Establishment of an in vivo Streptozotocin-Induced Type 1 Establishment of an in vivo Streptozotocin-Induced Type 1 Diabetes Model Recapitulating Early Brain and Retinal Fibrosis Diabetes Model Recapitulating Early Brain and Retinal Fibrosis

INTRODUCTION: Diabetes has risen to one of the top American public health concerns. The hyperglycemic state of chronic diabetes leads to microvascular and macrovascular changes that predispose patients to delayed wound healing and organ fibrosis. The validation of models to specifically detect early, quantifiable fibrotic changes seen in the diabetic state is of fundamental importance for understanding the diabetic pathophysiology and exploring earlier management options. Here, we investigated if we could detect early signs of internal fibrosis in a streptozotocin (STZ) diabetic mouse model by quantifying α -SMA expression in various organs using flow cytometry. METHODS: We used a low-dose STZ-induced T1DM model. T1DM was confirmed via sustained hyperglycemia (>250mg/dl) over 8-10 weeks. Delayed healing of full thickness wounds was confirmed by tracking wound healing progression over two weeks. Wounded and unwounded skin samples were analyzed histologically to quantify collagen deposition as a sign of fibrosis. Organ fibrosis was assessed in a semi-high-throughput manner using flow cytometry to quantify the percentage of alpha-Smooth Muscle Actin ( α -SMA) positive cells in diabetic versus normoglycemic controls. RESULTS: Combining STZ with post-injection glucose treatment yielded highly efficient 100% pathogenesis with 100% survival. Diabetic mice showed signs of hyperglycemia, polyuria, and delayed wound healing. Histological analysis indicated a greater increase in epidermal height and lower levels of collagen deposition in diabetic


INTRODUCTION
Type 1 Diabetes Mellitus (T1DM) and Type 2 Diabetes Mellitus (T2DM) have risen to one of the top American public health concerns, with a combined estimated prevalence rate of 11% (30.3 million cases) in 2015 1,2 . These numbers are predicted to rise by over 50% by 2030 1 . Although the prevalence rate of T1DM is highest among Caucasian populations, the steepest rise in incidence between 2000-2010 was seen in pediatric Hispanic populations 1−5 . Prevalence and incidence of T2DM are highest among non-Hispanic black populations 3,4 . Lower income levels and academic backgrounds are associated with higher diabetes prevalence and disease-related complications 6−9 . There is a growing need to lower the disease burden for these vulnerable populations.
Diabetes is a metabolic disorder caused by a functional insulin deficit, leading to an inability to properly absorb glucose 10 . T1DM accounts for 10% of diabetic cases. It is caused by the T-cell mediated autoimmune destruction of pancreatic islet β-cells, initiated by genetic mutation, viral infection, or environmental factors. Loss of β-cells depletes the endogenous insulin supply. T1DM is associated with autoimmune thyroid disease, celiac disease, autoimmune gastritis and Addison's disease 11 . T2DM is an acquired disorder caused by insulin resistance due to either polygenetic inheritance or metabolic overload brought on by obesity or sedentary lifestyle. Because some insulin activity is maintained, T2DM is generally a milder disease than T1DM. T2DM is associated with hypertension, dyslipidemia, metabolic syndrome, or polycystic ovarian syndrome.
In both T1DM and T2DM, hyperglycemia increases osmolarity and oxidative stress. This leads to neuropathies that impair peripheral sensation, motor control, and autonomics. Loss of functional insulin, which normally acts as a weak vasodilator, leads to increased vasoconstriction, peripheral vascular disease (PVD), and atherosclerosis of medium-and large-vessels 10 . This comorbidity of neuropathy and PVD puts patients at increased risk for chronic foot ulceration with impaired surface wound healing, which is seen in 25% of diabetic patients 12 . These ulcers often go unnoticed and become infected, which leads to increased medical costs, limb amputation, or death 12,13 . Diabetes also causes microvascular disease due the irreversible deposition of cross-linked advanced glycation end-product (AGE) proteins 14 . These AGE proteins create space-occupying vessel obstructions, resulting in decreased perfusion, growth inhibition, apoptosis, and ultimately fibrosis of small vessels, seen in diabetic retinopathy. Diabetic hypoperfusion-induced ischemia and hyperglycemic oxidative stress also leads to increased organ fibrosis 15,16 . Fibrosis is characterized by increased extracellular matrix collagen deposition and the presence of myofibroblasts, which ultimately lead to loss of functional tissue and contraction 17,18 . Diabetic complications resulting from fibrosis include nephropathy, cardiomyopathy, coronary heart disease, stroke, nonalcoholic fatty liver disease, and pulmonary fibrosis.
