The authors would like to express their gratitude to the Figdraw scientific research drawing platform of the Home for Researchers for providing the material used to create Figure 2 in this paper, with authorized license ID: URUUO5699e. This work has received support from grants provided by the National Natural Science Foundation of China (81873380, 82074566), the Natural Science Foundation of Hubei Province (2022CFCO43), and the National Famous Traditional Chinese Medicine Heritage Studio Construction Project of the National Administration of Traditional Chinese Medicine ([2022] No.5, [2022] No.75).

All procedures conducted on the rats in this study adhered to the National Standard for Laboratory Animal Welfare and Ethics Review Guidelines (2018) officially adopted by the People&#39;s Republic of China and Guide for the Care and Use of Laboratory Animals from the National Academies Press15. This protocol is composed of three main steps. Firstly, the rat jackets are tailored and fitted based on the individual body shapes of rats with different weights. Next, acupuncture manipulation is carried out following standard procedures. Lastly, the rats are suspended in their jackets while the needles remain in place to accommodate the need for simultaneous interventions among rats within the same group.
1. Pre-needling preparation
<ol>
	<li>Production of the rat jacket
	<ol>
		<li>Weigh the rats and measure their body shape before making their jackets. To verify the suitability of this protocol for rats of varying weights, select rats weighing 220 g, 300 g, 400 g, and 520 g for independent measurements of their bodily dimensions, encompassing measurements from a to f, as depicted in Figure 2. 
		NOTE: The correlation between the bodily proportions of rats and their corresponding jacket sizes is also presented in Figure 2. The pertinent body length parameters for rats of different weights are recorded in Table 1.</li>
		<li>Cut a rectangular piece of thick, dark fabric to make a rat jacket. Use the fabric with unfolded width equal to the circumference measured around the thickest part of the rat&#39;s abdomen. The length of the fabric is equal to the distance from the rat&#39;s nose tip to the base of its tail (see Figure 2).</li>
		<li>Fold the fabric in half and cut off the top left corner at an angle of about 45&#176;-65&#176; to form a right trapezoid (see Figure 3A).</li>
		<li>Sew the two sides on the left tightly with cotton thread. Refer to Figure 1B and Figure 3B for guidance.</li>
		<li>Cut two small holes, one on each side of the fabric, for the rat&#39;s upper and lower limbs to pass through (see Figure 3C). The distance between the two upper limbs determines the spacing between the upper holes of the rat jacket. The distance between the two lower limbs determines the spacing between the lower holes of the rat jacket.</li>
		<li>Use the longitudinal distance between the rat&#39;s armpit and the front of the ilium to serve as the spacing between the upper and lower holes of the rat jacket. Use the circumference of the shoulder joint to determine the circumference of the upper hole in the rat jacket. Use the circumference of the hip joint for the circumference of the lower hole in the rat jacket.</li>
		<li>Beside each hole, sew a long cloth strap that can be crossed and wrapped tightly around the body from the opening end of the jacket to fix the rat.</li>
	</ol>
	</li>
	<li>Rat jacket-wearing method
	<ol>
		<li>Wear gloves before proceeding to dress the rat in the jacket. Begin by holding the middle and back part of the rat jacket with one hand on the table. With the other hand, hold the rat&#39;s tail from its feeding cage and gently place it into the jacket.</li>
		<li>As the rat moves forward, its head will reach the headgear of the jacket. At this point, use both hands to carefully pull its left and right upper limbs out of the holes in the jacket.</li>
		<li>Take the tying rope from the opening end of the jacket, cross it over from left to right, and wrap it securely around the rat&#39;s trunk. Tie it tightly on the opposite side of the trunk to ensure proper fixation. Following the restraint of the rat&#39;s upper limbs, repeat the same procedure to fix its lower limbs.</li>
		<li>Take precautions to avoid getting bitten or scratched by the rat&#39;s claws when pulling its limbs out of the jacket holes. Ensure that the tightness of the rope is appropriately adjusted, i.e., not too tight to cause local limb ischemia and hypoxia in rats, and not too loose, allowing the rat to escape easily (see Video 1).</li>
	</ol>
	</li>
	<li>Fixation rack and S-shaped iron hooks
	<ol>
		<li>The fixation rack consists of two heavy iron trays (24 cm in length, 12 cm in width, and 2 cm in height) and an iron column (60 cm in length) that is firmly welded on each tray. Fix a solid iron stick (145 cm in length) at the same height on the two iron columns using a screw connection structure that can be adjusted and maintained at a fixed height (see Figure 4A).</li>
