailed in the next chapter (Animal Models of Diabetic Retinopathy Part 2).

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

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


**Type of** 

**Hyperglycemia** 

**Cellular, morphological and vascular features of human DR displayed in mouse models**

**(Age at which correlates are first reported unless otherwise specified)**

**+Time post treatment: diabetes, galactosemia, or induction of VEGF overexpression)**

**PDR features**

*• 6*

*mo*: disordered focal

proliferation of new vessels

[85]

**Functional changes (ERG)**

**diabetes**

**onset**

**(**

**NPDR features**

NOD

1

Female mice:

*• 3*

*wks+*: arteriolar vasoconstriction (in close proximity to

*wks+#:* ganglion cell, pericyte, endothelial cell apoptosis

initial onset at

venules) [83]

12–14 wks of

*• 4*

age [81]; 80%

[84]

reaching hyperglycemia at 30

Retinal capillary BM thickening [84]

Perivascular edema [84]

wks

*• 6*

*mo:* retinal microvessel loss [85]

Major vessel vasoconstriction or degeneration [85]

#changes became more obvious after 12

hyperglycemia

db/db

2

4–8 wks of age

*• 8*

*wks*: reduced number of RGCs [88]

*• 15* proliferation [87]

*mo*: retinal capillary

*• 8 wks:* *•* Progressive

reduced c-wave

amplitude [90]

*•* Reduction in fast

oscillation amplitude [90]

*• 12 wks* *•* Reduction in off

response amplitude [90]

*• 16 and 24 wks*

*•* Increased b-wave

implicit time [88]

*•* Reduced b-wave

amplitude [88, 90]

*•* Increased

oscillatory potential (OP)

implicit time (scotopic

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

conditions) [88]

*•* Reduced OP

amplitude (scotopic

conditions) [88]

*• 24 wks* *•* Reduced a-wave

amplitude [90]

23

Increased apoptotic cells in GCL [88] (15 mo) [87], INL

Glial activation (increased GFAP expression in Müller

cells) [88, 89] (15 mo) [87]

ONL thinning [88]

DNA fragmentation in photoreceptors [88]

Increased glutamate levels and reduced GLAST content

[88]

BRB disruption [89] (*19*

*• 16* thickness [88]

*• 18* *• 18–20*

[94]

*• 22* *• 26* *• 31*

*wks*: pericyte loss [92] (*15*

 *mo*) [87] *wks*: increased endothelial cell/pericyte ratio [112]

Acellular capillaries [112] (*34 wks*) [92]

*wks:* retinal capillary BM thickening [93]

*wks*: increased RBC velocity [91]

*wks:* increased VEGF and decreased PEDF in vitreous

*wks*: reduced central and peripheral total retinal

*weeks*) [111] (*15*

 *mo*) [87]

[89] and GCL [89]

[86]

weeks of


**Type of** 

**Hyperglycemia** 

**Cellular, morphological and vascular features of human DR displayed in mouse models**

**(Age at which correlates are first reported unless otherwise specified)**

**+Time post treatment: diabetes, galactosemia, or induction of VEGF overexpression)**

**PDR features**

**Functional changes (ERG)**

**diabetes**

**onset**

**(**

**NPDR features**

Galactose-fed /

/

*• 11* *• 13* *mo*) [33, 69]

*• 21*

*mo+*: saccular microaneurysms [33]

Increased capillary BM thickness [33]

\*50% galactose diet (remaining

*Ins2Akita*

1

4 wks of age

*• 8*

*wks*: retinal apoptosis [71–74]

Increased leukocytes adherent to vessels [71]

Activated microglia [71] (*21 wks* [76])

*wks*: increased vascular permeability\* [71] (*9 mo* [73])

Reduced total retinal and outer retina thickness (*in vivo)*

*• 12*

[77]

*• 22*

*• 6*

[77]

*• 25* Microgliosis [76]

*• 7* *31–36* *• 6–9*

*• 9*

*mo:* amacrine cell apoptosis [74]

*wks*: increased number of acellular capillaries [71]

*mo*: microaneurysm formation [73]

*mo:* increased capillary BM thickness [73]

*\**Conflicting results from alternate studies

^In vivo imaging techniques failed to reveal inner retinal

thinning [76, 78]

*wks*: reduced INL and IPL thickness (*ex vivo*)^\* [71]

Reduced number of RGCs\* [71, 72, 75] (*9 mo* [77])

*mo:* reduced inner retinal thickness (INL-NFL)*(in vivo)*

*wks*: increased GFAP expression in Müller cells\* [76]

(male mice)

 =

30% galactose diet)

*• 6–9*

*mo*: retinal

*• 3* amplitude

*9 mo:*

*•* Reduced scotopic

b-waves [73]

*•* Reduced a, b-wave

amplitude [77]

*•* Increased a, b-wave

implicit time [77]

*•* Reduced OP

amplitude [77]

*•* Increased OP implicit

time [77]

*•* Reduced b/a-wave

ratio [77]

*mo*: reduced b-wave

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

neovascularization [73]

*• 7*

*mo*: decreased retinal

blood flow rates [79]

*mo+*: reduced number of endothelial cells [67] (*22 mo*) [69]

Pericyte loss [67] (*22 mo*) [69] (*26 mo*) [33]

*mo+*: acellular capillaries [68] (*15*

*mo\**) [33] (*20 mo*) [67] (*21* 


**Type of** 

**Hyperglycemia** 

**Cellular, morphological and vascular features of human DR displayed in mouse models**

**(Age at which correlates are first reported unless otherwise specified)**

**+Time post treatment: diabetes, galactosemia, or induction of VEGF overexpression)**

**PDR features**

*• 8 wks:* *•* Retinal detachment

[78]

*•* Neovascularization

**Functional changes (ERG)**

**diabetes**

**onset**

**(**

**NPDR features**

Akimba

/

/

*• 8* Pericyte loss [103]

