This study was financially supported by the National Natural Science Foundation of China (82104877).

The protocol was conducted following the Declaration of Helsinki, and all animal experiments were approved by the Management Committee from Chengdu University of Traditional Chinese Medicine (Record No. 2021-13).
1. Preparation of the streptozotocin injection
<ol>
	<li>Select 15 SD male specific pathogen-free (SPF) rats aged 8 weeks and weighing 220 g &#177; 20 g. Using the simple randomization method, divide the rats into a diabetic group (n = 10) and a normal group (n = 5).</li>
	<li>Measure the initial weight of the rats, and determine the dosage of STZ through the administration of 55 mg/kg. 
	NOTE: Based on pre-experiments, 55 mg/kg is the optimal STZ dose.</li>
	<li>Weigh the STZ powder accurately, and place it in a light-proof container.</li>
	<li>Add an appropriate amount of 0.1 mol/L sodium citrate buffer (pH 4.5) to dissolve the STZ to a concentration of 1 %. 
	NOTE: The sodium citrate buffer must be pre-cooled for 2 h in a 4 &#176;C refrigerator before use.&#160;The preparation of the STZ solution must assure sterility.</li>
	<li>Shake for 30 s using a drug oscillator. Place in an ice box, and set aside. 
	&#8203;NOTE: The injection should be used within 15 min.</li>
</ol>
2. Induction of the T1DM model
<ol>
	<li>Before the STZ injection, fast the rats for 18 h, and allow free access to water.</li>
	<li>Perform an intraperitoneal injection of 1% STZ solution.
	<ol>
		<li>Grasp the rat, and expose the abdominal skin and the injection site (the intersection of the line connecting the roots of the two thighs and the midline of the abdomen).</li>
		<li>Disinfect the injection site twice using a cotton ball soaked in 75% alcohol (once clockwise and once counterclockwise). Place the rat&#39;s head below the abdomen.</li>
		<li>Insert the needle parallel to the abdominal midline at an angle of 45&#176;, and after piercing the skin, reduce the needle angle to 30&#176;, and then insert the needle 2-3 mm. Gently pull the needle plug, ensuring no blood or fluid is sucked into the syringe. Inject the STZ solution, pull out the needle, and stop the bleeding with a cotton swab. 
		NOTE: If a yellow fluid is drawn back into the syringe, the needle may have penetrated the bladder, and if a dark green fluid is drawn, the needle could have penetrated the large intestine or caecum. In either case, the needle should be removed immediately. The animal should be evaluated by veterinary personnel.</li>
	</ol>
	</li>
	<li>Measure the casual (non-fasting or fasting) blood glucose levels at 09:00 am on day 3 and day 7 after STZ induction. 
	NOTE: The time for random the blood glucose measurement is fixed. In this protocol, it is fixed at 09:00 am. However, it is not the only time used. Blood collected from the caudal vein by a needle puncture is less susceptible to tissue fluid than blood taken from a severed tail, so blood glucose values are more accurate.
	<ol>
		<li>Immobilize the rat using a rat fixator (Figure 1).</li>
		<li>Find the location of the caudal vein. Disinfect the tail of the rat twice using a cotton ball soaked in 75% alcohol.</li>
		<li>Puncture the caudal vein to induce bleeding, and measure the blood glucose using a glucometer. Stop the bleeding with a cotton swab. 
		NOTE: A glucose level higher than 16.7 mmol/L on day 7 after the STZ injection is considered T1DM.</li>
	</ol>
	</li>
	<li>Weigh the rats weekly, and measure the blood glucose levels and other parameters, including diet, water intake, and urine output.</li>
	<li>Feed the animals normally for 8 weeks after STZ induction.</li>
</ol>
3. Construction of the wound model
<ol>
	<li>Shave the rats with an electric razor 1 day before wound modeling. A shaved area of 5 cm x 5 cm on the dorsum side of the rat is generally ideal.</li>
	<li>Wipe the shaved area with a warm normal saline cotton ball, allow it to dry, and then apply depilatory cream for 5 min. Clean the area with gauze, and wash any residual depilatory cream with warm normal saline.</li>
	<li>Weigh the rats, and calculate the required dose of Nembutal based on the 35 mg/kg standard. Dissolve the Nembutal using normal saline to a concentration of 3%.&#160;Other general anesthetics such as ketamine/xylazine or isoflurane can be used for this procedure. Please work with Institutional Animal Care and Use Committees to ensure what is best. 
	NOTE: To ensure efficacy, the solution should be freshly prepared, and the Nembutal powder and solution should be protected from light.</li>
	<li>Fast the rats for 12 h before anesthetization. Inject the anesthesia intraperitoneally. Use tetracycline eye ointment or a general eye lubricant to prevent eye dryness following the anesthesia administration. 
	NOTE: Anesthesia was considered moderate when the rat&#39;s muscles were relatively relaxed, the eye movements disappeared, the breathing was regular, and the response to painful stimuli was small.</li>
	<li>Disinfect the dorsal skin twice using cotton balls soaked in iodine (once clockwise and once counterclockwise) and 75% alcohol (alternative rounds).</li>
	<li>After drying, cut the skin with a 20 mm diameter circular biopsy punch.</li>
	<li>Tent the skin with sterile forceps, and then use sterile surgical scissors to remove the full-thickness skin along the punch cut marks. Stop the bleeding with a normal saline cotton ball. 
	NOTE: The wound&#39;s superior border should be 5-10 mm below the lower scapular border and 5-10 mm to the right/left of the spine of the rat (Figure 2). The wounds are symmetrical along the spine when two wounds are constructed.</li>
	<li>Use Vaseline gauze to cover the wounds, and wrap them with a gauze and breathable bandage held in place with rubber tape. Inject&#160;carprofen subcutaneously (5 mg/kg) once daily to relieve postoperative pain. Change the wound bandage once a day (use of carprofen for pain relief). 