		<li>For connecting the rat wearing the jacket to the iron stick, use the S-shaped hooks (11 cm in diameter). Wrap one end of the hook around the strap on the jacket while the other end is hung on the iron stick. Use two S-shaped hooks to secure the rat onto the stick. Please refer to Figure 1D and Figure 4B for the overall structure.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65652/65652fig02.jpg" /> 
Figure 2: Corresponding diagram of rat body shape and matched rat jacket size. In the diagram, the parameter a represents the transverse distance at the back of the joint between the upper limbs and the rat&#39;s trunk. This value is utilized as the spacing measurement between the penetrating holes for the upper limbs of the rat jacket. Similarly, parameter b represents the transverse distance on the back where the bilateral lower limbs of the rat connect with the trunk. This measurement determines the spacing of the penetrating holes for the lower limbs of the rat jacket. The parameter c signifies the circumference of the thickest part of the rat&#39;s abdomen, which can be utilized as the width measurement for the fabric of the rat&#39;s jacket. The parameter d represents the distance from the rat&#39;s armpit to the front of its ilium, determining the longitudinal distance between the upper and lower holes of the jacket. The parameter e corresponds to the longitudinal distance from the rat&#39;s nose tip to the base of its tail, serving as the length measurement for the rat&#39;s jacket cloth. The parameter f denotes the circumference of the articulation connecting the upper limb and the rat&#39;s trunk, serving as the circumference of the hole through which the upper limbs pass. The parameter g represents the circumference of the joint connecting the lower limbs and the rat&#39;s trunk, determining the circumference of the hole through which the lower limbs pass. <a href="https://www.jove.com/files/ftp_upload/65652/65652fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Body Weight (g)</td>
			<td colspan="7">&#160;Body Shape Parameters (cm)</td>
		</tr>
		<tr>
			<td>a</td>
			<td>b</td>
			<td>c</td>
			<td>d</td>
			<td>e</td>
			<td>f</td>
			<td>g</td>
		</tr>
		<tr>
			<td>220</td>
			<td>6.0</td>
			<td>7.0</td>
			<td>15.0</td>
			<td>8.0</td>
			<td>19.0</td>
			<td>5.0</td>
			<td>7.0</td>
		</tr>
		<tr>
			<td>300</td>
			<td>7.0</td>
			<td>7.5</td>
			<td>17.0</td>
			<td>10.0</td>
			<td>21.0</td>
			<td>6.0</td>
			<td>8.0</td>
		</tr>
		<tr>
			<td>400</td>
			<td>8.0</td>
			<td>8.5</td>
			<td>19.0</td>
			<td>11.5</td>
			<td>23.0</td>
			<td>6.5</td>
			<td>9.0</td>
		</tr>
		<tr>
			<td>520</td>
			<td>8.0</td>
			<td>10.0</td>
			<td>20.0</td>
			<td>12.5</td>
			<td>26.0</td>
			<td>7.0</td>
			<td>9.5</td>
		</tr>
	</tbody>
</table>
Table 1. The measurement results of body shape parameters of rats with different body weights.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65652/65652fig03.jpg" /> 
Figure 3: Diagram depicting the physical appearance and usage of the rat jacket. (A) Plane exterior view of the rat jacket. The shape of the rat&#39;s jacket resembles a right-angled trapezoid, with separate openings on the left and right sides for the rat&#39;s upper and lower limbs to pass through. (B) Unfolded exterior view of the rat jacket.&#160;The closure of the headgear creates a semi-closed cavity, which serves as a crucial component to entice rats to enter. (C) Side view of a rat clothed with the jacket. Once the rat&#39;s limbs pass through the openings in the clothing, the straps are crossed over the ventral surface of the trunk and securely fastened at the back with appropriate tension. This ensures that the rat is relatively immobilized. (D) Abdominal view of a rat clothed with the rat jacket. The ventral surface of the jacket exposes the rat&#39;s abdominal skin, enabling convenient access for acupuncture at ventral points. <a href="https://www.jove.com/files/ftp_upload/65652/65652fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Video 1: Putting on the rat jacket. This video shows a typical process of dressing the rat in the jacket. <a href="https://www.jove.com/files/ftp_upload/65652/Video 1.Putting on the rat jacket.mp4" target="_blank">Please click here to download this Video.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65652/65652fig04.jpg" /> 
Figure 4: Diagram depicting the physical appearance and usage of the elevated fixation rack. (A) Elevated fixation rack. This rack consists primarily of two vertical iron columns (60 cm in length) welded to each tray, along with a long horizontal iron stick (145 cm in length) placed parallel to the ground. Screws are used to secure the horizontal stick at the same height as the vertical columns. (B) Rat suspension on the fixation rack. Rats wearing the rat jackets are securely fastened to the fixation rack using two S-shaped hooks per rat. (C) Simultaneous electroacupuncture treatment in multiple rats. To prevent any potential interference resulting from tail movement, it was necessary to maintain proper spacing among the rats, equivalent to the length of a rat&#39;s tail. <a href="https://www.jove.com/files/ftp_upload/65652/65652fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Electroacupuncture procedure
<ol>
	<li>Routine preparation
	<ol>
		<li>Place the fixation rack on a horizontal desktop or ground.</li>