Microaneurysms [78]

Hemorrhage [78]

Vascular leakage (cessation at 20

Reduced endothelial junction protein levels [103]

Vessel tortuosity, dilatation, constriction, beading;

Capillary dropout and capillary non-perfusion [78]

Retinal edema [78]

*• 24*

*wks:* RGC loss [78]

Neural retina thinning [78]

TgIGF-I

/

/

*• 2*

*mo:* pericyte loss [107]

*•* ≥*6 mo*

*• 7.5 mo* *•* Reduced scotopic

b-wave amplitude and

oscillatory potential

amplitude [114]

*•* Retina and vitreous

neovascularization [107]

*•* Retinal detachment

[107]

*•* Neovascular

glaucoma [107]

Retinal capillary BM thickening [107]

Acellular capillaries [107]

*mo:* Increased GFAP expression in Müller cells and

*• 3* astrocytes [107]

Increased VEGF [107]

*•* ≥*6 mo*: venule dilatation [107]

IRMAs [107]

BRB disruption

*7.6*

Intraocular

/

/

*• 2–4*

*wks\*:* venous dilatation

*• 12 wks\** *•* Increase in number of

retinal blood vessels in INL

Animal Models of Diabetic Retinopathy (Part 1) http://dx.doi.org/10.5772/intechopen.70238 25

Microaneurysm

*• 8*

*wks\*:* vascular leakage

\*post VEGF injection

Summary of the cellular, molecular and morphological features displayed in mouse models of DR.

This table has been modified from a review by Lai and Lo [32].

VEGF

injection [108]

**Table 3.**

*mo:* reduced ONL and INL thickness [114]

venous loops [78]

weeks) [78]

[78]

*wks*: uneven retinal thickness on OCT [78]


**Type of** 

**Hyperglycemia** 

**Cellular, morphological and vascular features of human DR displayed in mouse models**

**(Age at which correlates are first reported unless otherwise specified)**

**+Time post treatment: diabetes, galactosemia, or induction of VEGF overexpression)**

**PDR features**

**Functional changes (ERG)**

**diabetes**

**onset**

**(**

**NPDR features**

KKAy

OIR Kimba

/

/

*• P7* reduced total, INL, ONL thickness [101]

*• P28* reduced IPL and outer segment thickness [101]

Microaneurysms [101, 113] (*10 wks*) [100]

Vascular leakage [101] (moderate phenotypes

displaying decline in leakage at 9

leakage at 19 wks (mild and moderate phenotypes)) [102]

Tortuous vessels [101] (*9–19*

dropout [101]

*• 6* *• 9*

*wks*: pericyte loss\* [102]

Acellular capillaries\* [102]

Reduced vessel length\* [102]

Reduced area coverage by vessels\* [102]

Reduced number of crossing points\* [102]

*wks*: capillary non-perfusion [100]

\*for Kimba mice displaying moderate signs of retinopathy; the

observed changes were observed at 24

with a mild phenotype

weeks of age for those

*• 10*

*wks*: increased leucocyte adhesion and leucostasis [102]

*wks*) [102], capillary

weeks and cessation of

(trVEGF-029)

/

/

2

5 wks of age

*• 3 mo*

[98]

*•* Increased capillary BM thickness [98]

*• P18* [99] Reduced IPL and total retinal thickness

*• P18*

[99]

*•* Intravitreal

neovascularization across

all retinal eccentricities

*•* Decreased vessel

profiles in deep plexus

amplitude

*•* Absence of vessels in

the inner retinal plexus

*• P28*

*•* Neovascularization

[100]

*(postnatal day 18)*

*• p18* [99]

*•* Reduced a-wave,

b-wave amplitude

*•* Increased b-wave

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

implicit time

*•* Reduced OP3, OP4

Decreased outer segment length

Müller cell gliosis (increased GFAP expression)

Activated microglia

*•* Retinal neuronal cell apoptosis in GCL and medial INL

[96]

**Table 3.** Summary of the cellular, molecular and morphological features displayed in mouse models of DR. This table has been modified from a review by Lai and Lo [32].

#### **4.1. Mouse models**

#### *4.1.1. Pharmacological*

#### *4.1.1.1. STZ induced*

STZ-induced mice are one of the most commonly used DR models for DR characterization and therapeutic drug studies. The mice develop hyperglycemia within 1 week after being injected with a dose of STZ.

this model may be needed, the FOT\_FT mouse may represent a novel resource for the study of DR related genes and for testing of therapeutic interventions targeting vascular, neural and

Animal Models of Diabetic Retinopathy (Part 1) http://dx.doi.org/10.5772/intechopen.70238 27

Few studies have examined neuronal and vascular DR features of alloxan-induced diabetic C57BL/6 or albino mice, perhaps due to the absence of demonstrable lesions. About 3 months of alloxan-induced diabetes in C57BL/6 mice failed to induce neuronal apoptosis, glial activation, and microaneurysm and hemorrhage formation [64]. Only functional changes on ERG were observed, with decreased b-wave amplitudes at 3 weeks in albino mice [65, 66] and decreased b/a-wave amplitude ratio and increased OP latency at 3 months of hyperglycemia in C57BL/6 mice [64]. Morphologically, shortened dendrites and thickened proximal processes of microglia suggested the activation of microglia after 3 months of diabetes [64]. In the less conventionally used FOT\_FB mouse strain, the study reported disorganized capillaries within 7 days of diabetes induction [63]. By 21 days of diabetes, microaneurysms, IRMAs and capillary dilatation with preretinal neovascular lesions were found in the mice retinae [63].

