A Diabetic Mice Model for Studying Skin Wound Healing

Advances in wound treatment depend on the availability of animal models that reflect key aspects of human wound healing physiology. To this date, the accepted mouse models do not reflect defects in the healing process for chronic wounds that are associated with type two diabetic skin ulcers. The long term, systemic physiologic stress that occurs in middle aged or older type 2 diabetic patients is difficult to simulate in preclinical animal model. We have strived to incorporate the essential elements of this stress in a manageable mouse model: long term metabolic stress from obesity to include the effects of middle age and thereafter onset of diabetes. At six-weeks age, male C57BL/6 mice were separated into groups fed a Chow and High-Fat Diet for 0.5, 3, and 6 months. Treatment groups included long term, obesity stressed mice with induction of diabetes by streptozotocin at 5 months, and further physiologic evaluation at 8 months old. We show that this model results in a severe metabolic phenotype with insulin resistance and glucose intolerance associated with obesity and, more importantly, skin changes. The phenotype of this older age mouse model included a transcriptional signature of gene expression in skin that overlapped that observed with elderly patients who develop diabetic foot ulcers. This unique old age phenotype result contrasts with current non-obesity stressed mice models with induced diabetes at 4 months or younger which do not reflect the pathologic type 2 human phenotype.


INTRODUCTION
Diabetes mellitus (DM) is a chronic metabolic disease characterized by hyperglycemia that results from deficiencies in insulin secretion and/or action. It is one of the major global health problems affecting over 463 million people worldwide and projecting an increase to 578 million by the end of 2023 (1). As a chronic and refractory disease, DM affects every tissue and organ in the body, including the skin. Studies show that up to two-thirds of diabetic patients have skin problems at some point during their lifetime (2). There are several mechanisms behind DM-associated skin abnormalities, which include, but are not restricted to, oxidative stress, abnormal regulation of inflammatory products, impaired angiogenesis, and impaired growth factor production (3).
During the course of DM, two features seem central to explain the greater risk for development of skin disease: i, an accelerated skin aging (2,4) and ii, an increased risk for development of secondary infections, which is particularly important in diabetic foot ulcers (DFU). DFU is the most common complication affecting DM patients; it increases the risk for development of osteomyelitis, which can lead to lower extremity amputations (5,6) .
Treating DFU has proven difficult as it results from a complex pathophysiology (6).
Moreover, due to ethical concerns, trials for new therapeutic interventions in humans are limited, which delays the development of effective strategies to treat this condition.
Therefore, testing new approaches to treat DFU must rely, at least during early phases, on the existence of appropriate experimental models.
Over the years, several experimental models have been employed in studies aimed at evaluating interventions for treating DFU. These include spontaneous autoimmune DM in rodents, genetically induced DM models; diet-induced DM models and pharmacologically induced DM models (7)(8)(9). Regardless of the benefits obtained with each of these models, they all have particular limitations. One important limitation that occurs in virtually all models employed to date relies on the fact that they do not exhibit simultaneously two important features that are common to most patients with DFU; severe metabolic dysregulation and accelerated skin aging. Ideally, an experimental model for studying DFU should have close similarities to the clinical and pathological landscape of human DFU. In this study, we describe a model in which DM and aging result in skin alterations that display great similarities with those found in aging humans.

Diabetes induction
5-month old C57BL/6J mice were fed on HFD for 3 months when five low-dose injections of streptozotocin (STZ) (50 mg/kg, i.p.) were performed (Fig. 1a). Our results showed that after four weeks from STZ injections, HFD diabetic (HFD DM+) mice increased blood glucose levels up to 500mg/dL (489.7 mg/dL HFD DM+ vs 143 mg/dL age-matched non-diabetic mice, p value <0.0001). In addition, our result showed Chow diet did not affect fasting glycemia during the 8-month of analysis (Figure 1b, black bars). However, the 8-month old mice fed on HFD increased fasting glycemia compared to diet-paired 2.5-month old group (from 166 mg/dL in young to 206 mg/dL in older mice, p value 0.0268) (Figure 1b, blue bars). Moreover, the 8-month old HFD DM+ group increased even more fasting glycemia (3 fold more after 3 months streptozotocin injections) compared to diet-paired 2.5-month old counterparts (from 166 mg/dL younger to 490 mg/dL in older mice, p value <0.0001) ( Figure   1b, red bars).