	NOTE:&#160;Observe the rat&#39;s movement and respiration for any abnormalities after the bandage is completed, and ensure that the breathable bandage is appropriately tight.</li>
	<li>Place a ruler under the wound, and photograph the wound with a digital camera until day 14. Euthanize the rats on day 14 according to the institutional animal care and use guidelines. Cut the wound skin tissue 5 mm from the wound edge. Divide the tissue sample into two parts, wash them with PBS to remove visible blood stains, and then fix them with 4% paraformaldehyde solution.</li>
</ol>
4. Calculation of the wound area with ImageJ software
<ol>
	<li>Click on the File button after opening the software, and then drop down and click on Open to open the wound pictures.</li>
	<li>Select the Straight tool, and draw a straight line of 1 cm along the ruler in the wound pictures.</li>
	<li>Click on the Set Scale command in the Analyze menu, and set the Known distance to 1.</li>
	<li>Select the Freehand selections tool, and sketch the outline of the wound on the picture.</li>
	<li>Click on the Measure command in the Analyze menu, and read the Area value after the result pops up.</li>
</ol>
5. Hematoxylin and eosin (H&#38;E) staining
<ol>
	<li>Remove the skin tissue from the fixative, cut it into thin sections with a scalpel in a fume hood, and place it in a dehydration cassette.</li>
	<li>Put the dehydration cassette in a dehydration machine, and dehydrate the tissues in the following steps: 75% alcohol for 4 h; 85% alcohol for 2 h; 90% alcohol for 2 h; 95% alcohol for 1 h; anhydrous ethanol I and II for 30 min each; alcohol benzene for 5-10 min; xylene I and II for 5-10 min each; and wax I, II, and III for 1 h each.</li>
	<li>Embed the tissues in wax. Cool at &#8722;20&#176; on a freezing table, and correct the wax block neatly.</li>
	<li>Cut the wax blocks longitudinally using a paraffin sectioning machine into 3 &#181;m thick sections.</li>
	<li>Sequentially soak the section in xylene I and II for 20 min each, anhydrous ethanol I and II for 5 min each, and tap water for 5 min.</li>
	<li>Flood the tissues with hematoxylin stain for 3-5 min, 0.5% aqueous hydrochloric acid solution differentiation, 0.5% aqueous ammonia solution back to blue, and rinse with water.</li>
	<li>Dehydrate the tissue sections with 85% and 95% alcohol. Flood the tissues with eosin staining solution for 5 min. 
	NOTE: Normally, eosin staining takes 30 s to 2 min, and the time can be adjusted according to the staining results and requirements.</li>
	<li>Dehydrate the sections sequentially with the following solutions: anhydrous ethanol I, anhydrous ethanol II, anhydrous ethanol III, xylene I, and xylene II, each for 5 min. Finally, cover the glass slides with neutral balsam.</li>
	<li>Examine the H&#38;E-stained tissues under the microscope at 40x, 20x, and 10x, and take pictures to retain representative images of each slide.</li>
</ol>
6. CD31 immunofluorescence staining
<ol>
	<li>Soak the tissue sections in xylene I and II for 15 min each, anhydrous ethanol I and II for 5 min each, 85% alcohol for 5 min, and 75% alcohol for 5 min, and rinse with distilled water.</li>
	<li>Antigen repair
	<ol>
		<li>Add a suitable 10 mM citric acid buffer of pH 6.0 to a microwave oven container, heat it to boiling on high, and then place the glass slide into it.</li>
		<li>Boil for 8 min on a medium heat, stop for 8 min, and then boil again at a medium-low heat for 7 min.</li>
		<li>Allow the slides to cool, place them in PBS (pH 7.4), and wash them three times for 5 min each using a decolorization shaker.</li>
	</ol>
	</li>
	<li>Add 5% goat serum dropwise in the circle, and incubate for 30 min.</li>
	<li>Gently shake off the closure solution (5% goat serum), and add rabbit anti-CD31 antibody (diluted using PBS at a ratio of 1:200) dropwise onto the sections. Place the sections in a wet box, and incubate overnight at 4&#176;C.</li>
	<li>Wash the slides three times with PBS (pH 7.4) on a decolorization shaker for 5 min each. Lightly shake the sections to dry them, and then cover them with a circular drop of FITC-labeled goat anti-rabbit IgG. Incubate at room temperature for 50 min in darkness.</li>
	<li>Wash the slides three times with PBS (pH 7.4) on a decolorization shaker for 5 min each. Air-dry the sections with light shaking, and add DAPI staining solution. Incubate the sections in the dark for 10 min at room temperature.</li>
	<li>After drying the sections, draw circles around the tissue with the PepPen (to prevent the loss of antibodies), add an autofluorescence quenching agent (0.3% Sudan Black B) to the circles for 5 min, and thereafter rinse them under running water for 10 min.</li>
	<li>Wash the slides three times with PBS (pH 7.4) on a decolorization shaker for 5 min each. Shake the sections slightly, and seal them with an antifade mounting medium.</li>
	<li>Observe and photograph the sections under a fluorescence microscope at 40x, 20x, and 10x. 
	&#8203;NOTE: The DAPI UV excitation wavelength is 330-380 nm, and the emission wavelength is 420 nm (blue light). The FITC excitation wavelength is 465-495 nm, and the emission wavelength is 515-555 nm (green light).</li>
</ol>
7. Statistical analysis
<ol>
	<li>Collect and analyze the data using SPSS.</li>
	<li>Report the data as mean &#177; standard deviation.</li>
	<li>Use an independent samples t-test to analyze the differences between the diabetic and normal groups.</li>
	<li>Set the statistical significance at **p &#60; 0.01 and *p &#60; 0.05.</li>
</ol>

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</ol>

All authors declare that this manuscript has no conflicts of interest.

