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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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.

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			<td>Southern Biotechnology Associates, Inc.</td>
			<td>0100-01</td>
			<td></td>
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			<td>AutoFluo Quencher</td>
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			<td>G1221</td>
			<td></td>
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			<td>Automatic slide stainer</td>
			<td>Thermo Fisher Scientific Inc.</td>
			<td>Varistain&#8482; Gemini ES</td>
			<td></td>
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			<td>CD31</td>
			<td>Servicebio Technology co., Ltd.</td>
			<td>GB11063-2</td>
			<td></td>
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			<td>Citrate antigen retrieval solution</td>
			<td>Servicebio Technology co., Ltd.</td>
			<td>G1201</td>
			<td></td>
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			<td>Cover glass</td>
			<td>Citotest Labware Manufacturing Co., Ltd.</td>
			<td>10212432C</td>
			<td></td>
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			<td>DAPI</td>
			<td>Servicebio Technology co., Ltd.</td>
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			<td>Decolorization shaker</td>
			<td>Scilogex</td>
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			<td>Guangzhou Ruixin Biotechnology Co., Ltd.</td>
			<td>&#8212;</td>
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			<td>Dimethyl benzene</td>
			<td>Chengdu Kelong Chemical Co., Ltd.</td>
			<td>64-17-5</td>
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			<td>Drug oscillator</td>
			<td>Shenzhen Jiashi Technology Co., Ltd.</td>
			<td>VM-370</td>
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			<td>Electric razor</td>
			<td>Shanghai Flyco Electrical Appliance Co., Ltd.</td>
			<td>FC5908</td>
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			<td>Embedding machine</td>
			<td>Wuhan Junjie Electronics Co., Ltd.</td>
			<td>JB-P5</td>
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			<td>Ethanol absolute</td>
			<td>Chengdu Kelong Chemical Co., Ltd.</td>
			<td>1330-20-7</td>
			<td></td>
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			<td>Fitc-labeled goat anti-rabbit IgG</td>
			<td>Servicebio Technology co., Ltd.</td>
			<td>GB22303</td>
			<td></td>
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			<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>&#8212;</td>
			<td></td>
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			<td>Microwave oven</td>
			<td>Midea Group Co., Ltd.&#160;</td>
			<td>M1-L213B</td>
			<td></td>
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			<td>Mini centrifuge</td>
			<td>Scilogex</td>
			<td>D1008</td>
			<td></td>
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			<td>Neutral balsam</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10004160</td>
			<td></td>
		</tr>
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			<td>PBS buffer</td>
			<td>Biosharp</td>
			<td>G4202</td>
			<td></td>
		</tr>
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			<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 &#38; Technology Co., Ltd.</td>
			<td>c1013</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptozotocin</td>
			<td>Sigma</td>
			<td>57654595</td>
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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>

Watch this Scientific Journal Video about A Protocol for Constructing a Rat Wound Model of Type 1 Diabetes at JoVE.com

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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3.3K 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 ...

A Zebrafish Model of Diabetes Mellitus and Metabolic Memory

Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory and large scale clinical trials has revealed that diabetic complications progress unimpeded via the phenomenon of metabolic memory even when glycemic control is pharmaceutically achieved. Gene expression can be stably altered through epigenetic changes which not only allow cells and organisms to quickly respond to changing environmental stimuli but also confer the ability of the cell to "memorize" these encounters once the stimulus is removed. As such, the roles that these mechanisms play in the metabolic memory phenomenon are currently being examined.
We have recently reported the development of a zebrafish model of type I diabetes mellitus and characterized this model to show that diabetic zebrafish not only display the known secondary complications including the changes associated with diabetic retinopathy, diabetic nephropathy and impaired wound healing but also exhibit impaired caudal fin regeneration. This model is unique in that the zebrafish is capable to regenerate its damaged pancreas and restore a euglycemic state similar to what would be expected in post-transplant human patients. Moreover, multiple rounds of caudal fin amputation allow for the separation and study of pure epigenetic effects in an in vivo system without potential complicating factors from the previous diabetic state. Although euglycemia is achieved following pancreatic regeneration, the diabetic secondary complication of fin regeneration and skin wound healing persists indefinitely. In the case of impaired fin regeneration, this pathology is retained even after multiple rounds of fin regeneration in the daughter fin tissues. These observations point to an underlying epigenetic process existing in the metabolic memory state. Here we present the methods needed to successfully generate the diabetic and metabolic memory groups of fish and discuss the advantages of this model.

