Manifestation of Pathology in Animal Models of Diabetic Retinopathy Is Delayed from the Onset of Diabetes

Diabetic retinopathy (DR) is the most common complication that develops in patients with diabetes mellitus (DM) and is the leading cause of blindness worldwide. Fortunately, sight-threatening forms of DR develop only after several decades of DM. This well-documented resilience to DR suggests that the retina is capable of protecting itself from DM-related damage and also that accumulation of such damage occurs only after deterioration of this resilience. Despite the enormous translational significance of this phenomenon, very little is known regarding the nature of resilience to DR. Rodent models of DR have been used extensively to study the nature of the DM-induced damage, i.e., cardinal features of DR. Many of these same animal models can be used to investigate resilience because DR is delayed from the onset of DM by several weeks or months. The purpose of this review is to provide a comprehensive overview of the literature describing the use of rodent models of DR in type-1 and type-2 diabetic animals, which most clearly document the delay between the onset of DM and the appearance of DR. These readily available experimental settings can be used to advance our current understanding of resilience to DR and thereby identify biomarkers and targets for novel, prevention-based approaches to manage patients at risk for developing DR.


Introduction
Diabetic retinopathy (DR) is a chronic sequelae of diabetes mellitus (DM) and a leading cause of blindness worldwide [1][2][3].Early detection of DR symptoms is crucial for timely intervention and prevention of irreversible vision loss [2].In humans, the diagnosis of DR is based exclusively on the presence of retinal vascular abnormalities such as microaneurysms, hemorrhages, exudates, leakage resulting in edema, and neovascularization [1][2][3][4].Although neural and visual abnormalities also develop, they are not considered in the diagnosis [1,2].
In contrast, all types of retinal dysfunction (vascular, neural, and visual) are used to assess DR in animal models [4].Common pathologic features of DR in rodents affect both the vascular and neural compartments of the retina.Vascular abnormalities include inflammation of the endothelium, increased retinal vascular permeability, and manifestation of acellular capillaries.Within the neural retina, diagnostic features of DR include the death of various neural cell types (thinning of the retina), reactive gliosis, and neural dysfunction.These outcomes are typically detected using immunofluorescence, immunohistochemical staining, electroretinogram (ERG), and optical coherence tomography (OCT).While rodent models develop the early stages of DR, they do not develop the advanced, proliferative stage of DR, which involves neovascularization of the retina.Retinal neovascularization can be modeled with the oxygen-induced retinopathy protocol [5]; however, animals are not DM in this experimental setting and, hence, constitute a major difference from the clinical scenario.Thus, DM induces the early stages of retinal damage in experimental animals, similar to what occurs in humans.however, animals are not DM in this experimental setting and, hence, constitute a difference from the clinical scenario.Thus, DM induces the early stages of retinal d in experimental animals, similar to what occurs in humans.
Rodent models are some of the most useful models for studying the pathology Not only does the pathogenesis closely mimic the retinopathy-free period and then phases of DR in a fraction of the time (Figure 1), but rodents are also extraordinari satile when it comes to gene modification.Rodent models have been and continu instrumental in documenting the duration of DM necessary for first detecting DMdamage in both the vascular and neural compartments of the retina, and the prog nature of such damage as the duration of DM is prolonged.Such descriptive stud the bedrock for both mechanistic studies seeking to understand the nature of DM-in damage as well as translational efforts seeking to identify agents that prevent an reverse such damage.This large body of the literature, which is focused on DR, i.e age to the retina that becomes detectable after weeks or months of DM and progre the duration of DM is prolonged, only indirectly addresses resilience to DR.
Recent studies focused on resilience to DR have provided additional evidence existence and have begun to elucidate the underlying mechanism [6,7].In both s zotocin-induced type 1 diabetic (T1D) and db/db genetically modified type 2 d (T2D) mice, durations of DM that were insufficient to cause detectable damage inc expression of NAD(P)H Quinone Dehydrogenase 1, Sod2, Gclc, and other antioxid fense genes within the retina [7].Furthermore, the retinal vessels of these DM mice b resistant to oxidative stress-induced death.As the duration of DM increased, res was lost-the expression of antioxidant defense genes declined; the vasculature b more (instead of less) vulnerable to death, and both neural and vascular hallmarks appeared.Thus, the delay in developing DR corresponded to a period of resilience t associated damage.DR pathogenesis involves loss of resilience, which is a prerequisite for the subs accumulation of damage within the retina.In both humans and experimental animals, a re thy-free period precedes the appearance of DR.Recent discoveries in both T1D and T2D mi cate that the retinopathy-free period is associated with enhanced tolerance of insults that cau age to the retina, such as increased mitochondrial oxidative stress and cytokine production [ of resilience sets the stage for progressive accumulation of retinal damage, which indicates istence of DR.
Of the two commonly used rodent models of DR, mice are used more often tha While the eye of a rat is larger than a mouse's eye, the lower cost and greater selec genetically modified mice are some of the reasons why mice are more popular th in the field of DR research.DR pathogenesis involves loss of resilience, which is a prerequisite for the subsequent accumulation of damage within the retina.In both humans and experimental animals, a retinopathyfree period precedes the appearance of DR.Recent discoveries in both T1D and T2D mice indicate that the retinopathy-free period is associated with enhanced tolerance of insults that cause damage to the retina, such as increased mitochondrial oxidative stress and cytokine production [7].Loss of resilience sets the stage for progressive accumulation of retinal damage, which indicates the existence of DR.
Of the two commonly used rodent models of DR, mice are used more often than rats.While the eye of a rat is larger than a mouse's eye, the lower cost and greater selection of genetically modified mice are some of the reasons why mice are more popular than rats in the field of DR research.
In this review, we provide a comprehensive overview of the current literature demonstrating that DR does not develop coincident with the onset of DM in murine and rat models of T1D and T2D.Rather, DR becomes detectable after weeks or months of DM, whereupon its severity increases as the duration of DM is prolonged.The progressive nature of DR suggests the existence of resilience, which protects from and/or repairs DM-induced damage, and that deterioration of such systems is an essential component of DR pathogenesis.