Mice fed with a 30% galactose diet were found to have reduced endothelial cells and pericyte loss beginning as early as at 11 months of hypergalactosemia [67]. With prolonged hypergalactosemia, the number of acellular capillaries increased [33, 67–69]. By 21–22 months, microvascular changes, including saccular microaneurysms and capillary BM thickening, were present [33]. Variations in age of reported features exist depending on the strain of mice used and the percentage of galactose incorporated into the mice's diet. The majority of reports used

The *Ins2Akita* mouse is a T1D mouse model carrying an endogenous point mutation in the *Mody4* locus (i.e. Insulin 2 gene) with an autosomal dominant mode of inheritance. The mutation results in misfolding of the insulin protein, leading to beta-cell death and decreased insulin secretion, with subsequent development of hypoinsulinemia and hyperglycemia at around 4 weeks of age in male mice. Female mice are less commonly used for DR studies due to their remission to a mild to moderate hyperglycemic state after sexual maturation following transient hyperglycemia during puberty [70]. Males, on the other hand, develop progres-

Early subclinical DR features in heterozygous *Ins2Akita* mice retinae have been consistently reported by numerous studies. Cellular changes observed in humans, including increased retinal apoptosis [71–74] and activated microglia, have been documented in mice as early as at 8weeks of age. RGC loss by 22 weeks has also been evidenced by several groups [71, 72, 75].

sive hyperglycemia, resulting in a shortened average survival time of 305 days [70].

inflammation-mediated damage in DR.

*4.1.1.2. Alloxan induced*

*4.1.2. Diet induced*

mice on a 30% galactose-fed diet.

*4.1.3. Transgenic diabetic mice*

*4.1.3.1. Ins2Akita mouse*

STZ-induced mice have been reported to exhibit various NPDR features. Signs of neuronal degeneration, including a decrease in RGC number and reactive gliosis, were observed as early as at 5–6 weeks post-hyperglycemia induction [47–50]. Thinning of the GCL, IPL, OPL and total retinal thickness occurred at 3–4 weeks of hyperglycemia [51], with INL and outer nuclear layer (ONL) thinning by 10 weeks [50]. Microvascular changes included increased vascular permeability within 8 weeks of hyperglycemia, pericyte loss as early as at 2 months [52–54], capillary basal lamina thickening at 17 weeks [55], capillary apoptosis [53] and increased acellular capillaries by 6–9 months [51, 53, 54]. Persistent inflammation resulted in leukostasis at 2–3 months of hyperglycemia [51, 54, 56, 57] with an increased number of leukocytes in the microvasculature at 3 months [54]. Hemodynamic changes have also been documented. There was a decrease in arteriolar and venular velocity, shear rates, blood flow rates and diameter at 4 weeks of hyperglycemia [58, 59]. However, the changes in the arteriolar and venular diameters were no longer apparent at 8 weeks of hyperglycemia and hence may not be a reproducible feature of the model. ERG demonstrated decreased total OP and OP3 amplitudes with prolonged OP2-3 implicit times at 4 weeks of hyperglycemia [60, 61]. One study also noted decreased a- and b-wave amplitudes, though this was not evident in the majority of reports [51].

Evidence regarding diabetes-induced RGC apoptosis and loss remain controversial. Some studies reported increased RGC apoptosis within 2 weeks of diabetes induction [50] and decreased RGC numbers by 6–10 weeks of diabetes [48, 50]. Others found no evidence of RGC apoptosis or GCL cell loss after up to 10 months of hyperglycemia [51, 56, 62]. The transient increase in neural apoptosis and astrocyte activation that regressed after a longer duration of diabetes in one study suggested that such changes may have been induced by STZ toxicity [53]. Variations in the onset of DR features may be attributable to the use of different strains of mice (despite most using C57BL/6 mice) or differing STZ-injection protocols.

More recently, in a study of various inbred strains of mice selected using "The Collaborative Cross" mouse resource, the FOT\_FB strain was identified to exhibit a wide range of NPDR and PDR lesions within a significantly shorter duration of hyperglycemia induction. Classical features of neurodegeneration including Müller cell gliosis and RGC loss were displayed 7 days after diabetes induction. Other lesions included IRMAs, dilated vessels resembling venous dilatation and venous beading, increased acellular capillaries, and signs of vessel invasion into the avascular vitreous cavity [63]. The presence of PDR features absent in conventional strains of mice with STZ-induced diabetes may be attributable to the expression of genes implicated in DR in the FOB\_FT strain [63]. Though further characterization studies on this model may be needed, the FOT\_FT mouse may represent a novel resource for the study of DR related genes and for testing of therapeutic interventions targeting vascular, neural and inflammation-mediated damage in DR.

#### *4.1.1.2. Alloxan induced*

**4.1. Mouse models**

*4.1.1. Pharmacological*

*4.1.1.1. STZ induced*

injected with a dose of STZ.

majority of reports [51].

STZ-induced mice are one of the most commonly used DR models for DR characterization and therapeutic drug studies. The mice develop hyperglycemia within 1 week after being

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

STZ-induced mice have been reported to exhibit various NPDR features. Signs of neuronal degeneration, including a decrease in RGC number and reactive gliosis, were observed as early as at 5–6 weeks post-hyperglycemia induction [47–50]. Thinning of the GCL, IPL, OPL and total retinal thickness occurred at 3–4 weeks of hyperglycemia [51], with INL and outer nuclear layer (ONL) thinning by 10 weeks [50]. Microvascular changes included increased vascular permeability within 8 weeks of hyperglycemia, pericyte loss as early as at 2 months [52–54], capillary basal lamina thickening at 17 weeks [55], capillary apoptosis [53] and increased acellular capillaries by 6–9 months [51, 53, 54]. Persistent inflammation resulted in leukostasis at 2–3 months of hyperglycemia [51, 54, 56, 57] with an increased number of leukocytes in the microvasculature at 3 months [54]. Hemodynamic changes have also been documented. There was a decrease in arteriolar and venular velocity, shear rates, blood flow rates and diameter at 4 weeks of hyperglycemia [58, 59]. However, the changes in the arteriolar and venular diameters were no longer apparent at 8 weeks of hyperglycemia and hence may not be a reproducible feature of the model. ERG demonstrated decreased total OP and OP3 amplitudes with prolonged OP2-3 implicit times at 4 weeks of hyperglycemia [60, 61]. One study also noted decreased a- and b-wave amplitudes, though this was not evident in the