Aging increases body mass and white adipose tissue deposition in mice.
Aging affects body mass (Figure 1c). Independently of food characteristics, mice fed on Chow or HFD increased Body mass during the 8 months of analysis (Figure 1c and Table 1 In addition, we found increased epididymal white adipose tissue (WAT) mass in older 5-and 8-month old mice fed with Chow diet compared to younger 2.5-month old mice fed on Chow (from 0.16g to 0.64g, p value <0.0001). This increase represents a 4-fold WAT mass in the 8-month old group fed with Chow diet compared with their younger counterpart (Figure 1d, black bars). We found this WAT mass increase following the Body Mass increase pattern (Supplementary 1a). Additionally, mice fed on HFD increased even more WAT mass ( Figure   1d, blue bars). We found increased WAT in older 8-month old animals fed with HFD compared with younger HFD 2.5-month and 5-month old groups (from 0.48g to 1.94g, p value 0.0001). In the same way that Chow fed animals, the increased WAT mass represents 4-fold the WAT mass of younger mice fed on HFD ( Figure 1d). As well as the Chow group, we found this increase in WAT mass also follows the Body Mass increment pattern (Supplementary 1a).
However, the biggest differences were found in older 8-month old HFD DM+. HFD DM+ mice decreased WAT mass compared to younger 5-month old diabetic mice (from 1.7g to 0.7g, p value 0.0005) representing 2.3-fold less WAT mass (Figure 1d, red bars).
Interestingly, over the course of 3 months with induced diabetes beginning at 5 months' age, this decrease in WAT mass also follows the Body Mass decline pattern of the HFD DM+ animals (Supplementary 1a).

DM+.
We explored how aging affects insulin levels and polyphagia. In 2.5-month old mice fed on chow diet showed 0.4 ng/mL insulin levels while 2.5-month old HFD mice showed 0.9 ng/mL insulin levels with no polyphagia. In 8-month old mice fed on chow diet showed 0.35 ng/mL insulin levels while 8-month old mice fed on HFD presented as 1.1 ng/mL insulin levels with no polyphagia. However, 8-month HFD-diabetic mice (HFD DM+) presented 0.3ng/mL insulin levels ( Figure 1e) with polyphagia ( Figure 1f). 8-month old HFD mice increased Food intake compared to 2.5-month old HFD mice (from 3g/day to 4g/day, p value 0.0232, Figure 1f). 8-month old HFD DM+ mice further increased Food intake to 5g/day (p value <0.0001, Figure 1f). In contrast, 8-month old Chow diet mice showed no differences in Food intake ( Figure 1f).

Aging impacts Insulin and Glucose tolerance in mice fed on HFD diet.
We investigated the contribution of aging to insulin sensitivity by Intraperitoneal injection of insulin (ITT) and glucose metabolism by Intraperitoneal injection of glucose (GTT) in Chow, HFD, HFD DM+ mice. Our results showed aging has a progressive and detrimental effect on insulin sensitivity and glucose metabolism in 8-month old HFD mice (Figure 1h

Aging impacts gene expression associated with insulin pathway, obesity and aging.
To better understand the effect of aging in the skin of Chow, HFD and HFD DM+ mice, we screened 3 different gene arrays: Aging, Insulin Pathway and Obesity.
Column C. Figure 1J shows the expression of Obesity-associated genes in the skin of

Aging affects pancreatic Insulin 1 and 2 expressions in mice fed on HFD.
Ins1 and Ins2 genes encode for insulin 1 and 2, peptides that are vital in the regulation of carbohydrate and lipid metabolism. Our result showed that total Ins1&2 gene expression increased in pancreatic tissue in 5-month old mice fed on HFD (Figure 1k). After streptozotocin injections, the HFD DM+ mice decreased pancreatic total Ins1&2 gene expression ( Figure 1k).