Several in vivo mouse models have been used to investigate the pathophysiology of diabetes. The chemical agent streptozotocin (STZ) has been widely used to induce a type 1 diabetic state 19,20 . STZ is a toxic glucose analogue that enters pancreatic islet β-cells via Glut-2 transporter and increases reactive oxygen and nitrogen species 19,21 . This results in cell death and prohibits the biosynthesis and release of insulin, leading to sustained hyperglycemia 19,22 . STZ-induced diabetic rodents exhibit many hyperglycemic complications, including delayed wound healing, allodynia, hyperalgesia, visual impairment, renal toxicity, bradycardia, hypotension and cardiomyopathy 23,24 . However, its use to study fibrosis has been limited due to the long time-course of at least six months before the overt manifestations of fibrotic collagen depositions are apparent on histopathology 25 . Further, STZ-induced diabetes model needs to be coupled with costly genetic deficiencies to recapitulate renal fibrosis, and it requires at least six months of hyperglycemia to reproduce early signs of diabetic retinopathy, such as loss of retinal pericytes and capillaries, thickening of the vascular basement membrane, vascular occlusion and increased vascular permeability 25−29 .
Extracellular matrix depositions and the appearance of myofibroblasts are thought to be hallmarks of fibrosis and wound healing across many tissues, including retinal, brain, renal, and lung samples 30−35 .
Alpha-Smooth Muscle Actin (α-SMA) is an intracellular protein expressed in these myofibroblasts that is responsible for the contractility of these cells 36 . As such, α-SMA represents a key marker of the myofibroblastic phenotype. Nonetheless, the pathogenesis of fibrosis and appearance of myofibroblasts is complex, and α-SMA as a fibrotic marker has been inconsistent in pulmonary, renal and muscle fibrosis 37,38 . These inconsistences could be attributed to differences in tissue properties, disease stages, experimental models or varied etiologies of the fibrotic disease 39,40 .
The validation of methods and reliable markers to specifically detect early, quantifiable fibrotic changes seen in the diabetic state is of fundamental importance for understanding the diabetic pathophysiology and exploring earlier management options. Here, we investigated if we could detect early signs of internal fibrosis in an STZ-induced diabetic mouse model by measuring α-SMA expression in various organs by flow cytometry. Flow cytometry offers unique advantages over tissue histology alone, including quick individual cell analysis and cell sorting for further molecular studies. This method would be useful to characterize molecular and cellular changes that precede histological manifestations of fibrosis and overt disease burden seen in patients.

METHODS
This study was carried out in strict accordance with the recommendations in the Guide for the Care and  Figure 1A, B). STZ was resuspended in sodium-citrate buffer solution (pH 4.5) and injected within 10 min from preparation. Mice were injected daily intraperitoneally with 50 mg/kg STZ after fasting for 4-6 hours. STZ-driven β-cell destruction may cause severe hypoglycemia, which may be lethal. This was prevented using 10% sucrose solution for 48 hours immediately following STZ initiation 19 . Normoglycemic mice were injected with sodium-citrate buffer only. Blood glucose has been shown to peak and remain stable at three days post-STZ treatment 41 .
Glycemia was checked after 5-10 days. Mice with glucose <250 mg/dL were re-injected. Hyperglycemia was sustained for 8-10 weeks prior to wounding.
Glycemia measurement: Prior to measurement, mice were food-starved for 4 hours with water ad libitum.
A scalpel was used to pierce the tail and collect a drop of blood for glucose measurement using a Bayer Tissue dissociation and flow cytometry: Whole organs, including brain, retinas, heart, lung, liver, kidney, pancreas, and peritoneal fat were collected from 8 normoglycemic and 8 hyperglycemic mice at the end of the experiment and dissociated using gentleMACS TM Dissociator (Miltenyi Biotec) ( Figure 3A). Tissues were cut in small pieces with a scalpel and dissociated with tissue-specific programs of gentleMACS. 8 mice were used to generate 2 retina samples by pooling the retinas from 4 mice in each sample, for a total n=2 for both diabetic and non-diabetic samples. All other organs were analyzed individually, for a total n=4-8. Intracellular staining for alpha-smooth muscle actin (α-SMA) was performed on single cell suspensions. First, cells were fixed and permeabilized using Transcription Factor Buffer Set (BD Biosciences, Jose, CA). Then cells were incubated with 2% mouse normal sera to block non-specific binding, followed by incubation with Alexa 488 conjugated anti-human α-SMA antibodies (Invitrogen, clone 1A4). Matching isotype control was used to determine background fluorescence level. Cells were analyzed by Flow cytometry to quantify α-SMA protein levels using S1000EX Flow cytometer (Stratedigm, San Jose, CA). Data were analyzed using CellCapture software (Stratedigm).