This protocol clarifies the disputed operations in T1DM wound modeling. Concerns on the STZ injection protocols, T1DM induction success criteria, blood glucose stabilization time, and wound location and size have been addressed in this work. Furthermore, the pathological characteristics and measurable parameters for T1DM wound healing assessment have been clarified.
The rats fasted for 18 h before the STZ injection to avoid the competitive binding of glucose or its analogs to &#946;-cells, which could affect the efficacy of STZ. The most commonly used method to induce T1DM is a single high dose of STZ, which increases blood glucose by damaging the islets and decreasing insulin secretion21. Pre-experimental trials revealed that the optimal STZ dose for a high success rate and a low mortality rate was 55 mg/kg, which is lower than the optimal doses reported in previous studies22,23,24. In this protocol, T1DM was induced using a single intraperitoneal injection of 55 mg/kg STZ.
The blood glucose levels were all higher than 16.7 mmol/L 3 days after the STZ injection. However, a blood glucose level higher than 16.7 mmol/L on day 7 after STZ injection is the recommended criterion for successful T1DM modeling, because the extent of islet damage varies among rats, and an appropriate extension of the diagnostic time can reduce the false-negative rate. In addition, the blood glucose fluctuations stabilized 5 weeks after the STZ injection, and the rats gradually gained weight during this period, consistent with previous findings25,26. This indicates that the blood glucose level in the T1DM model should be stabilized for at least 6 weeks, and an increase in rat weight after 6 weeks reduces the mortality rates during the wound modeling. Hence, this protocol conducted wound modeling 8 weeks after the STZ injection.
The wound closure rate on day 7 and day 14 after wounding was significantly lower in the diabetic than in the normal wound group, indicating slow healing. Moreover, wound re-epithelialization and angiogenesis were significantly lower in the diabetic than in the normal group. This demonstrates that the T1DM wound model shows slower wound healing and delayed re-epithelialization than in normal rats, which may be related to the pathological changes of reduced wound angiogenesis. However, on day 14, the T1DM wound healing rate was also above 90%, which is different from the chronic non-healing characteristic of human diabetic wounds. This could be because rodents&#39; physiological mechanisms for wound healing differ from those of humans27. Consequently, the best wound diameter is at least 20 mm, which is large enough to allow time to assess an intervention&#39;s efficacy in a diabetic wound study. The wound location should avoid the scapula and spine, as continuous motion in these two sites could disrupt wound healing.
In conclusion, the construction of the T1DM wound model using the method of this protocol is effective. The protocol replicates some of the characteristics of chronic diabetic wounds, such as slower wound healing, delayed re-epithelialization, and reduced angiogenesis compared to normal rat wounds. However, it is unknown whether the model can replicate other chronic phenotypes of diabetic wounds. Furthermore, this protocol describes the most fundamental and widely used method, which does not account for the issue of skin contraction in rats. Future research can incorporate the use of wound splints into this protocol or explore additional models of chronic diabetic wounds, which will be a significant challenge for researchers in the future.

Type 1 diabetes mellitus (T1DM) is a chronic metabolic disease characterized by hyperglycemia and the destruction of pancreatic &#946;-cells1. A T1DM wound is a chronic non-healing wound and the most common and devastating complication of diabetes in humans2,3. Animal models are the most appropriate prototypes for studying the pathological changes during wound healing and the safety and efficacy of potential therapeutic agents4. Compared to other types, male Sprague-Dawley (SD) rats are more sensitive to streptozotocin (STZ) and show a lower related mortality rate, making them popular in diabetic wound research5,6.
Numerous methods for constructing T1DM wound models have been described. Regarding the T1DM model, studies have primarily focused on the effect of the STZ injection method on the success rate of diabetes induction7,8. However, the modeling process suffers from the inconsistent operation of this same step. In one study, rats fasted for 18 h before the STZ injection; rats with blood glucose levels higher than 16.67 mmol/L 1 week after the STZ injection were deemed diabetic, and the diabetic wound was introduced after 3 weeks9. Conversely, in a related study, Zhu et al. fasted rats for 12 h before the STZ injection; rats with blood glucose levels higher than 16.7 mmol/L at 72 h after the injection were considered diabetic, and the diabetic wound was introduced after 4 weeks10. Overall, there are inconsistencies in the STZ injection protocols, diabetes diagnosis criteria, and wound introduction times.
In terms of wound modeling, in most studies, the full thickness of the dorsal skin is excised to construct T1DM wounds after successful diabetes induction11,12,13. Although this model is susceptible to skin contracture in rats, it is the most commonly used model in wound healing research because it is less labor-intensive and is cheap14,15. Nevertheless, method-guided research on this full-thickness excision technique is lacking. Furthermore, there are no uniform standards in existing studies regarding wound size and location12,16. The size and location of the wound can indirectly affect the consistency of the experimental design and the scientific validity of the results. Therefore, there is an urgent need for a standard protocol for T1DM induction and wound modeling as a reference for researchers. The goal of this study is to visualize a specific protocol for T1DM wound modeling that can be used as a reference for T1DM wound studies.