Aging affects the accumulation of intrahepatic and dermal fat in HFD and Chow fed mice.
Both the accumulation of intrahepatic fat (Hepatic steatosis) and insulin resistance are associated with liver metabolic dysfunction (10,11). For this reason, we explored intrahepatic fat percentage in HFD DM+. Figure 1m shows the histological presence of intrahepatic fat. After 8 months on Chow diet, intrahepatic fat increased from 7% to 11% when comparing to 2-month old mice (p value <0.0001). 8-month old HFD mice had increased intrahepatic fat percentage from 19% to 38% (p value <0.0001). HFD DM+ animals increased from 19% to 25% (p value 0.0148).
Dermal white adipose tissue (dWAT), occurs in the dermis underlying the reticular dermis (12), and participates in thermogenesis, wound healing and immune defense against infection (13). We investigated dermal fat deposition in mice fed on Chow, HFD and HFD DM+. Figure 1n shows the histological presence of dWAT. 8 months Chow and HFD animals increased dWAT relative to both 2-and 5-month old mice (p value <0.0001). In contrast, HFD DM+ mice decreased dWAT relative to 5-month old HFD mice (p value <0.0001).

HFD and hyperglycemia affects body mass in HFD DM+ mice.
In our previous studies of mice less than 5-month age, we have shown that HFD affects body mass (14-17) (). Figure 2a shows that after only 2 weeks on HFD, mice had 13% increased body mass as compared to the Chow diet group (24.6g HFD vs 21.9g Chow, p value <0.0001 ). 3 months additional feeding on HFD resulted an increase of 35% of body mass compared to Chow diet (37.8g HFD vs 27.9g Chow, p value 0.0024). A total of 6 months HFD increased body mass by 47% compared to Chow diet mice (49.1g vs 33.5g, p value <0.0001).
In contrast, HFD DM+ after a total of 6 months on HFD had decreased 39% body mass compared with their non-diabetic HFD counterparts (30.1g HFD DM+ vs 49.1g HFD, p value <0.0001).

HFD DM+ have both decreased insulin expression and circulating insulin.
Our result showed that after post Streptozotocin treatment, HFD DM+ decreased Pancreatic  We found mice fed on HFD for 3 months increased 37% Fasting blood glucose compared to Chow diet mice (190mg/dL vs 139mg/dL, p value 0.0021) (Figure 2d). After 6 months on HFD, mice had a 41% increased glucose levels compared to Chow mice (201mg/dL vs 142mg/dL, p value <0.0001). In contrast, HFD DM+ had 144% and 243% increased Fasting blood glucose when compared with age and HFD-and Chow, respectively (490mg/dL vs 201mg/dL vs 142mg/dL, p value <0.0001).

Diabetic mice showed polyphagic food intake and polydipsic water intake.
Our results showed HFD DM+ mice increased daily Food intake ( Figure 2e). HFD DM+ mice displayed a 25% increased daily Food intake compared with their non-diabetic HFD counterparts (5g vs 4g, p value 0.0021). We found no differences in water intake (9mL vs 6mL, p value 0.0718) between Chow diet and HFD mice (Figure 2f). However, HFD DM+ showed polydipsic water intake compared either to Chow diet or their non-diabetic HFD counterparts (16g vs 9g, p value <0.0001) (Figure 2f). This difference in diabetic mice represents a 44% more daily water intake compared to the HFD mice and 63% more water intake compared to mice fed on Chow diet.