The Literature Review
2.1.Murine Models of Type-1 Diabetic-Induced Retinopathy T1D rodent models mimic the autoimmune destruction of pancreatic β-cells, leading to insulin deficiency.Mouse models such as the streptozotocin (STZ)-induced diabetic mouse and rat, non-obese diabetic (NOD) mouse, and alloxan-induced mouse models have been extensively employed in investigations of DR.STZ and alloxan are chemical compounds commonly used to induce T1D in mice through intravenous or intraperitoneal injection.Although the exact mechanism behind alloxan's effects is not fully understood, both of these chemicals preferentially affect the pancreatic β-cells due to the cells' elevated levels of the GLUT2 glucose transporter.STZ and alloxan enter β-cells through GLUT2 and are metabolized into toxic side-products that damage DNA, cause oxidative stress, and ultimately lead to the death of these insulin-producing cells.At a concentration of STZ that kills Glut2-expressing cells, it is non-cytotoxic toward cells that do not express its transporter [8].Multiple injections of moderate doses of STZ cause gradual death of beta cells that results from the combined action of STZ-mediated cytotoxicity (alkylating DNA) and immune attack [9][10][11].It is this destruction of insulin-producing cells by which STZ induces DM; insulin supplementation or a pancreatic islet transplant prevents DM in STZ-treated animals [12].Similarly, the breakdown of the blood-retinal barrier does not occur in STZ-treated animals that are given insulin [12][13][14].While STZ has effects unrelated to eliminating the insulin-producing cells, such side effects are more common in single, high-dose STZ protocol that also induces DM [15].NOD mice, on the other hand, exhibit T1D-like phenotypes due to the interplay of several factors that result in genetic susceptibility, autoimmune dysregulation, inflammatory infiltration, and environmental triggers that lead to the destruction of pancreatic β-cells [4,16].Unlike the way DR is diagnosed in humans (based exclusively on vascular parameters), the presence of DR in T1D is evaluated considering vascular, neural, and visual dysfunction.The earliest signs of DR in T1D mice have been reported as early as one week after DM onset, as can be seen in Table 1.
Throughout the literature on T1D mouse models, there has been consistent notice of a delay between the onset of DM and outcomes seen in DR, although of variable length.Once these outcomes have developed, they persist and intensify as DM and DR continue to progress, with the exception of leukostasis and inflammation [17].Consequently, the longer the animal has DM, the greater the differences will be between the DM and non-DM groups.For this reason, investigators focused on DM-induced damage of the retina, prolonging the duration of DM until after resilience had deteriorated.The earliest signs of vascular dysfunction were seen following one week of DM and consisted of degenerated pericytes, acellular capillaries, and minor vessel proliferation [18][19][20][21][22][23][24][25][26][27][28][29].Neural dysfunction, which was measured with electroretinogram (ERG), was absent after 1 week of DM and detectable two weeks after the onset of DM.Once outcomes such as visual acuity decreases, reduced contrast sensitivity, ERG changes, NFL-GCL thinning, RGC and amacrine cell neurodegeneration, and gliosis have developed, they persist and intensify with the duration of DM, with the exception of leukostasis and inflammation [17].Although a variety of markers have been used throughout the literature to study the role of inflammation in DR, some of the more common markers included an elevated number of leukocytes, leukocyte adhesion, microglial infiltration, production of cytokines, eNOS, and several caspases [20,32,33,38,[43][44][45]. Every murine model of DR shows the progression of the disease, i.e., the extent and nature of the damage of the retina increases, but not the transition from NPDR to PDR.