		<li>Prior to the needling procedure, remove the fur around the acupoints using an electric shaver to ensure complete exposure. When the acupoint to be punctured is located on the abdomen of the rat, such as Guanyuan (CV4, about 25 mm below the navel), shorten the width of rat clothing appropriately to expose abdominal acupoints.</li>
		<li>Locate the acupoint from the gap on the midline of the abdomen that is not covered by the rat jacket (see Figure 3D) and then inject the needle.</li>
		<li>When the acupoints to be punctured are on the head of the rat, such as Baihui (GV20, located at the middle of the parietal bone), make a small incision at the corresponding position on the head to fully expose the skin around GV20 (See Figure 1C).</li>
		<li>When acupuncturing acupoints on the limbs of rats, such as Neiguan (PC6, on the inner side of the forelimb, between the radial and ulnar sutures about 3 mm away from the wrist joint) in the upper limb or Zusanli (ST36, on the posterolateral side of the knee joint, about 2 mm below the fibula head) in the lower limb, due to the obvious exposure of the acupoints, use one hand to assist in fixing the limb, while the other can directly perform the acupuncture operation.</li>
		<li>When the acupoint is located on the back or waist of a rat, such as Shenshu (BL23, below the 2nd lumbar vertebrae, 6 mm apart from the midline of the back), cut an opening approximately 2 cm wide in the center of the rat&#39;s jacket to fully expose the skin of the corresponding site (See Figure 1C).</li>
		<li>Dress the rat in the jacket. After disinfecting the local skin of acupoints with alcohol swabs, begin the acupuncture procedure.</li>
	</ol>
	</li>
	<li>Needling technique: Locate the acupoint accurately. Insert a sterile disposable acupuncture needle (0.16 mm x 7 mm) into the rat&#39;s acupoint at the appropriate depth.</li>
	<li>Electroacupuncture operation
	<ol>
		<li>Wrap the electrode wire around the horizontal iron rod. This step reduces the risk of the needle falling out due to gravitational force acting on the electroacupuncture clip. Then, attach the end of the electrode wire securely onto the needle handle.</li>
		<li>Switch on the power and select the appropriate frequency, current, and time parameters. In this experiment, the parameters were set to 2 Hz, 1 mA, and 15 min. Once the parameters are adjusted, commence the electroacupuncture treatment process (See Video 2 and Video 3).</li>
		<li>Simultaneous electroacupuncture on rats: During treatment, suspend the rats in the same group together on the iron stick using the S-shaped hooks and let them undergo electroacupuncture intervention simultaneously.</li>
		<li>NOTE: The iron stick should not only be sturdy but also long enough to treat a group of rats at the same time. In this experiment, while ensuring appropriate spacing and no interference among the rats, 3-4 rats can be treated simultaneously (see Figure 4C).</li>
	</ol>
	</li>
	<li>Observation of rat responses during electroacupuncture treatment: Evaluate the suitability of this fixation method for electroacupuncture in rats based on the following factors, such as the number of personnel required to complete the operation, time taken to fix a rat, the rats&#39; response during the electroacupuncture treatment, the number of needles dropped during the procedure, and any accidental deaths that might occur during the operation.</li>
	<li>Removal of the needles
	<ol>
		<li>Once the pre-set time is completed, the electroacupuncture apparatus will automatically shut off. Remove the small clip that is connected to the needle handle and carefully remove the acupuncture needle from beneath the rat&#39;s skin, thereby concluding the electroacupuncture treatment process.</li>
		<li>Loosen the straps, lift the head cover of the rat&#39;s jacket, and position the rat above the cage. The rat will then be free to release itself from the jacket and enter the cage independently (see Video 4).</li>
	</ol>
	</li>
</ol>
Video 2. Needling acupoints in the lumbar region. This video demonstrates the comprehensive and coherent procedure encompassing the attiring of the rat in the jacket, the insertion of acupuncture needles into acupoints at the waist, and the subsequent connection of an electroacupuncture apparatus. <a href="https://www.jove.com/files/ftp_upload/65652/Video 2. Needling acupoints in lumbar region.mp4" target="_blank">Please click here to download this Video.</a>
Video 3. Needling acupoints in the abdomen and lower limb. This video demonstrates the comprehensive and coherent procedure encompassing the attiring of the rat in the jacket, the insertion of acupuncture needles into acupoints located in the abdomen and the lower limb, and the subsequent connection of an electroacupuncture apparatus. <a href="https://www.jove.com/files/ftp_upload/65652/Video 3.Needling acupoints in abdomen and lower limb.mp4" target="_blank">Please click here to download this Video.</a>
Video 4. Removing the rat jacket. This video showcases the customary practice of removing the jacket from the rat. <a href="https://www.jove.com/files/ftp_upload/65652/Video 4.Removing the rat jacket.mp4" target="_blank">Please click here to download this Video.</a>