RESULTS
The T1DM model yielded highly efficient pathogenesis with 100% survival following STZ treatment with sucrose supplement. 90% of mice had sustained hyperglycemia (>250 mg/dL) at two weeks following one serial 5-day low-dose STZ treatment. The remaining 10% were given a second serial 5-day low-dose STZ treatment, after which all STZ-treated mice demonstrated sustained hyperglycemia (>250 mg/dL) ( Figure   1C). Control mice injected with sodium-citrate buffer demonstrated 100% survival and normoglycemia (<150 mg/dL). Diabetic mice also demonstrated characteristic weight loss over the study period ( Figure   1D). Diabetic mice demonstrated polyuria requiring more frequent cage changes (data not shown), as previously reported 47,48 .
Full thickness excisional wounds with splints were photographed and measured for two weeks. A representative image demonstrates the delay in wound healing seen grossly in diabetic mice, with measurements confirming delayed wound healing (Figure 2A, B). H&E histology stain demonstrated morphological differences in cellular architecture between unwounded and wounded tissues ( Figure 2C).
Unwounded tissue samples demonstrated no significant difference in dermal height in diabetic tissue compared to normoglycemic tissues, although unwounded diabetic tissues demonstrated thinner dermal height than all other samples ( Figure 2F). Epidermal height for both groups was significantly increased post-injury ( Figure 2G, p<0.0001). Diabetic wound tissues showed a larger increase in epidermal height than normoglycemic wound tissue (mean fold-change normoglycemic = 2.3; mean fold-change diabetic = 6.0). Trichrome analysis showed similar baseline collagen content in diabetic and normoglycemic unwounded tissues ( Figure 2H). Post-injury, diabetic wounds demonstrated significantly impaired collagen deposition relative to normoglycemic controls (p<0.05).
Cell flow cytometry analysis has shown a significant increase in the percentage of α-SMA-positive cells only in brain and retinal tissues of diabetic mice compared to normoglycemic ones ( Figure 3B). Although the fraction of α-SMA-positive cells did not increase in kidney and heart tissues of diabetic mice, we observed an increase in cellular levels of α-SMA expression, as demonstrated by increased α-SMA mean fluorescent intensity of these samples (data not shown). No difference was observed between normoglycemic and diabetic lung, liver, pancreas, skin, or peritoneal fat in numbers of α-SMA-positive cells or α-SMA mean fluorescent intensity.

DISCUSSION
Given the high prevalence of diabetes and its complications, we reproduced a low-dose STZ-induced T1DM mouse model with didactic and research purposes. STZ-induced diabetes is a valuable method to study hyperglycemic complications seen in both T1DM and T2DM, including peripheral vascular disease and wound healing delay 23,24 . In the past, this model has presented cumbersome, costly, and time-intensive limitations for the study of hyperglycemic complications including pulmonary fibrosis, cardiomyopathy, retinopathy, and kidney fibrosis 25,26,49,50 . Here, we investigated if we could detect early signs of fibrosis by quantifying the number of α-SMA-positive cells across organs using flow cytometry, paired with complementary histologic analysis of surface wounds to corroborate our diabetic state.
We combined several known protocols to achieve the highest rate of diabetes induction. We successfully established a T1DM mouse model with 100% efficiency and survival, confirmed by sustained hyperglycemia, polyuria, weight loss and delayed wound closure rates compared to normoglycemic controls ( Figures 1C, 1D and 2B). This is consistent with the disease manifestations seen in diabetic Histologic analysis demonstrated a greater increase in diabetic post-injury epidermal height than in normoglycemic tissue, indicating greater inflammation. Unwounded dermal height is smaller in diabetic mice than normoglycemic mice, but post-injury diabetic dermal height increased to match normoglycemic dermal height. Post-injury collagen deposition was decreased in diabetic mice. This is consistent with previous studies of the impaired re-epithelialization of diabetic wounds in both rat models and diabetic patients, which found that hyperglycemic tissues have altered collagen and elastin deposition 23,51,52 . These histologic findings further validate the diabetic state in our model system and offer a robust method for tracking the architectural changes that take place on a cellular scale in the hyperglycemic state.
The cellular analysis using flow cytometry demonstrated increased number of α-SMA-positive cells in the brain and eyes of diabetic mice ( Figure 3B). α-SMA is a well-known marker of myofibroblasts, the cells implicated in fibrosis 30 . Diabetes is known to cause macrovascular disease by direct hyperglycemic injury to the vascular endothelium and by inhibition of nitric oxide, resulting in vasoconstriction, increased intravascular inflammation, and atherosclerosis 10 . The increase of α-SMA in brain tissue may indicate early atherosclerosis preceding diabetic stroke 53 . It is also possible this increase in α-SMA plays a role in dementia pathogenesis, although the exact mechanism for the increased prevalence of dementia among diabetic patients is unknown 54,55 .