Evidence regarding diabetes-induced RGC apoptosis and loss remain controversial. Some studies reported increased RGC apoptosis within 2 weeks of diabetes induction [50] and decreased RGC numbers by 6–10 weeks of diabetes [48, 50]. Others found no evidence of RGC apoptosis or GCL cell loss after up to 10 months of hyperglycemia [51, 56, 62]. The transient increase in neural apoptosis and astrocyte activation that regressed after a longer duration of diabetes in one study suggested that such changes may have been induced by STZ toxicity [53]. Variations in the onset of DR features may be attributable to the use of different strains of

More recently, in a study of various inbred strains of mice selected using "The Collaborative Cross" mouse resource, the FOT\_FB strain was identified to exhibit a wide range of NPDR and PDR lesions within a significantly shorter duration of hyperglycemia induction. Classical features of neurodegeneration including Müller cell gliosis and RGC loss were displayed 7 days after diabetes induction. Other lesions included IRMAs, dilated vessels resembling venous dilatation and venous beading, increased acellular capillaries, and signs of vessel invasion into the avascular vitreous cavity [63]. The presence of PDR features absent in conventional strains of mice with STZ-induced diabetes may be attributable to the expression of genes implicated in DR in the FOB\_FT strain [63]. Though further characterization studies on

mice (despite most using C57BL/6 mice) or differing STZ-injection protocols.

Few studies have examined neuronal and vascular DR features of alloxan-induced diabetic C57BL/6 or albino mice, perhaps due to the absence of demonstrable lesions. About 3 months of alloxan-induced diabetes in C57BL/6 mice failed to induce neuronal apoptosis, glial activation, and microaneurysm and hemorrhage formation [64]. Only functional changes on ERG were observed, with decreased b-wave amplitudes at 3 weeks in albino mice [65, 66] and decreased b/a-wave amplitude ratio and increased OP latency at 3 months of hyperglycemia in C57BL/6 mice [64]. Morphologically, shortened dendrites and thickened proximal processes of microglia suggested the activation of microglia after 3 months of diabetes [64]. In the less conventionally used FOT\_FB mouse strain, the study reported disorganized capillaries within 7 days of diabetes induction [63]. By 21 days of diabetes, microaneurysms, IRMAs and capillary dilatation with preretinal neovascular lesions were found in the mice retinae [63].

#### *4.1.2. Diet induced*

Mice fed with a 30% galactose diet were found to have reduced endothelial cells and pericyte loss beginning as early as at 11 months of hypergalactosemia [67]. With prolonged hypergalactosemia, the number of acellular capillaries increased [33, 67–69]. By 21–22 months, microvascular changes, including saccular microaneurysms and capillary BM thickening, were present [33]. Variations in age of reported features exist depending on the strain of mice used and the percentage of galactose incorporated into the mice's diet. The majority of reports used mice on a 30% galactose-fed diet.

#### *4.1.3. Transgenic diabetic mice*

#### *4.1.3.1. Ins2Akita mouse*

The *Ins2Akita* mouse is a T1D mouse model carrying an endogenous point mutation in the *Mody4* locus (i.e. Insulin 2 gene) with an autosomal dominant mode of inheritance. The mutation results in misfolding of the insulin protein, leading to beta-cell death and decreased insulin secretion, with subsequent development of hypoinsulinemia and hyperglycemia at around 4 weeks of age in male mice. Female mice are less commonly used for DR studies due to their remission to a mild to moderate hyperglycemic state after sexual maturation following transient hyperglycemia during puberty [70]. Males, on the other hand, develop progressive hyperglycemia, resulting in a shortened average survival time of 305 days [70].

Early subclinical DR features in heterozygous *Ins2Akita* mice retinae have been consistently reported by numerous studies. Cellular changes observed in humans, including increased retinal apoptosis [71–74] and activated microglia, have been documented in mice as early as at 8weeks of age. RGC loss by 22 weeks has also been evidenced by several groups [71, 72, 75]. Morphologically, there was abnormal swelling in RGC somas, axons and dendrites, with increased dendritic length in ON-type RGCs in three-month old mice [75]. One study revealed increased GFAP expression in Müller cells in 25-week-old mice [76], yet another only found increased GFAP immunoreactivity in astrocytes [71].

Only female mice were used in the studies due to the inconsistent and low rates of hyperglycemic induction in males. However, estrogen is speculated to play a protective role in DR. This may arguably affect the interpretation of potential therapeutic drug studies [32]. Although the NOD mouse represents an autoimmune diabetic model similar to the pathogenesis of human T1D, the onset of hyperglycemia is highly variable, making it a less reliable

The C57BL/KsJ-*db/db* or *Leprdb/db* (db/db) mouse is a T2D model carrying a mutation of recessive inheritance in the leptin receptor gene. Homozygotes develop obesity at 3–4 weeks of

The mice exhibited progressive neuronal cell loss [87], glial activation [87], neuroretinal thinning, BRB disruption and accumulating glutamate concentrations accompanied with downregulation of the glutamate/aspartate transporter (GLAST) as early as at 8 weeks of age [88, 89]. Progressively worsening retinal function and retinal pigment epithelium dysfunction with persistent hyperglycemia have been evidenced by ERG changes (a-wave, b-wave, c-wave and oscillatory potential changes) beginning at 8 or 16 weeks of age [88, 90]. Sustained hyperglycemia is also suggested to be associated with increased RBC velocity in these mice at the age of 18 weeks [91], though the nature of microcirculatory hemodynamic changes in diabetes remains controversial. Upon lowering of blood glucose levels by dietary restriction, many of the observed neurodegeneration abnormalities regressed or were arrested [88]. Such findings suggest that the observed neurodegeneration features are attributable to the effect of