Metabolic memory is the phenomenon by which diabetic complications persist and progress unimpeded even after euglycemia is achieved pharmaceutically. Here we describe a diabetes mellitus zebrafish model which is unique in that it allows for the examination of the mitotically transmissible epigenetic components of metabolic memory in vivo.

Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory ...

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. ...

A Model of Chronic Nutrient Infusion in the Rat

Chronic exposure to excessive levels of nutrients is postulated to affect the function of several organs and tissues and to contribute to the development of the many complications associated with obesity and the metabolic syndrome, including type 2 diabetes. To study the mechanisms by which excessive levels of glucose and fatty acids affect the pancreatic beta-cell and the secretion of insulin, we have established a chronic nutrient infusion model in the rat. The procedure consists of catheterizing the right jugular vein and left carotid artery under general anesthesia; allowing a 7-day recuperation period; connecting the catheters to the pumps using a swivel and counterweight system that enables the animal to move freely in the cage; and infusing glucose and/or Intralipid (a soybean oil emulsion which generates a mixture of approximately 80% unsaturated/20% saturated fatty acids when infused with heparin) for 72 hr. This model offers several advantages, including the possibility to finely modulate the target levels of circulating glucose and fatty acids; the option to co-infuse pharmacological compounds; and the relatively short time frame as opposed to dietary models. It can be used to examine the mechanisms of nutrient-induced dysfunction in a variety of organs and to test the effectiveness of drugs in this context.

A protocol for chronic infusions of glucose and Intralipid in rats is described. This model can be used to study the impact of nutrient excess on organ function and physiological parameters.

Chronic exposure to excessive levels of nutrients is postulated to  affect the function of several organs and tissues and to contribute to the  ...

A Mouse Fetal Skin Model of Scarless Wound Repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

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 ...

A High-Throughput Electrochemiluminescence 7-Plex Assay Simultaneously Screening for Type 1 Diabetes and Multiple Autoimmune Diseases

Islet autoantibodies (IAbs) are widely used in type 1 diabetes (T1D) diagnosis and prediction. Four major IAbs to insulin (IAA), glutamate decarboxylase-65 (GADA), insulinoma antigen-2 (IA-2A), and zinc transporter-8 (ZnT8A) are equally important in disease prediction. Presently, up to 40% of patients diagnosed with T1D go on to develop other autoimmune disorders. Unfortunately, current screening methods using a single autoantibody for measurement are laborious and inefficient for large scale screening studies. We recently developed a simple multiplexed electrochemiluminescence (ECL) assay to address these current issues. The assay combines all 7 autoantibody tests into one well. Each well includes three IAbs (IAA, GADA, and IA-2A), autoantibodies to thyroid peroxidase (TPOA) and thyroid globulin (ThGA) to detect autoimmune thyroid disease, autoantibodies to tissue transglutaminase (TGA) for celiac disease, and autoantibodies to interferon alpha (IFN&#945;A) for autoimmune polyglandular syndrome-1 (APS-1); all of which screen for T1D and other relevant autoimmune diseases, simultaneously. The multiplex ECL assay is based on the single ECL assay platform, but instead uses the multiplex plate combining multiple autoantibody assays, up to 10, into a single well. The main difference from the single ECL assay is that each antibody-antigen complex formed in the fluid-phase is restrained onto a specific spot on each well through a linker system on the multiplex plate. The 7-Plex ECL assay, in the present study, is validated against standard radio-binding assays (RBA) and single ECL assays, using a large cohort of newly diagnosed T1D patients and age-matched healthy controls, resulting in excellent assay sensitivity and specificity.