Murine Models of Type-2 Diabetic-Induced Retinopathy
T2D rodent models mimic insulin resistance and impaired glucose metabolism.The T2D models rely on various genetic modifications that lead to DM and thus show a much wider variation between strains.The leptin receptor-deficient (db/db) mouse and highsugar diet-induced models are some of the commonly used models in this realm [4,16].Because the leptin receptor in db/db mice is non-functional, the leptin signaling pathway is disrupted and leads to dysregulation of appetite and satiety [4,16].Such mice become obese, resistant to insulin, hyperinsulinemic, and develop pancreatic β-cell dysfunction that causes T2D symptoms.In a similar fashion, high-sugar diet-induced models of T2D are fed specific diets that lead to obesity and glucose dysregulation, causing overt diabetic phenotypes [4].Db/db mice develop DM between 4-12 weeks of life, but commonly at 8 weeks.In contrast to the terminology found in the literature on T1D STZ-induced mouse models, the literature studying the db/db model uses "weeks of age" instead of time from DM onset.This is because the onset of DM is more difficult to pinpoint in this T2D mouse model as compared to the STZ-induced T1D models.In these scenarios, the approximate duration of DM is 8 weeks shorter than the age of the mice.The section earliest detection of diabetic retinopathy, of Table 2 depicts this shift in terminology.
Damage to the retina is time-sensitive, with prolonged DM resulting in worsening manifestations of DR without any worsening of DM, suggesting the progressive nature of DR.In T2D mice, the earliest indication of retinal dysfunction occurred at 10 weeks of age (after approximately 2 weeks of DM) and included signs of apoptotic changes in the INL, ONL, and GCL retina layers, along with increased vascular permeability and leakage [41, [56][57][58].Once outcomes such as visual acuity decreases, reduced contrast sensitivity, ERG changes, NFL-GCL thinning, RGC and amacrine cell neurodegeneration, and gliosis have developed, they persist and intensify with the duration of DM, with the exception of leukostasis and inflammation [17].Although a variety of markers have been used throughout the literature to study the role of inflammation in DR, some of the more common markers included the elevated number of leukocytes, leukocyte adhesion, microglial infiltration, production of cytokines, eNOS, and several caspases [20,32,33,38,[43][44][45]. Every murine model of DR shows the progression of the disease, i.e., the extent and nature of the damage of the retina increases, but not the transition from NPDR to PDR.

Rat Model of Type-1 Diabetic-Induced Retinopathy
The rat model has also been in use for some time by researchers and has been shown to reproduce a similar pathology to human DM.Most commonly, STZ injections have been used to induce a non-autoimmune form of T1D.The rat model has also shown a delay in the detection of diabetic retinopathy after induction/onset of diabetes; however, it may be less than that seen in T1D mice.In contrast to the STZ-induced mice, the STZ-induced DM rats demonstrated leakage of retinal blood vessels to very small molecules such as fluorescein after as early as 2 days of DM, as can be seen in Table 3 [12,14,74].A shorter delay in the T1D rats may suggest that they are less appropriate models for studying resilience to DR when compared to T1D mouse models.However, this conclusion is weak because different outcomes were performed in different labs to assess vascular damage.Once outcomes such as visual acuity decreases, reduced contrast sensitivity, ERG changes, NFL-GCL thinning, RGC and amacrine cell neurodegeneration, and gliosis have developed, they persist and intensify with the duration of DM, with the exception of leukostasis and inflammation [17].Although a variety of markers have been used throughout the literature to study the role of inflammation in DR, some of the more common markers included the elevated number of leukocytes, leukocyte adhesion, microglial infiltration, production of cytokines, eNOS, and several caspases [20,32,33,38,[43][44][45]. Every murine model of DR shows the progression of the disease, i.e., the extent and nature of the damage of the retina increases, but not the transition from NPDR to PDR.