HFD and HFD DM+ mice present chronic Insulin resistance.
To determine the whole-body sensitivity to insulin, we measured blood glucose levels after intraperitoneal insulin administration (Figure l-m). ITT results showed that after 0.5 months of HFD plasma glucose levels increased at 5 min (175mg/dL vs 142mg/dL) and 15 min (108mg/dL vs 91mg/dL) compared to mice fed on Chow diet (p value <0.05) (Figure l). After 3 months on HFD the plasma glucose levels increased at 0 min (180mg/dL vs 153mg/dL), 5 min (214mg/dL vs 164mg/dL), and also 10 min (177mg/dL vs 122mg/dL) (p value <0.05).
After 6 months on HFD the plasma glucose levels were increased at all time points (p value 0.0001). 8-month old HFD DM+ mice (6 months on HFD) also presented higher plasma glucose levels at all time points (p value <0.0001). A constant rate of glucose disappearance was observed (kITT) (Figure 2m) where higher values indicate greater tissue insulin resistance (18,19). As results, no kITT differences were observed after 0.5 months of HFD feeding (p value 0.0783) (Figure 2m) but kITT increased after 3 months on HFD (p value 0.0071) (Figure 2m). This kITT difference represents a 41% more in insulin resistance in HFD animals compared to the Chow group. HFD DM+ showed increased kITT values compared to age-matched Chow diet groups (p value 0.0274), but not when compared to the HFD group (Figure 2m). This kITT difference in HFD DM+ mice represents a 133% increase in insulin resistance compared to Chow diet mice but no difference with the HFD group (p valor 0.7867) (Figure 2m).

Diet and glycemia impact gene expression associated with insulin pathway, obesity and aging.
Column A. Figure 2i shows the expression of insulin-associated genes in the skin of 8-month old HFD animals relative to 8-month old Chow counterparts. After 2 weeks on HFD, skin tissue increased Igfbp1 (3.2-fold), G6pc (2.5-fold), and Ins1 (2-fold) more transcripts than their age-matched Chow diet counterparts. After 6 months on HFD, skin tissue overexpressed G6pc2 (8.7-fold), Lep (8.5-fold), and Ins1 (6 fold) more transcripts as well as downregulation of Akt1 (0.01 fold) when compared to their age-matched Chow diet counterparts (Figure 2i).
Column B. Figure 2i

HFD and hyperglycemia affect intrahepatic fat deposition in mice.
Our results showed that HFD and hyperglycemia both are associated with accumulation of intrahepatic fat percentage in mice (Figure 3a,c). Mice fed for 0.5 months on HFD had increased intrahepatic fat compared to age-matched Chow fed mice (19% vs 11% , p value <0.0001). Mice fed for 3 months on HFD also had increased intrahepatic fat compared to age-matched Chow fed mice (21% vs 13%, p value <0.0001). After 6 months on HFD, mice further increased intrahepatic fat compared to age-matched Chow fed mice (38% vs 9.3%, p value <0.0001). In contrast, HFD DM+ had decreased intrahepatic fat percentage as compared to age-matched HFD fed mice (38% vs 25% , <0.0001) (Figure 3a,c).

HFD and hyperglycemia affect dermal fat deposition in mice.
Our histological analysis showed that HFD and HFD DM+ had different levels of accumulated dWAT (Figure 3b,d). Both mice with 0.5 months and 3 months on HFD had increased dWAT as compared to the Chow fed mice (p value <0.05). In contrast, HFD DM+ mice had decreased dWAT as compared to HFD group (p value <0.0001) (Figure 3b,d). We compared this 8-month old HFD DM+ model data to previously published data of 4month old STZ-induced diabetic non-HFD mice. We identified 4 common genes increased between older and younger STZ-induced diabetic mice: Pparg, Dok3, Vega and Grb2 ( Figure   4a).