<table><tbody><tr><td>Antifade mounting medium</td><td>Southern Biotechnology Associates, Inc.</td><td>0100-01</td><td></td></tr><tr><td>AutoFluo Quencher</td><td>Servicebio Technology co., Ltd.</td><td>G1221</td><td></td></tr><tr><td>Automatic slide stainer</td><td>Thermo Fisher Scientific Inc.</td><td>Varistain&amp;trade; Gemini ES</td><td></td></tr><tr><td>CD31</td><td>Servicebio Technology co., Ltd.</td><td>GB11063-2</td><td></td></tr><tr><td>Citrate antigen retrieval solution</td><td>Servicebio Technology co., Ltd.</td><td>G1201</td><td></td></tr><tr><td>Cover glass</td><td>Citotest Labware Manufacturing Co., Ltd.</td><td>10212432C</td><td></td></tr><tr><td>DAPI</td><td>Servicebio Technology co., Ltd.</td><td>G1012</td><td></td></tr><tr><td>Decolorization shaker</td><td>Scilogex</td><td>S1010E</td><td></td></tr><tr><td>Depilatory cream</td><td>Guangzhou Ruixin Biotechnology Co., Ltd.</td><td>&amp;mdash;</td><td></td></tr><tr><td>Dimethyl benzene</td><td>Chengdu Kelong Chemical Co., Ltd.</td><td>64-17-5</td><td></td></tr><tr><td>Drug oscillator</td><td>Shenzhen Jiashi Technology Co., Ltd.</td><td>VM-370</td><td></td></tr><tr><td>Electric razor</td><td>Shanghai Flyco Electrical Appliance Co., Ltd.</td><td>FC5908</td><td></td></tr><tr><td>Embedding machine</td><td>Wuhan Junjie Electronics Co., Ltd.</td><td>JB-P5</td><td></td></tr><tr><td>Ethanol absolute</td><td>Chengdu Kelong Chemical Co., Ltd.</td><td>1330-20-7</td><td></td></tr><tr><td>Fitc-labeled goat anti-rabbit IgG</td><td>Servicebio Technology co., Ltd.</td><td>GB22303</td><td></td></tr><tr><td>Goat serum</td><td>Thermo Fisher Scientific Inc.</td><td>16210064</td><td></td></tr><tr><td>Hematoxylin and eosin staining solution</td><td>Beijing Regan Biotechnology Co., Ltd.</td><td>DH0020</td><td></td></tr><tr><td>Image J software</td><td>National Institutes of Health</td><td>&amp;mdash;</td><td></td></tr><tr><td>Microwave oven</td><td>Midea Group Co., Ltd.&amp;nbsp;</td><td>M1-L213B</td><td></td></tr><tr><td>Mini centrifuge</td><td>Scilogex</td><td>D1008</td><td></td></tr><tr><td>Neutral balsam</td><td>Sinopharm Chemical Reagent Co., Ltd</td><td>10004160</td><td></td></tr><tr><td>PBS buffer</td><td>Biosharp</td><td>G4202</td><td></td></tr><tr><td>Portable blood glucose meter</td><td>Sinocare Inc.</td><td>GA-3</td><td></td></tr><tr><td>Rapid tissue processor</td><td>Thermo Fisher Scientific Inc.</td><td>STP420 ES</td><td></td></tr><tr><td>Rat fixator</td><td>Globalebio (Beijing) Technology co., Ltd</td><td>GEGD-Q10G1</td><td></td></tr><tr><td>Slicing machine</td><td>Thermo Fisher Scientific Inc.</td><td>HM325</td><td></td></tr><tr><td>Slides glass</td><td>Citotest Labware Manufacturing Co., Ltd.</td><td>80312-3181</td><td></td></tr><tr><td>sodium citrate buffer</td><td>Beijing Solarbio Science &amp;amp; Technology Co., Ltd.</td><td>c1013</td><td></td></tr><tr><td>Streptozotocin</td><td>Sigma</td><td>57654595</td><td></td></tr></tbody></table>

a protocol for constructing a rat wound model of type 1 diabetes

A single high dose of streptozotocin injection followed by full-thickness skin excision on the dorsum of rats is a common method for constructing animal models of type 1 diabetic wounds. However, improper manipulation can lead to model instability and high mortality in rats. Unfortunately, there are few existing guidelines on type 1 diabetic wound modeling, and they lack detail and do not present specific reference strategies. Therefore, this protocol details the complete procedure for constructing a type 1 diabetic wound model and analyzes the progression and angiogenic characteristics of the diabetic wounds. Type 1 diabetic wound modeling involves the following steps: preparation of the streptozotocin injection, induction of type 1 diabetes mellitus, and construction of the wound model. The wound area was measured on day 7 and day 14 after wounding, and the skin tissues of the rats were extracted for histopathological and immunofluorescence analysis. The results revealed that type 1 diabetes mellitus induced by 55 mg/kg streptozotocin was associated with lower mortality and a high success rate. The blood glucose levels were relatively stable after 5 weeks of induction. The diabetic wound healing rate was significantly lower than that of normal wounds on day 7 and day 14 (p &#60; 0.05), but both could reach more than 90% on day 14. Compared with the normal group, the epidermal layer closure of diabetic wounds on day 14 was incomplete and had delayed re-epithelialization and significantly lower angiogenesis (p &#60; 0.01). The type 1 diabetic wound model constructed based on this protocol has the characteristics of chronic wound healing, including poor closure, delayed re-epithelialization, and decreased angiogenesis compared to normal rat wounds.