We model a simple multiplexed ECL assay that combines 7 autoantibody assays together. The assay is capable of screening for T1D and multiple other autoimmune diseases, simultaneously, including celiac disease, autoimmune thyroid disease, and autoimmune polyglandular syndrome 1.

Islet autoantibodies (IAbs) are widely used in type 1 diabetes (T1D) diagnosis and prediction. Four major IAbs to insulin (IAA), glutamate ...

Fiber Type and Subcellular-Specific Analysis of Lipid Droplet Content in Skeletal Muscle

Skeletal muscle lipid infiltration, known as myosteatosis, increases with obesity and ageing. Myosteatosis has also recently been discovered as a negative prognostic factor for several other disorders such as cardiovascular disease and cancer. Excessive lipid infiltration decreases muscle mass and strength. It also results in lipotoxicity and insulin resistance depending on total intramyocellular lipid content, lipid droplet (LD) morphology, and subcellular distribution. Fiber type (oxidative vs glycolytic) is also important, since oxidative fibers have a greater capacity to utilize lipids. Because of their crucial implications in pathophysiology, in-depth studies on LD dynamics and function in a fiber type-specific manner are warranted.
Herein, a complete protocol is presented for the quantification of intramyocellular lipid content and analysis of LD morphology and subcellular distribution in a fiber type-specific manner. To this end, serial muscle cryosections were stained with the fluorescent dye Bodipy and antibodies against myosin heavy chain isoforms. This protocol enables the simultaneous processing of different muscles, saving time and avoiding possible artifacts and, thanks to a personalized macro created in Fiji, the automatization of LD analysis is also possible.

Increasing evidence indicates that excessive infiltration of lipids inside skeletal muscle results in lipotoxicity and diabetes. Here, we present a complete protocol, including tissue processing, staining with Bodipy, image acquisition, and analysis, to quantify the size, density, and subcellular distribution of lipid droplets in a fiber-type specific manner.

Skeletal muscle lipid infiltration, known as myosteatosis, increases with obesity and ageing. Myosteatosis has also recently been discovered as a negative ...

Tuina Therapy for Frozen Shoulders in a Rat Model

Tuina in a Frozen Shoulder Rat Model: An Efficient and Reproducible Protocol

Frozen shoulder (FS) is a common condition with no defined optimal therapy. Tuina therapy, a traditional Chinese medicine (TCM) technique used to treat FS patients in Chinese hospitals, has demonstrated excellent results, but its mechanisms are not fully understood. Building on a previous study, this work aimed to develop a Tuina protocol for an FS rat model. We randomly divided 20 SD rats into control (C; n = 5), FS model (M; n = 5), FS model Tuina treatment (MT; n = 5), and FS model oral treatment (MO; n = 5) groups. This study used the cast immobilization method to establish the FS rat model. The effect of Tuina and oral dexamethasone on the glenohumeral range of motion (ROM) was evaluated, and the histological findings were assessed. Our study showed that Tuina and oral dexamethasone were able to improve shoulder active ROM and preserve the structure of the capsule, with Tuina therapy proving to be more effective than oral dexamethasone. In conclusion, the Tuina protocol established in this study was highly effective for FS.

Author Spotlight: Investigating the Effectiveness of Tuina Therapy for Frozen Shoulder 

This study develops an efficient and reproducible Tuina protocol for treating frozen shoulder established in a rat model. This approach will help to study the Tuina therapy treatment method for frozen shoulders.

Frozen shoulder (FS) is a common condition with no defined optimal therapy. Tuina therapy, a traditional Chinese medicine (TCM) technique used to treat FS ...