Microvascular complications, such as pericyte loss [87, 92], presence of acellular capillaries [92] and thickening of the capillary BM [93], were also displayed in this model. Retinal angiogenesis dysregulation in these mice is further supported by corresponding associated biochemical changes in the vitreous and retina associated with DR pathogenesis (increased VEGF) and decreased pigment epithelium-derived factor (PEDF)) [94, 95]. The presence of more advanced features of DR,

While the model confers signs of retinal neurodegeneration, the mice have a shortened life span and do not breed well [86]. Homozygote females are infertile and homozygote males have low fecundity. Despite such limitations, with numerous reports characterizing structural abnormalities and increasing studies examining its functional deficits in recent years, the

traits were inherited by polygenes [96]. The mice develop hyperglycemia, hyperinsulinemia and obesity beginning at around 5 weeks of age and display marked hyperglycemia by 16 weeks of age [96]. At the age of 40 weeks, the mouse reverts back to normal [97]. Only one

mouse (or Yellow KK mouse) is a congenic strain of the KK mouse. It was created

) into KK mice, on the basis that diabetic

Animal Models of Diabetic Retinopathy (Part 1) http://dx.doi.org/10.5772/intechopen.70238 29

however, is limited to the proliferation of retinal capillaries at 15 months of age [87].

db/db mouse remains an extensively used model for therapeutic drug research.

model for DR studies.

age, and hyperglycemia at 4–8 weeks [86].

diabetes as opposed to genetic factors.

*4.1.3.4. KKAy*

The KKA<sup>y</sup>

 *mouse*

through the transfer of the yellow obese gene (Ay

*4.1.3.3. Db/db mouse*

Retinal microvascular changes consistent with clinical NPDR have been documented in *Ins2Akita* mice. It is important to note that advanced DR clinical correlates of proliferative DR, such as preretinal neovascularization, have not yet been detected in this model. Studies have reported increased leucocyte adhesion to retinal vessels in eight-week-old mice [71] with increased retinal vascular permeability [71, 73] and presence of acellular capillaries [71] in older mice. *Ex vivo* and *in vivo* histological analyses demonstrated inner retinal thinning at 22 weeks [71, 74] and 6 months, respectively [77], conceivably due to dopaminergic and cholinergic amacrine cell loss or dendritic atrophy [74]. Total and outer retinal thinning had been evidenced earlier on at 3 months of age [77]. By 9 months, there was increased capillary BM thickness, with evidence of neovascularization and worsening microaneurysm formation [73]. The use of *in vivo* imaging techniques (OCT) in other studies, however, failed to show evidence of retinal thinning [76, 78] and neovascularization (both by histology and *in vivo* imaging techniques) in 25-week-old mice [76]. Vascular function assessments revealed significantly reduced retinal blood flow rates, blood cell velocity and vascular wall shear rates without signs of increased hypoxia in mice after 26 weeks of hyperglycemia [79]. Corresponding functional deficits, as documented by significantly reduced scotopic a-wave, b-wave and OP amplitudes, increased a-wave, b-wave and OP implicit times, and reduced b/a-wave ratio have also been found in mice 9 months of age [73, 77]. It has been suggested that differences in reported DR morphological features may be due to the potential presence of *rd8* mutations in the *Crb1* gene in C57BL/6 N mice used for the generation of *Ins2Akita* mice. Affected mice have been described to display signs of retinal degeneration and ocular lesions due to the presence of *rd8* unrelated to the mutated genes of transgenic mice [80].

Despite its short average lifespan [70], the *Ins2Akita* mouse is a well-characterized model of T1D exhibiting changes associated with early DR. It's stable insulin-deficient diabetic state that does not require exogenous administration of insulin and lack of systemic immunologic modifications makes it ideal for DR therapy testing. However, it still fails to display preretinal neovascularization and other features of advanced-stage DR.

#### *4.1.3.2. Non-obese diabetic (NOD) mouse*

The Non-obese diabetic (NOD) mouse spontaneously develops T1D beginning from 12 to 30 weeks of age. An autoimmune process involving CD4+ and CD8+ cells triggers insulitis and subsequent overt T1D in 80% of female and 20% of male mice by the age of 30 weeks [81, 82].

After 3 weeks of hyperglycemia, constriction of retinal arterioles in close proximity to venules was observed in NOD mice [83]. There was evident degeneration of RGCs, endothelial cell and pericyte apoptosis, retinal capillary BM thickening, perivascular edema and microvascular occlusion by 12 weeks of hyperglycemia (pathological changes initially arose after 4 weeks of hyperglycemia) [84]. Six-month-old mice exhibited further vascular changes, including retinal microvessel loss, vasoconstriction or degeneration of major vessels and focal proliferation of new vessels [85].

Only female mice were used in the studies due to the inconsistent and low rates of hyperglycemic induction in males. However, estrogen is speculated to play a protective role in DR. This may arguably affect the interpretation of potential therapeutic drug studies [32]. Although the NOD mouse represents an autoimmune diabetic model similar to the pathogenesis of human T1D, the onset of hyperglycemia is highly variable, making it a less reliable model for DR studies.