A total of 10 SD rats received a single STZ intraperitoneal injection to induce the T1DM model. One rat prematurely died (10%), but diabetes was induced in all the rats (100%). After 3 days of STZ injection, the blood glucose levels of all the rats were higher than 16.7 mmol/L, and the blood glucose levels stabilized 5 weeks after induction (Figure 3A). The weight of the diabetic group increased gradually after the STZ injection but decreased in week 3 and then slowly increased again from week 4 (Figure 3B). In contrast, the weight of rats in the normal group increased steadily, and their mean weight 3 days after diabetes induction was higher than that of the diabetic group (Figure 3B). The diabetic rats all exhibited typical symptoms of thirst, polyuria, and weight loss, similar to the findings of Hao et al.17.
On day 7 and day 14 after wounding, the macroscopic analysis revealed that re-epithelialization was more pronounced in rats in the normal group than in the diabetic group (Figure 4A). The quantitative results revealed that the wound healing rate was significantly lower in the diabetic group than in the normal group on day 7 and day 14 (p &#60; 0.01). However, on day 14, the wound healing rates could also be above 90% in the diabetic group (p &#60; 0.05, Figure 4B). This suggests that the T1DM wound model is characterized by poor closure but not to the extent of the chronic non-healing seen in human diabetic wounds.
H&#38;E staining on day 14 of wound healing revealed an incomplete wound epidermis, slow proliferation of keratinocytes, and delayed re-epithelialization in the diabetic group compared to the normal group. The diabetic wounds showed partial loss of the hair follicles and sebaceous glands. There were also fewer visible capillaries (Figure 5).
Diabetes causes endothelial cell dysfunction, glycosylation of the extracellular matrix proteins, and vascular denervation18. These complications result in lower-than-normal wound angiogenesis in diabetic wounds18. Angiogenesis is necessary for wound healing, and wound angiogenesis is frequently analyzed by CD31 immunostaining (Figure 6A)19,20. Based on the average optical density (AOD) of CD31 expression, angiogenesis at the wound site was significantly higher in the normal than in the diabetic group (p &#60; 0.01, Figure 6B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64914/64914fig01.jpg" /> 
Figure 1: Picture of rats immobilized by fixators. <a href="https://www.jove.com/files/ftp_upload/64914/64914fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64914/64914fig02.jpg" /> 
Figure 2: Diagram of the rat wound location. <a href="https://www.jove.com/files/ftp_upload/64914/64914fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64914/64914fig03.jpg" /> 
Figure 3: Blood glucose levels and weights of the experimental rats. <a href="https://www.jove.com/files/ftp_upload/64914/64914fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64914/64914fig04.jpg" /> 
Figure 4: Full-thickness skin wounds (20 mm in diameter) on the backs of the experimental rats. (A) The macroscopic appearance of the wounds on day 0, day 7, and day 14. The wound morphology images on day 0, day 7, and day 14 were captured with a digital camera. (B) The wound area was measured using ImageJ software and was used to calculate the wound healing rate. The wound healing rate (%) was calculated as follows: (initial wound area &#8722; wound area at the indicated time point)/initial wound area &#215; 100. The values are presented as mean &#177; SD (n = 14). Statistical significance was set at ** p &#60; 0.01 and * p &#60; 0.05. <a href="https://www.jove.com/files/ftp_upload/64914/64914fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64914/64914fig05.jpg" /> 
Figure 5: Representative histopathological H&#38;E images on day 14 after wound establishment. The blue arrows indicate capillaries. The red arrows show the proliferation of keratinocytes. Left scale: one bar = 200 &#181;m; right scale: one bar = 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64914/64914fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/64914/64914fig06v2.jpg" /> 
Figure 6: Immunofluorescence staining analysis for the expression of CD31. CD31 levels were used to determine the state of angiogenesis.&#160;(A)&#160;Representative images of CD31 immunofluorescence staining in the diabetic and normal groups. The integrated optical density (IOD) value and the pixel area (AREA) for each skin sample were calculated with Image-Pro Plus 6.0 software. The average optical density (AOD) value (AOD = IOD/AREA) was also derived. The AOD value was directly proportional to the positive expression of CD31. (B) Quantitative comparison of CD31 positive expression in the diabetic and normal groups. Data are presented as mean &#177; SD. ** p &#60; 0.01. Scale: one bar = 200 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64914/64914fig06largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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A Protocol for Constructing a Rat Wound Model of Type 1 Diabetes

The streptozotocin-induced diabetic wound model in male SD rats is currently the most widely used model for studying wound healing in type I diabetes mellitus. This protocol describes the methods used to construct this model. It also presents and addresses potential challenges and examines the progression and angiogenic characteristics of diabetic wounds.

A single high dose of streptozotocin injection followed by full-thickness skin excision on the dorsum of rats is a common method for constructing animal ...