Rat Jackets to Enhance Suspension Fixation During Electroacupuncture on Rats

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All authors have declared no potential conflicts of interest.

Electroacupuncture has been shown to have good therapeutic effects on various diseases such as cognitive impairment16,17, myocardial injury18, and inflammatory pain19. To understand its mechanism of action, animal experiments primarily using rats have been conducted. Fixation of the animals during the procedures is crucial for accurate results. While many studies have administered electroacupuncture subsequent to an...

Acupuncture is a traditional therapy characterized by needling at specific acupoints or other positions to prevent and treat diseases. Electroacupuncture combines filiform needles with electrical stimulation1, which allows for precise control over the amount of stimulation by adjusting relevant parameters, promoting the standardization of acupuncture treatment2. In recent years, research on the mechanisms of electroacupuncture has continued to expand and deepen3,4, which has led to an annual increase in studies focusing on the mechanisms of electroacupuncture5.
In basic research on electroacupuncture, rats are the most commonly used animals. During electroacupuncture treatment, the recipient must remain still to ensure a smooth procedure. Unlike in clinical trials with human subjects, small animals used in animal experiments cannot follow commands consciously during 15-30 min lengthy electroacupuncture procedures. This presents researchers with a primary challenge of achieving stable and reliable fixation. Improper fixation can lead to negative emotions such as anxiety, depression, and fear in animals6, which not only increases the risk of injury for rats and the chance of being bitten for the experimental personnel but also affects the stability and persuasiveness of research results.
In previous studies, researchers have often used anesthesia fixation7, high platform fixation8, binding fixation6,9, or rat coat fixation10 before acupuncture or electroacupuncture operations. However, given the effects of anesthesia on the body11 and brain function12, and considering that acupuncture is performed on conscious patients in clinical cases, anesthesia may impact research results to a certain extent. Therefore, compared with anesthesia fixation, comfortable fixation of rats in an awake state is a better option.
However, in the non-anesthetized state, various fixation methods also possess their respective limitations, including restricted acupoint exposure due to coverage of the rat coat on rats10, the necessity of at least two individuals to perform the operation13, non-adjustable fixator size14, or the stressed state of rats in a forced position6. To address these technical issues, we designed a rat jacket and a fixation rack suitable for acupuncture manipulation in most parts of the rat body. This can help researchers efficiently carry out acupuncture procedures with rats.
The rationale for this protocol is as follows: firstly, the closed headgear on the rat&#39;s jacket takes advantage of the rats&#39; natural inclination for darkness and burrowing, thus restricting their range of activities. Secondly, the binding ropes near the joints of the rats&#39; limbs provide physical compression restraint, imposing certain limitations on their movements. However, rats are still able to jump and move horizontally even while wearing the jacket. Lastly, since rats cannot touch the ground without a fixed force point when suspended, we added a suspension fixation device to the rat jacket, further restricting their movements and ensuring needle retention. The composition of the fixation device is shown in Figure 1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65652/65652fig01.jpg" /> 
Figure 1: Schematic diagram of the rat jacket and suspension fixation. (A) The general appearance of the rat jacket in a flat view. (B) Three-dimensional view of the rat jacket. (C) Specially tailored rat jacket with an incision near the acupoints on the head and back waist. (D) Illustration of multiple rats simultaneously suspended on the elevated rack. 1: Fabric overall view; 2: Forelimb outlet; 3: Hind limb outlet; 4: Tying rope; 5: Cavity formed after suturing; 6: Incision for exposing head acupoints; 7: Incision for exposing back and waist acupoints. 8: Suture position. <a href="https://www.jove.com/files/ftp_upload/65652/65652fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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			<td>75% alcohol</td>
			<td>Shandong Lircon medical technology co., ltd</td>
			<td>2002-0059</td>
			<td>Disinfection</td>
		</tr>
		<tr>
			<td>acupuncture needles</td>
			<td>Beijing Luo ya shan chuan medical equipment co., Ltd.</td>
			<td></td>
			<td>needling</td>
		</tr>
		<tr>
			<td>Adult Sprague-Dawley (SD) rats</td>
			<td>Liaoning Changsheng biotechnology co., Ltd.</td>
			<td>SCXK 2020-0001</td>
			<td>The recipient&#160; subjected to fixation</td>
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		<tr>
			<td>an elevated fixation rack&#160;</td>
			<td>Taizhou shiai instruments and equipments for scientific and teaching research&#160; Co., ltd</td>
			<td>/</td>
			<td>Fix rats</td>
		</tr>
		<tr>
			<td>Hans electronic acupuncture apparatus</td>
			<td>Beijing Huayun Ante Science and Technology Co. Ltd.&#160;</td>
			<td>HANS-200A</td>
			<td>electroacupuncture</td>
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		<tr>
			<td>Medical cotton ball</td>
			<td>Jiangsu Misawa Medical Supplies Co., Ltd.</td>
			<td>20172142357</td>
			<td>Disinfection</td>
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			<td>Pet electric clipper</td>
			<td>Fenghua Xikou Lubao Knives &#38; Scissors Electrical Appliance Factory</td>
			<td>/</td>
			<td>Shave the hair of rats</td>
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			<td>Tweezers</td>
			<td>Qingdao Schultz Biotechnology Co., ltd</td>
			<td></td>
			<td>Pinch cotton balls</td>
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improved suspension fixation using rat jacket in rat acupuncture experiments