#### *4.1.3.3. Db/db mouse*

Morphologically, there was abnormal swelling in RGC somas, axons and dendrites, with increased dendritic length in ON-type RGCs in three-month old mice [75]. One study revealed increased GFAP expression in Müller cells in 25-week-old mice [76], yet another

Retinal microvascular changes consistent with clinical NPDR have been documented in *Ins2Akita* mice. It is important to note that advanced DR clinical correlates of proliferative DR, such as preretinal neovascularization, have not yet been detected in this model. Studies have reported increased leucocyte adhesion to retinal vessels in eight-week-old mice [71] with increased retinal vascular permeability [71, 73] and presence of acellular capillaries [71] in older mice. *Ex vivo* and *in vivo* histological analyses demonstrated inner retinal thinning at 22 weeks [71, 74] and 6 months, respectively [77], conceivably due to dopaminergic and cholinergic amacrine cell loss or dendritic atrophy [74]. Total and outer retinal thinning had been evidenced earlier on at 3 months of age [77]. By 9 months, there was increased capillary BM thickness, with evidence of neovascularization and worsening microaneurysm formation [73]. The use of *in vivo* imaging techniques (OCT) in other studies, however, failed to show evidence of retinal thinning [76, 78] and neovascularization (both by histology and *in vivo* imaging techniques) in 25-week-old mice [76]. Vascular function assessments revealed significantly reduced retinal blood flow rates, blood cell velocity and vascular wall shear rates without signs of increased hypoxia in mice after 26 weeks of hyperglycemia [79]. Corresponding functional deficits, as documented by significantly reduced scotopic a-wave, b-wave and OP amplitudes, increased a-wave, b-wave and OP implicit times, and reduced b/a-wave ratio have also been found in mice 9 months of age [73, 77]. It has been suggested that differences in reported DR morphological features may be due to the potential presence of *rd8* mutations in the *Crb1* gene in C57BL/6 N mice used for the generation of *Ins2Akita* mice. Affected mice have been described to display signs of retinal degeneration and ocular lesions due to the presence of *rd8* unrelated to the mutated genes of

Despite its short average lifespan [70], the *Ins2Akita* mouse is a well-characterized model of T1D exhibiting changes associated with early DR. It's stable insulin-deficient diabetic state that does not require exogenous administration of insulin and lack of systemic immunologic modifications makes it ideal for DR therapy testing. However, it still fails to display preretinal

The Non-obese diabetic (NOD) mouse spontaneously develops T1D beginning from 12 to

subsequent overt T1D in 80% of female and 20% of male mice by the age of 30 weeks [81, 82]. After 3 weeks of hyperglycemia, constriction of retinal arterioles in close proximity to venules was observed in NOD mice [83]. There was evident degeneration of RGCs, endothelial cell and pericyte apoptosis, retinal capillary BM thickening, perivascular edema and microvascular occlusion by 12 weeks of hyperglycemia (pathological changes initially arose after 4 weeks of hyperglycemia) [84]. Six-month-old mice exhibited further vascular changes, including retinal microvessel loss, vasoconstriction or degeneration of major vessels and focal proliferation of new vessels [85].

and CD8+

cells triggers insulitis and

neovascularization and other features of advanced-stage DR.

30 weeks of age. An autoimmune process involving CD4+

*4.1.3.2. Non-obese diabetic (NOD) mouse*

only found increased GFAP immunoreactivity in astrocytes [71].

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

transgenic mice [80].

The C57BL/KsJ-*db/db* or *Leprdb/db* (db/db) mouse is a T2D model carrying a mutation of recessive inheritance in the leptin receptor gene. Homozygotes develop obesity at 3–4 weeks of age, and hyperglycemia at 4–8 weeks [86].

The mice exhibited progressive neuronal cell loss [87], glial activation [87], neuroretinal thinning, BRB disruption and accumulating glutamate concentrations accompanied with downregulation of the glutamate/aspartate transporter (GLAST) as early as at 8 weeks of age [88, 89]. Progressively worsening retinal function and retinal pigment epithelium dysfunction with persistent hyperglycemia have been evidenced by ERG changes (a-wave, b-wave, c-wave and oscillatory potential changes) beginning at 8 or 16 weeks of age [88, 90]. Sustained hyperglycemia is also suggested to be associated with increased RBC velocity in these mice at the age of 18 weeks [91], though the nature of microcirculatory hemodynamic changes in diabetes remains controversial. Upon lowering of blood glucose levels by dietary restriction, many of the observed neurodegeneration abnormalities regressed or were arrested [88]. Such findings suggest that the observed neurodegeneration features are attributable to the effect of diabetes as opposed to genetic factors.

Microvascular complications, such as pericyte loss [87, 92], presence of acellular capillaries [92] and thickening of the capillary BM [93], were also displayed in this model. Retinal angiogenesis dysregulation in these mice is further supported by corresponding associated biochemical changes in the vitreous and retina associated with DR pathogenesis (increased VEGF) and decreased pigment epithelium-derived factor (PEDF)) [94, 95]. The presence of more advanced features of DR, however, is limited to the proliferation of retinal capillaries at 15 months of age [87].

While the model confers signs of retinal neurodegeneration, the mice have a shortened life span and do not breed well [86]. Homozygote females are infertile and homozygote males have low fecundity. Despite such limitations, with numerous reports characterizing structural abnormalities and increasing studies examining its functional deficits in recent years, the db/db mouse remains an extensively used model for therapeutic drug research.

#### *4.1.3.4. KKAy mouse*

The KKA<sup>y</sup> mouse (or Yellow KK mouse) is a congenic strain of the KK mouse. It was created through the transfer of the yellow obese gene (Ay ) into KK mice, on the basis that diabetic traits were inherited by polygenes [96]. The mice develop hyperglycemia, hyperinsulinemia and obesity beginning at around 5 weeks of age and display marked hyperglycemia by 16 weeks of age [96]. At the age of 40 weeks, the mouse reverts back to normal [97]. Only one study to date has documented retinal changes in the KKA<sup>y</sup> mouse. The study reported retinal neuronal cell apoptosis in the GCL and inner INL [98] with capillary BM thickening [32] after 3 months of hyperglycemia.