DISCUSSION
Here, we described the metabolic phenotype of an experimental model that can be used to study skin wound healing in aging and DM. We showed that an experimental model that associates diet-induced obesity, aging and pharmacological induction of diabetes mellitus results in a severe metabolic phenotype with insulin resistance, reduced b-cell insulin expression and glucose intolerance and this is associated with changes in the skin that match the skin of aging humans with DM.
DM is a chronic metabolic disease that is associated with accelerated aging resulting in the damage of several tissues and organs in the body including steatosis of the liver (20)(21)(22). The skin is an important target of the metabolic, vascular, immunological, and neural abnormalities that result from a poorly controlled DM. The processes for the development of these problems are not yet entirely elucidated (4,23). Diminished lamellar body synthesis, lipid production of epidermal cells, and decreased hydration have also been correlated with persistent hyperglycemia, take the lead to the deterioration of the cutaneous barrier function, and turning the skin prone to dryness and infections (24,25) .Furthermore, increased oxidative stress and low-grade inflammation are associated with the development of skin lesions from diabetes (10.2174/157339911797415585). Thus, in our animal model, we observed changes in the transcripts of important aging intermediaries that could result in alterations in the skin (Figure 4a). Moreover, we observed decreased in transcripts involved in insulin action and obesity. Pro-inflammatory cytokines are crucial in regulating the inflammatory process during cutaneous wound healing, that can prolong the inflammatory phase and inhibit healing progression (6,26,27). Furthermore, some transcripts associate with diet-induced obesity, aging and pharmacological induction of diabetes, as a Gsk3b, was observed in this work ( Figure 5). Gsk3b performs as a negative controller in Wnt signaling (28). Wound healing stimulate Wnt signaling and this canonical signaling contributes to each sequential period of the healing process from the management of the inflammatory phase and programmed cell death to the mobilization of stem cell pools within the wound (29).
Likewise, c-Jun and Serpina-1 were shown to be modulated in the skin of this animal model ( figure 5). C-Jun and Serpina-1 are intimately involved in the inflammatory as well as the proliferative phase of the wound healing process (30).
DFU is the most common skin condition associated with diabetes and, because of the lack of optimal therapeutic interventions, it leads to prolonged periods of inactivity, high risk of infections and eventually the need for amputations (31)(32)(33). The development of new therapeutic interventions that could promote faster and more effective wound healing could improve life quality of affected patients, reducing the risk for severe outcomes. However, testing new agents that could improve the therapeutics of DFU is frequently a problem as ethical issues impose a number of restrictions. Thus, at least in early phases of testing, animal models can provide a fast and effective approach to identify candidates with high chance of success.
Most animal models used to date do not match clinical conditions frequently seen in humans with DM and DFU. Usually mice models with severe diabetes, develop such condition early in life, thus, the important component of aging is missing (7)(8)(9). Conversely, in models of DM induced by the consumption of energy dense diets lead to a chronic condition that can be sustained until late life; however, under these circumstances, metabolic abnormalities are generally mild (34)(35)(36).

CONCLUSION
Here we present an old age, obesity stressed mouse model with old age onset of diabetes. We showed that mice submitted to a 8 month long physiologic stress developed severe metabolic abnormalities associated with aging, long-term obesity and subsequent onset of diabetes. For example, tissue abnormalities such as hepatic steatosis occurred but the most striking was a skin phenotype which histologically and transcriptionally could be compared to aging DM patients. Thus, this unique model is perhaps useful to therapeutic development for the treatment of human decubitus ulcers.

RT2 Profiler PCR arrays
Gene expression was analyzed as previously described (37) . Briefly, Gene expression was obtained using 3 different 96-well RT2 Profiler PCR Arrays, Mouse Wound Healing (REF.

Dietary interventions
Eight-week-old mice were separated into seven groups. Three groups fed with Chow diet (base-line control). The remaining four groups were fed with High-Fat diet (HFD) for 2, 12 or 24 weeks (diets composition in Table 1, experimental design in Figure 1). Mice were analyzed for Intraperitoneal Insulin Tolerance test (ITT) and Intraperitoneal Glucose Tolerance Test (GTT). Then, mice were subjected to lethal anesthesia and tissues specimens were extracted for analyses.