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Research

JoVE Journal

Medicine

3.7K Views.  Chengdu University of Traditional Chinese Medicine. The streptozotocin-induced diabetic wound model in male SD rats is currently the most widely used model for studying wound healing in type I diabetes mellitus. This protocol describes the methods used to construct this model. It also presents and addresses potential challenges and examines the progression and angiogenic characteristics of diabetic wounds. 

Video: A Protocol for Constructing a Rat Wound Model of Type 1 Diabetes

A Simplified Technique for Producing an Ischemic Wound Model

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work well in non-ischemic wounds may not work in human diabetic wounds because vasculopathy is one major factor that hinders healing of these wounds. We published an article in 2007 describing a rabbit ear ischemic wound model created by a minimally invasive surgical technique. Since then, we have further simplified the procedure for easier operation. On one ear, three small skin incisions were made on the vascular pedicles, 1-2 cm from the ear base. The central artery was ligated and cut along with the nerve. The whole cranial bundle was cut and ligated, leaving only the caudal branch intact. A circumferential subcutaneous tunnel was made through the incisions, to cut subcutaneous tissues, muscles, nerves, and small vessels. The other ear was used as a non-ischemic control. Four wounds were made on the ventral side of each ear. This technique produces 4 ischemic wounds and 4 non-ischemic wounds in one animal for paired comparisons. After surgery, the ischemic ear was cool and cyanotic, and showed reduced movement and a lack of pulse in the ear artery. Skin temperature of the ischemic ear was 1-10 &deg;C lower than that on the normal ear and this difference was maintained for more than one month. Ear tissue high-energy phosphate contents were lower in the ischemic ear than the control ear. Wound healing times were longer in the ischemic ear than in the non-ischemic ear when the same treatment was used. The technique has now been used on more than 80 rabbits in which 23 were diabetic (diabetes time ranging from 2 weeks to 2 years). No single rabbit has developed any surgical complications such as bleeding, infection, or rupture in the skin incisions. The model has many advantages, such as little skin disruption, longer ischemic time, and higher success rate, when compared to many other models. It can be safely used in animals with reduced resistance, and can also be modified to meet different testing requirements.

We have developed a minimally invasive technique to create a rabbit ischemic ear wound model by dividing the central artery and nerve and the cranial neurovascular bundle. A subcutaneous tunnel then cuts all subcutaneous tissues. This procedure causes minimal skin disruption and can be safely used in diabetic animals.

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work ...

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

Demonstration of the Rat Ischemic Skin Wound Model

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and unrelieved pressure due to immobility. Animal models have been developed that attempt to mimic these conditions for the purpose of furthering our understanding of the complexity of chronic wounds. The model described herein is a rat ischemic skin flap model that permits a prolonged reduction of blood flow resulting in wounds that become ischemic and resemble a chronic wound phenotype (reduced vascularization, increased inflammation and delayed wound closure). It consists of a bipedicled dorsal flap with 2 ischemic wounds placed centrally and 2 non-ischemic wounds lateral to the flap as controls. A novel addition to this ischemic skin flap model is the placement of a silicone sheet beneath the flap that functions as a barrier and a splint to prevent revascularization and reduce contraction as the wounds heal. Despite the debate of using rats for wound healing studies due to their quite distinct anatomic and physiologic differences compared to humans (i.e., the presence of a panniculus carnosus muscle, short life-span, increased number of hair follicles, and their ability to heal infected wounds) the modifications employed in this model make it a valuable alternative to previously developed ischemic skin flap models.

The rat, due to its size, availability, and rather docile behavior, has been utilized as a research model for many years. The goal of this protocol is to utilize the rat as an ischemic skin wound healing model to provide valuable insight into the pathophysiology of chronic wounds.

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and ...

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. Chronic mouse wounds models have been used to understand the underlying interactions between the microorganisms and the host. The models developed to date rely on the use of haired animals and terminal collection of wound tissue for determination of viable bacteria. While significant insight has been gained with these models, this experimental procedure requires a large number of animals and sampling is time consuming. We have developed a novel murine model that incorporates several optimal innovations to evaluate biofilm progression in chronic wounds: a) it utilizes hairless mice, eliminating the need for hair removal; b) applies pre-formed biofilms to the wounds allowing for the immediate evaluation of persistence and effect of these communities on host; c) monitors biofilm progression by quantifying light production by a genetically engineered bioluminescent strain of Pseudomonas aeruginosa, allowing real-time monitoring of the infection thus reducing the number of animals required per study. In this model, a single full-depth wound is produced on the back of STZ-induced diabetic hairless mice and inoculated with biofilms of the P. aeruginosa bioluminescent strain Xen 41. Light output from the wounds is recorded daily in an in vivo imaging system, allowing for in vivo and in situ rapid biofilm visualization and localization of biofilm bacteria within the wounds. This novel method is flexible as it can be used to study other microorganisms, including genetically engineered species and multi-species biofilms, and may be of special value in testing anti-biofilm strategies including antimicrobial occlusive dressings.

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

Here we describe a novel diabetic murine model utilizing hairless mice for real-time, non-invasive, monitoring of biofilm wound infections of bioluminescent Pseudomonas aeruginosa. This method can be adapted to evaluate infection of other bacterial species and genetically modified microorganisms, including multi-species biofilms, and test the efficacy of antibiofilm strategies.

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. ...