*4.1.4.3. Akimba mouse*

an advanced human DR retinal environment.

*4.1.4.4. TgIGF-I mouse*

linemic conditions [107].

*4.1.4.5. Intraocular injection of VEGF*

To create a hyperglycemic model displaying signs of PDR, the *Ins2Akita* mouse was crossbred with the Kimba mouse to generate the Akimba mouse (*Ins2AkitaVEGF+/−)*. With the inheritance of diabetic and retinal neovascular phenotypes from parental strains, the Akimba mouse exhibits most characteristic features of NDPR and PDR, with the exception of preretinal neovascularization. By 8 weeks of age, the Akimba mouse had developed major retinal microvascular abnormalities including vessel tortuosity, venous loups, vessel beading, vascular dilatation, microaneurysms and non-perfused capillaries [78]. Increased vascular leakage was accompanied with lowered levels of endothelial junction proteins [103]. Significant capillary drop out resulted in leakage cessation at 20 weeks of age [78]. Neural retinal thickness decreased with age [78]. Severe loss of ganglion cells and complete photoreceptor loss occurred in 24-weekold mice [78]. Retinal edema, neovascularization and retinal detachment were also present in the mice at an early age. The vascular changes observed here were more severe than those of Kimba mice [78], suggestive of the dual (and possibly synergistic) effects of simultaneous hyperglycemia and VEGF upregulation, and the potential use of this model to study the interaction of these two factors in DR. However, the vascular abnormalities may have developed predominantly due to VEGF upregulation rather than longstanding hyperglycemia as seen in human DR, making the model unsuitable for etiological studies. In spite of such dissimilarities in the sequential pathogenic processes, the Akimba mouse is a unique model simulating

Animal Models of Diabetic Retinopathy (Part 1) http://dx.doi.org/10.5772/intechopen.70238 31

Insulin-like growth factor I (IGF-I) is a VEGF inducer that has been associated with the pathogenesis of DR. Clinically, increased levels of IGF-I have been found in the vitreous of DR patients [104, 105]. To create a model of neovascularization via increased VEGF expression, the RIP/IGF-I chimeric gene was first introduced into mice with a C57BL/6-SJL background, and these mice were subsequently backcrossed to CD-1 mice to create transgenic mice overexpressing insulin-like growth factor I (TgIGF-I) [106]. The mice were reported to exhibit NPDR-like features at the age of 2 months, including pericyte loss, retinal capillary BM thickening and presence of acellular capillaries [107]. With increasing age, there was progressive development of venule dilatation, IRMAs, retinal and vitreous neovascularization, and subsequent retinal detachment [107]. The model has also been found to induce rubeosis iridis, neovascular glaucoma and cataract under normoglycemic and normoinsu-

As intraocular injections of VEGF are less feasible in rodent models, subretinal injection of a binary recombinant adeno-associated virus construct producing green fluorescent protein (GFP) and VEGF was used in one study. VEGF overexpression resulted in microaneurysm formation, venous dilatation and vascular leakage [108]. However, the model failed to induce the pronounced neovascularization seen in transgenic animals and was only able to manifest

#### *4.1.4. Angiogenesis models*

#### *4.1.4.1. Oxygen-induced retinopathy (OIR)*

Characterization of retinal features exhibited by mouse models of OIR has been performed on postnatal day 18-old (P18) mice [99]. Documented cellular features included reduced IPL, outer segment (central and mid-peripheral) and total (central) retinal thickness, and increased gliotic Müller cells and reactived microglia predominantly in areas where deep plexus vascularization was absent. Substantial intravitreal angiogenesis was present in all retinal eccentricities. The number of vessels was reduced in the inner and deep vascular plexues (central and mid-peripheral), with the central retina remaining fairly avascular. Corresponding functional changes on the ERG were also observed. A-wave, b-wave, OP3 and OP4 amplitudes were reduced and the b-wave implicit time was increased. The OIR model is not widely utilized for therapeutic drug studies for DR, owing to the spontaneous regression of neovascularization within a week of its development.

#### *4.1.4.2. Kimba mouse*

The Kimba trVEGF029 mouse (Kimba) is a neovascularization model whereby photoreceptorspecific human VEGF165 overexpression is induced using a truncated rhodopsin promoter [100]. The Kimba mouse line displays features most similar to NPDR or early PDR out of the four hVEGF-overexpressing transgenic mouse lines generated, while displaying stable mild to moderate retinopathy for at least 3 months [100]. The phenotypic observations discussed below correspond to the Kimba trVEGF029 individuals displaying mild or moderate retinopathy.

Vascular changes in this model have been documented as early as at postnatal day 7 (P7), with INL, ONL and total retinal thinning as one of the first features displayed. P28 mice exhibited classical features of NPDR (tortuous vessels, microaneurysms, vascular leakage and capillary hemorrhages) that progressed with increasing age [100–102]. The development of such retinal vascular abnormalities was accompanied by increasing adherent leucocyte numbers corresponding to the severity of the abnormality observed [102]. Counter intuitively, vascular leakage began to cease at 9 weeks among moderate phenotypes, but this is most likely due to the significant reduction in hVEGF165 expression. Mild neovascularization and altered retinal vasculature demonstrating reduced vessel length, coverage area and crossing points have also been reported in mice 9 weeks of age [102]. However, the observed neovascular changes in such VEGF models occur in the outer retina, as opposed to the inner retina as seen in DR. While new vessels typically grow into the vitreous in DR, vessel growth in this model occurs in the opposite direction, from the capillary bed to the ONL. There has not been widespread use of the Kimba mouse in DR studies perhaps as a result of the commercial unavailability of the mouse.

#### *4.1.4.3. Akimba mouse*

study to date has documented retinal changes in the KKA<sup>y</sup>

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

3 months of hyperglycemia.