Prior studies of STZ-induced hyperglycemia have confirmed retinopathy in mice after 24-64 weeks of sustained hyperglycemia 27−29 . Here we found increased number of α-SMA-positive cells in brain and retina tissues at 10-12 weeks after hyperglycemia onset. This suggests that pro-fibrotic α-SMA-positive myofibroblasts precede the appearance of overt clinical signs. Our observation is consistent with a study that identified elevated expression of N-cadherin, α-SMA, Snail, fibronectin and connective tissue growth factor in retinal tissue purified from patients with proliferative diabetic retinopathy 29 . Our finding of elevated α-SMA expression in diabetic retinal tissues is also consistent with the fact that microvascular retinopathy is one of the earliest complications in young patients 56 . This finding is unlikely to simply be the result of STZ toxicity to retinal ganglion cells, as similar levels of retinal fibrosis have been identified across many diabetic animal model systems as well as in diabetic patient autopsies 57,58 .
We were not able to confirm elevated α-SMA expression in the rest of the analyzed organs. The short diabetic disease state of 10-12 weeks at sustained hyperglycemia may be insufficient to lead to these late-stage disease complications that are often seen decades into the disease in humans. Further, although α-SMA has long been used as the standard marker for fibrosis, studies have shown that α-SMA-negative lung and kidneys are able to manifest fibrosis by other means 38,59 . Certain tissues express higher levels of alternate proteins during wound repair, such as Smad, N-cadherin, β-catenin, and Snail 15,29,30,60 . Another possible explanation for this discrepancy is that α-SMA expression may only be present in tissues that are actively expressing fibrotic components and laying down extracellular collagen and fibrin networks 61 .
After the fibrosis has taken place and collagen is laid down, α-SMA expression decreases and may not be detectable by flow cytometry. It would be useful to investigate the histology of these internal organs to visualize the extent of fibrosis. Further analysis of organ-level diabetic fibrosis would benefit from the use of additional molecular markers to account for the variety of fibrotic response in different tissues. Flow cytometry offers a convenient method to assess the number of cells positive for a marker of interest such as α-SMA. Several cell markers could be detected simultaneously to define a pre-fibrotic profile and cascade of molecular events. For example, epithelial to mesenchymal transition (EMT) has been indicated as a process that can generate myofibroblasts and has been implicated directly in fibrosis 62 . A parallel biopsy and histologic analysis could offer complementary evidence of fibrosis and hyperglycemia-induced changes.
In summary, we successfully established the use of flow cytometry to detect early changes in the number of α-SMA positive cells in brain and retina tissue of a T1DM murine model obtained with low-dose STZ protocol. The STZ protocol is a well-known, robust and reliable method to induce diabetic hyperglycemia.
We have coupled it with flow cytometry to demonstrate the feasibility of quantitative measurements of the number α-SMA-positive cells and observed changes in brain and retinal tissues. This cellular analysis is representative of increased vascular fibrosis in hyperglycemic mice central nervous system tissues, detectable as early as 10-12 weeks after hyperglycemia onset. Flow cytometry is a fast, reproducible, and reliable quantitative method that could be used to study organ fibrosis in this and other diabetic models.
The quantification of cell markers involved in fibrosis could be used to investigate early events preceding diabetic retinopathy, stroke or dementia.  (E) ImageJ Color Deconvolution image processing of normoglycemic G8-treated wound tissue Trichrome stain to isolate collagen stain. (F) Baseline dermal height was decreased in unwounded hyperglycemic mice compared to normoglycemic controls, although not statistically significant. Post-injury, no difference is seen in dermal height of normoglycemic and hyperglycemic tissue. Data was analyzed using unpaired 2-tailed student t-test. Ng unwounded and wounded n=16; Hg unwounded and wounded n=14. (G) Post-injury, epidermal height was significantly increased in both normoglycemic and hyperglycemic mice (P<0.0001). The fold-increase of unwounded to wounded epidermal height was larger in hyperglycemic mice (normoglycemic = 2.3, hyperglycemic = 6.0). Data was analyzed using unpaired 2-tail student t-test. Ng unwounded and wounded n=16; Hg unwounded and wounded n=14. (H) Decreased collagen deposition was seen in hyperglycemic wounds compared to unwounded normoglycemic tissue (P<0.05). Data was analyzed using unpaired 2-tail student t-test. Ng unwounded and wounded n=15; Hg unwounded and wounded n=14.