Immunology and Infection

Creation and Transplantation of an Adipose-derived Stem Cell (ASC) Sheet in a Diabetic Wound-healing Model

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients with impeded blood flow or with large wounds might be prolonged. Cell-based therapies have appeared as a new technique for the treatment of diabetic ulcers, and cell-sheet engineering has improved the efficacy of cell transplantation. A number of reports have suggested that adipose-derived stem cells (ASCs), a type of mesenchymal stromal cell (MSC), exhibit therapeutic potential due to their relative abundance in adipose tissue and their accessibility for collection when compared to MSCs from other tissues. Therefore, ASCs appear to be a good source of stem cells for therapeutic use. In this study, ASC sheets from the epididymal adipose fat of normal Lewis rats were successfully created using temperature-responsive culture dishes and normal culture medium containing ascorbic acid. The ASC sheets were transplanted into Zucker diabetic fatty (ZDF) rats, a rat model of type 2 diabetes and obesity, that exhibit diminished wound healing. A wound was created on the posterior cranial surface, ASC sheets were transplanted into the wound, and a bilayer artificial skin was used to cover the sheets. ZDF rats that received ASC sheets had better wound healing than ZDF rats without the transplantation of ASC sheets. This approach was limited because ASC sheets are sensitive to dry conditions, requiring the maintenance of a moist wound environment. Therefore, artificial skin was used to cover the ASC sheet to prevent drying. The allogenic transplantation of ASC sheets in combination with artificial skin might also be applicable to other intractable ulcers or burns, such as those observed with peripheral arterial disease and collagen disease, and might be administered to patients who are undernourished or are using steroids. Thus, this treatment might be the first step towards improving the therapeutic options for diabetic wound healing.

Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model

Adipose-derived stem cells (ASCs) are easily isolated and harvested from the fat of normal rats. ASC sheets can be created using cell-sheet engineering and can be transplanted into Zucker diabetic fatty rats exhibiting full-thickness skin defects with exposed bone and then covered with a bilayer of artificial skin.

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients ...

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the availability of the widest range of molecular probes and reagents to perform in vivo as well as in vitro studies. Based on our experience with various murine transplantation models, we developed a heterotopic pancreas transplantation model in mice with the intent to analyze mechanisms underlying severe ischemia reperfusion injury-associated early graft damage. In contrast to previously described techniques using suture techniques, herein we describe a new procedure using a non-suture cuff technique. 
In recent years, we have performed more than 300 pancreas transplantations in mice with an overall success rate of &gt;90%, a success rate never described before in mouse pancreas transplantation. The backbone of this non-suture cuff technique for graft revascularization consists of two major steps: (I) pulling the recipient vessel over a polyethylene/polyamide cuff and fixing it with a circumferential ligature, and (II) placing the donor vessel over the everted recipient vessel and fixing it with a second circumferential ligature. The resultant continuity of the endothelial layer results in less thrombogenic lesions with high patency rates and, finally, high success rates.
In this model, arterial anastomosis is achieved by pulling the abdominal aorta of the donor graft over the everted common carotid artery of the recipient animal. Venous drainage of the graft is achieved by pulling the portal vein of the graft over the everted external jugular vein of the recipient. This manuscript provides details and crucial steps of the organ recovery and organ implantation procedures, which will allow researchers with microsurgical skills to perform the transplantation successfully in their laboratories.

Among abdominal solid organ transplantation, pancreatic grafts are prone to develop severe ischemia reperfusion injury-associated graft damage, leading eventually to early graft loss. This protocol describes a model of murine pancreas transplantation using a non-suture cuff technique, ideally suited for analyzing these early, deleterious damages.

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the ...

Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease conditions1, 2. Network growth is commonly attributed to angiogenesis, defined as the growth of new vessels from pre-existing vessels. The angiogenic process is also directly linked to arteriogenesis, defined as the capillary acquisition of a perivascular cell coating and vessel enlargement. Needless to say, angiogenesis is complex and involves multiple players at the cellular and molecular level3. Understanding how a microvascular network grows requires identifying the spatial and temporal dynamics along the hierarchy of a network over the time course of angiogenesis. This information is critical for the development of therapies aimed at manipulating vessel growth.
The exteriorization model described in this article represents a simple, reproducible model for stimulating angiogenesis in the rat mesentery. It was adapted from wound-healing models in the rat mesentery4-7, and is an alternative to stimulate angiogenesis in the mesentery via i.p. injections of pro-angiogenic agents8, 9. The exteriorization model is attractive because it requires minimal surgical intervention and produces dramatic, reproducible increases in capillary sprouts, vascular area and vascular density over a relatively short time course in a tissue that allows for the two-dimensional visualization of entire microvascular networks down to single cell level. The stimulated growth reflects natural angiogenic responses in a physiological environment without interference of foreign angiogenic molecules. Using immunohistochemical labeling methods, this model has been proven extremely useful in identifying novel cellular events involved in angiogenesis. Investigators can readily correlate the angiogenic metrics during the time course of remodeling with time specific dynamics, such as cellular phenotypic changes or cellular interactions4, 5, 7, 10, 11.

This article describes a simple model for stimulating angiogenesis in the rat mesentery. The model produces dramatic increases in capillary sprouting, vascular area and vascular density over a relatively short time course in a tissue that allows en face visualization of entire microvascular networks down to the single cell level.

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease ...