Discussion
DR is a complex and progressive disease characterized by a spectrum of pathologic changes, which include retinal vascular abnormalities, inflammatory processes, and neurodegeneration.The delay between the onset of DM and the manifestation of retinal damage/dysfunction indicates the existence of resistance to DR, which must be overcome before outcomes of the disease appear.
There are multiple explanations for resilience, i.e., the absence of damage in the face of insults that are capable of causing such damage.It is possible that resilience relates to the level of sensitivity of current approaches to detect damage.Damage may commence with the onset of DM, but its extent is below the level of detection.This scenario predicts that lowering the detection limit will reduce or eliminate the period of resilience.However, technological advances that have been made to date have not demonstrated that DR manifests coincident with the onset of DM.
An alternative explanation of resilience is that cells adapt to hyperglycemia (HG) and, thereby, become resistant to its deleterious effects.Our recent publications provided compelling support for this possibility.In DM mice, resistance is transient, and its loss sets the stage for accumulation of damage, i.e., manifestation of DR.More specifically, the retinal vasculature of mice that had experienced a short duration of DM acquired resistance to oxidative stress/ischemia-or cytokine-induced death [7].As the duration of DM was prolonged, protection waned and was replaced by increased vulnerability; i.e., there was more insult-driven death in retinal vessels from DM mice as compared with age-matched non-DM mice [7].Finally, the appearance of vulnerability coincided with the manifestation of DR-vascular and neural dysfunctions [7].
The underlying mechanism of resilience involves increased mitophagy (clearance of dysfunctional mitochondria), which boosts mitochondrial functionality and prevents mitochondrial oxidative stress in the face of HG [6].It resides in endothelial cells but not in pericytes of human retinal capillaries [6].Whether resilience exists in other retinal cell types has not been investigated to date.The antioxidant component of resilience has been noted by other groups reporting that expression of RBP3 (retinol-binding protein 3), an antioxidant [89][90][91], is elevated in patients who are resistant to diabetic retinopathy [92,93].Taken together, it appears that retinopathy is held in check during the resilience phase because the endothelium has engaged in mitophagy-based defense against the deleterious effects of HG (Figure 2).Loss of resilience is a prerequisite for accumulation of damage in the retina, which is recognized as DR.
The appreciation of resilience, or the innate ability of the retina to remain healthy in the face of DM, raises essential questions regarding the potential role of genetic susceptibility and genetic protection in the development of DR.Moreover, it opens the door to a world of novel biomarkers and potential therapeutic targets of DR that must be further explored to manage people at risk of developing DR and resisting the deleterious effects of DM.Such alternatives are necessary because the current prophylactic options are not effective for all people.This is especially crucial when considering that 26% of the diabetic US population has some form of DR [94].
phase because the endothelium has engaged in mitophagy-based defense against the deleterious effects of HG (Figure 2).Loss of resilience is a prerequisite for accumulation of damage in the retina, which is recognized as DR.The appreciation of resilience, or the innate ability of the retina to remain healthy in the face of DM, raises essential questions regarding the potential role of genetic susceptibility and genetic protection in the development of DR.Moreover, it opens the door to a world of novel biomarkers and potential therapeutic targets of DR that must be further explored to manage people at risk of developing DR and resisting the deleterious effects of DM.Such alternatives are necessary because the current prophylactic options are not effective for all people.This is especially crucial when considering that 26% of the diabetic US population has some form of DR [94].

Figure 1 .
Figure1.DR pathogenesis involves loss of resilience, which is a prerequisite for the subs accumulation of damage within the retina.In both humans and experimental animals, a re thy-free period precedes the appearance of DR.Recent discoveries in both T1D and T2D mi cate that the retinopathy-free period is associated with enhanced tolerance of insults that cau age to the retina, such as increased mitochondrial oxidative stress and cytokine production [ of resilience sets the stage for progressive accumulation of retinal damage, which indicates istence of DR.

Figure 1 .
Figure1.DR pathogenesis involves loss of resilience, which is a prerequisite for the subsequent accumulation of damage within the retina.In both humans and experimental animals, a retinopathyfree period precedes the appearance of DR.Recent discoveries in both T1D and T2D mice indicate that the retinopathy-free period is associated with enhanced tolerance of insults that cause damage to the retina, such as increased mitochondrial oxidative stress and cytokine production[7].Loss of resilience sets the stage for progressive accumulation of retinal damage, which indicates the existence of DR.

Figure 2 .
Figure 2. Mitophagy is an essential component of resilience.The onset of DM, i.e., enduring elevation of blood sugar, triggers mitochondrial adaptation, which includes enhanced clearance of dysfunctional mitochondria.Deterioration of this innate defense mechanism results in self-perpetuating progress damage of the retina, which manifests as DR.

Figure 2 .
Figure 2. Mitophagy is an essential component of resilience.The onset of DM, i.e., enduring elevation of blood sugar, triggers mitochondrial adaptation, which includes enhanced clearance of dysfunctional mitochondria.Deterioration of this innate defense mechanism results in selfperpetuating progress damage of the retina, which manifests as DR.

Table 3 .
Articles of note discussing Type-1 diabetic rat models.