Metabolic characterizations
For ITT, mice were fasted as previously described (Bombassaro et al., 2019). Tail blood was collected for basal glucose evaluation. A solution of insulin (1.5 U/kg body weight) was administrated via intraperitoneal and blood was collected from tail vein after 5, 10, 15 and 20 minutes. For GTT, mice were submitted to fasting protocol according previously described (38). Basal glucose concentration was determined from collected tail blood. A solution of 20% glucose (2.0 g/kg body weight) was administrated via intraperitoneal and blood from tail vein was collected after 15, 30, 60, 90 and 120 minutes. In both tests, blood glucose concentration was measured using handheld glucometer.

Diabetes Mellitus induction protocol
At 22 weeks of age, one group on HFD was induced to diabetes mellitus (DM) with Streptozotocin (STZ). Mice were fasted for 4h before daily intraperitoneal injections of STZ (50 mg/kg). STZ was daily and freshly dissolved in 0.1 M sodium citrate buffer, pH 4.5, and the dose volumes for each mouse were calculated by Labinsane app (Jara et al., 2021) STZ injections were made for five consecutive days (low multiple-dose protocol). One group on HDF and one on chow were injected sodium citrate buffer and served as control.
Induced DM state was assessed after four weeks using an OptiumTM mini (Abbott Diabetes Care, Alameda, CA, USA) handheld glucometer with appropriate test strips. Blood glucose levels were measured by blood from tail vein. Mice whose blood glucose levels exceeded 300 mg/dL after treatment were considered diabetic.

Diary food intake and mice weight measurements (Renan)
One week before ITT and GTT experiments of each group, the mice were submitted to food and weight measurements to avoid more distress from the procedures that could interfere in their intake. The mice were fasted for 12 hours overnight and then weighed for five consecutive days at the same hour, their food was also weighed at the same time to analyze calories intake and cumulative weight gain. In addition, the 24-week group with mice that were induced to a DM state also had their water intake measured in the same period.

Tissue extraction
Mice were submitted to fasting protocol according to previously described (Nogueira et al, 2020). Subsequently, mice were anesthetized with lethal doses of xylocaine and ketamine calculated by Labinsane App. Pancreas and fragments of dorsal skin (8.0 mm punch), liver, epididymal white adipose tissue, were extracted and prepared for molecular analysis or histological staining. Plasma samples was obtained from whole blood samples collected in EDTA pre-coated tubes, followed by centrifugation (3500 RPM, 15 minutes, room temperature), and were stored at −80 °C.

Determination of glucose, and insulin
Serum glucose was determined by the glucose oxidase method, as previously described (17).
Serum insulin was determined by RIA, as described (18). Serum leptin was determined using an ELISA kit from Linco/Millipore (Billerica, MA) according to the recommendations of the manufacturer (39).

Intraperitoneal glucose tolerance test
After 6 h fasting, mice were anesthetized by an ip injection of sodium amobarbital (15 mg/kg body weight), and the experiments were initiated after the loss of corneal and pedal reflexes.
After collection of an unchallenged sample (time 0), a solution of 20% glucose (2.0 g/kg body weight) was administered into the peritoneal cavity. Blood samples were collected from the tail at 30, 60, 90, and 120 min for determination of glucose and insulin concentrations (39).

Insulin tolerance test
Insulin (1.5 U/kg) was administered by ip injection, and blood samples were collected at 0, 5, 10, 15, 20, 25, and 30 min for serum glucose determination. The rate constant for glucose disappearance during an insulin tolerance test (kITT) was calculated using the formula 0.693/t1/2. The glucose t1/2 was calculated from the slope of the least-square analysis of the plasma glucose concentrations during the linear decay phase (39).
For the histological analyses, fragments of liver, pancreas and skin tissues were processed and stained with hematoxylin and eosin as previously described.  Schematic representation of experimental design. C57BL/6 mice were fed either with Chow Diet or High Fat diet. Some 5-month old HFD animals were treated with streptozotocin for Diabetes induction (HFD DM+ group) generating 2 HFD groups: a) HFD DM+ (T2DM-like) and b) HFD non-diabetic.