Biology

Protocol to Create Chronic Wounds in Diabetic Mice

Chronic wounds develop as a result of defective regulation in one or more complex cellular and molecular processes involved in proper healing. They impact ~6.5M people and cost ~$40B/year in the US alone. Although a significant effort has been invested in understanding how chronic wounds develop in humans, fundamental questions remain unanswered. Recently, we developed a novel mouse model for diabetic chronic wounds that have many characteristics of human chronic wounds. Using db/db-/- mice, we can generate chronic wounds by inducing high levels of oxidative stress (OS) in the wound tissue immediately after wounding, using a one-time treatment with inhibitors specific to the antioxidant enzymes catalase and glutathione peroxidase. These wounds have high levels of OS, develop biofilm naturally, become fully chronic within 20 days after treatment and can remain open more for more than 60 days. This novel model has many features of diabetic chronic wounds in humans and therefore can contribute significantly to advancing fundamental understanding of how wounds become chronic. This is a major breakthrough because chronic wounds in humans cause significant pain and distress to patients and result in amputation if unresolved. Moreover, these wounds are very expensive and time-consuming to treat, and lead to significant loss of personal income to patients. Advancements in this field of study through the use of our chronic wound model can significantly improve health care for millions who suffer under this debilitating condition. In this protocol, we describe in great detail the procedure to cause acute wounds to become chronic, which has not been done before.

Chronic wounds are developed from acute wounds on a diabetic mouse model by inducing high levels of oxidative stress after a full-thickness cutaneous wound. The wound is treated with inhibitors for catalase and glutathione peroxidase, resulting in impaired healing and biofilm development by bacteria present in the skin microbiome.

Chronic wounds develop as a result of defective regulation in one or more complex cellular and molecular processes involved in proper healing. They impact ...

Laser Capture Microdissection on Surgical Tissues to Identify Aberrant Gene Expression in Impaired Wound Healing in Type 2 Diabetes

The global prevalence Type 2 diabetes mellitus (T2DM) is escalating at a rapid rate. Patients with T2DM suffer from a multitude of complications and one of these is impaired wound healing. This can lead to the development of non-healing sores or foot ulcers and ultimately to amputation. In healthy individuals, wound healing follows a controlled and overlapping sequence of events encompassing inflammation, proliferation, and remodelling. In T2DM, one or more of these steps becomes dysfunctional. Current models to study impaired wound healing in T2DM include in vitro scratch wound assays, skin equivalents, or animal models to examine molecular mechanisms underpinning wound healing and/or potential therapeutic options. However, these do not fully recapitulate the complex wound healing process in T2DM patients, and ex vivo human skin tests are problematic due to the ethics of taking punch biopsies from patients where it is known they will heal poorly. Here, a technique is described whereby expression profiles of the specific cells involved in the (dys)functional wound healing response in T2DM patients can be examined using surplus tissue discarded following amputation or elective cosmetic surgery. In this protocol samples of donated skin are collected, wounded, cultured ex vivo in the air liquid interface, fixed at different time points and sectioned. Specific cell types involved in wound healing (e.g., epidermal keratinocytes, dermal fibroblasts (papillary and reticular), the vasculature) are isolated using laser capture microdissection and differences in gene expression analyzed by sequencing or microarray, with genes of interest further validated by qPCR. This protocol can be used to identify inherent differences in gene expression between both poorly healing and intact skin, in patients with or without diabetes, using tissue ordinarily discarded following surgery. It will yield greater understanding of the molecular mechanisms contributing to T2DM chronic wounds and lower limb loss.

This technique provides a guide to inflicting ex vivo wounds, performing laser capture microdissection and quantifying changes in gene expression related to poor wound healing processes in diabetes using clinically relevant human tissue.

The global prevalence Type 2 diabetes mellitus (T2DM) is escalating at a rapid rate. Patients with T2DM suffer from a multitude of complications and one ...

Surgical Bone Implantation Technique for Rat Tibia Models of Diabetes and Osteoporosis

The rat has long served as a valuable animal model in implant dentistry and orthopedics, particularly in studying the interactions between biomaterials and bone tissue. The rat's tibia is frequently chosen due to its easy surgical access through thin tissue layers (skin and muscle) and the flattened shape of its medial face, facilitating the surgical insertion of intraosseous devices. Additionally, this model enables the induction of specific diseases, mimicking various clinical conditions to assess biological responses to different implant conditions like geometry, surface texture, or biological cues. However, despite its robust cortical structure, certain intraosseous devices may require adaptations in design and size for successful implantation. Therefore, establishing standardized surgical methods for manipulating both soft and hard tissues in the implantation region is essential for ensuring proper implant or screw device placement, particularly in fields like implant dentistry and orthopedics. This study included eighty Sprague Dawley rats divided into two groups based on their respective diseases: Group 1 with osteoporosis and Group 2 with Type 2 Diabetes. Implantations were performed at 4 weeks and 12 weeks, with the same surgeon following a consistent surgical technique. A positive biological response was observed, indicating complete osseointegration of all implants placed. These results validate the success of the surgical protocol, which can be replicated for other studies and serve as a benchmark for the biomaterials community. Notably, osseointegration values remained stable at both 4 weeks and 12 weeks for both disease models, demonstrating a durable integration of the implant over time and emphasizing the establishment of an intimate bone connection as early as 4 weeks.

The placement of implants in a rat model is an essential experimental procedure for clinical research. This study presents a comprehensive surgical protocol for implanting titanium implants into the tibia of rat models with diabetes and osteoporosis.

The rat has long served as a valuable animal model in implant dentistry and orthopedics, particularly in studying the interactions between biomaterials ...

A Protocol for Constructing a Rat Wound Model of Type 1 Diabetes

Summary

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Surgical Bone Implantation Technique for Rat Tibia Models of Diabetes and Osteoporosis