*4.1.4.1. Oxygen-induced retinopathy (OIR)*

within a week of its development.

*4.1.4.2. Kimba mouse*

retinopathy.

*4.1.4. Angiogenesis models*

neuronal cell apoptosis in the GCL and inner INL [98] with capillary BM thickening [32] after

Characterization of retinal features exhibited by mouse models of OIR has been performed on postnatal day 18-old (P18) mice [99]. Documented cellular features included reduced IPL, outer segment (central and mid-peripheral) and total (central) retinal thickness, and increased gliotic Müller cells and reactived microglia predominantly in areas where deep plexus vascularization was absent. Substantial intravitreal angiogenesis was present in all retinal eccentricities. The number of vessels was reduced in the inner and deep vascular plexues (central and mid-peripheral), with the central retina remaining fairly avascular. Corresponding functional changes on the ERG were also observed. A-wave, b-wave, OP3 and OP4 amplitudes were reduced and the b-wave implicit time was increased. The OIR model is not widely utilized for therapeutic drug studies for DR, owing to the spontaneous regression of neovascularization

The Kimba trVEGF029 mouse (Kimba) is a neovascularization model whereby photoreceptorspecific human VEGF165 overexpression is induced using a truncated rhodopsin promoter [100]. The Kimba mouse line displays features most similar to NPDR or early PDR out of the four hVEGF-overexpressing transgenic mouse lines generated, while displaying stable mild to moderate retinopathy for at least 3 months [100]. The phenotypic observations discussed below correspond to the Kimba trVEGF029 individuals displaying mild or moderate

Vascular changes in this model have been documented as early as at postnatal day 7 (P7), with INL, ONL and total retinal thinning as one of the first features displayed. P28 mice exhibited classical features of NPDR (tortuous vessels, microaneurysms, vascular leakage and capillary hemorrhages) that progressed with increasing age [100–102]. The development of such retinal vascular abnormalities was accompanied by increasing adherent leucocyte numbers corresponding to the severity of the abnormality observed [102]. Counter intuitively, vascular leakage began to cease at 9 weeks among moderate phenotypes, but this is most likely due to the significant reduction in hVEGF165 expression. Mild neovascularization and altered retinal vasculature demonstrating reduced vessel length, coverage area and crossing points have also been reported in mice 9 weeks of age [102]. However, the observed neovascular changes in such VEGF models occur in the outer retina, as opposed to the inner retina as seen in DR. While new vessels typically grow into the vitreous in DR, vessel growth in this model occurs in the opposite direction, from the capillary bed to the ONL. There has not been widespread use of the Kimba mouse in DR studies perhaps as a result of the commercial unavailability of the mouse.

mouse. The study reported retinal

To create a hyperglycemic model displaying signs of PDR, the *Ins2Akita* mouse was crossbred with the Kimba mouse to generate the Akimba mouse (*Ins2AkitaVEGF+/−)*. With the inheritance of diabetic and retinal neovascular phenotypes from parental strains, the Akimba mouse exhibits most characteristic features of NDPR and PDR, with the exception of preretinal neovascularization. By 8 weeks of age, the Akimba mouse had developed major retinal microvascular abnormalities including vessel tortuosity, venous loups, vessel beading, vascular dilatation, microaneurysms and non-perfused capillaries [78]. Increased vascular leakage was accompanied with lowered levels of endothelial junction proteins [103]. Significant capillary drop out resulted in leakage cessation at 20 weeks of age [78]. Neural retinal thickness decreased with age [78]. Severe loss of ganglion cells and complete photoreceptor loss occurred in 24-weekold mice [78]. Retinal edema, neovascularization and retinal detachment were also present in the mice at an early age. The vascular changes observed here were more severe than those of Kimba mice [78], suggestive of the dual (and possibly synergistic) effects of simultaneous hyperglycemia and VEGF upregulation, and the potential use of this model to study the interaction of these two factors in DR. However, the vascular abnormalities may have developed predominantly due to VEGF upregulation rather than longstanding hyperglycemia as seen in human DR, making the model unsuitable for etiological studies. In spite of such dissimilarities in the sequential pathogenic processes, the Akimba mouse is a unique model simulating an advanced human DR retinal environment.

#### *4.1.4.4. TgIGF-I mouse*

Insulin-like growth factor I (IGF-I) is a VEGF inducer that has been associated with the pathogenesis of DR. Clinically, increased levels of IGF-I have been found in the vitreous of DR patients [104, 105]. To create a model of neovascularization via increased VEGF expression, the RIP/IGF-I chimeric gene was first introduced into mice with a C57BL/6-SJL background, and these mice were subsequently backcrossed to CD-1 mice to create transgenic mice overexpressing insulin-like growth factor I (TgIGF-I) [106]. The mice were reported to exhibit NPDR-like features at the age of 2 months, including pericyte loss, retinal capillary BM thickening and presence of acellular capillaries [107]. With increasing age, there was progressive development of venule dilatation, IRMAs, retinal and vitreous neovascularization, and subsequent retinal detachment [107]. The model has also been found to induce rubeosis iridis, neovascular glaucoma and cataract under normoglycemic and normoinsulinemic conditions [107].

#### *4.1.4.5. Intraocular injection of VEGF*

As intraocular injections of VEGF are less feasible in rodent models, subretinal injection of a binary recombinant adeno-associated virus construct producing green fluorescent protein (GFP) and VEGF was used in one study. VEGF overexpression resulted in microaneurysm formation, venous dilatation and vascular leakage [108]. However, the model failed to induce the pronounced neovascularization seen in transgenic animals and was only able to manifest some features of NPDR. Significant new vessel formation was restricted to the INL of VEGF expression site. Only one mouse displayed signs of retinal degeneration with blood vessel growth into the subretinal space.

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