In basic experimental acupuncture research, rats are commonly used as laboratory animals. However, it is difficult for them to maintain a fixed posture. During electroacupuncture procedures, proper immobilization of rats is essential. Various methods of rat fixation are currently used, including anesthesia fixation, high-platform fixation, binding fixation, and fixation with a self-made rat coat. However, these methods have their limitations, which may affect the efficiency and operability of the experiment to some extent. This protocol introduces a method of suspending and fixing rats using rat clothes. Firstly, rats are clothed with rat jackets that match their body shape, taking advantage of their preference for darkness and burrowing. The needling operation can then be carried out after the rats have worn rat clothes. When suspended, the rats are relatively still, as their limbs cannot move. This fixation method offers not only economical and user-friendly benefits but also ensures a stable and reliable fixation of the rats in a comfortably relaxed position. It also effectively minimizes time consumption, experimental space, and manpower resources. Additionally, this method allows for the exposure of most acupoints used for acupuncture in rats. This article primarily concentrates on the device's composition, encompassing a specially designed rat jacket, an elevated fixation rack, and their connecting structures. Additionally, an illustrative example will be presented to demonstrate the application of the rat clothing-based suspension fixation method in rat electroacupuncture procedures.

In this experiment, a total of 12 rats were selected for electroacupuncture treatment, and their reactions under the fixation method were observed (as shown in Table 2). Among the 12 rats, only 1 rat exhibited intense struggle during the 15 min electroacupuncture needle retention period. As a result of its vigorous movements, the needle came off midway, accounting for 8.33% of the total number of rats. This outcome suggests that the fixation method used generally demonstrates a notable degree of stabilit...

Watch this Scientific Journal Video about Efficient Rat Clothing for Improved Rat Fixation During Electroacupuncture at JoVE.com

Author Spotlight: Efficient Rat Clothing for Improved Rat Fixation During Electroacupuncture 

Here, we present an improved suspension fixation method using a rat jacket to effectively immobilize rats during acupuncture experiments. Furthermore, the structure and application of this fixation apparatus, as well as instructions and evaluation for its utilization in electroacupuncture therapy, is elucidated.

Improved Suspension Fixation Using Rat Jacket in Rat Acupuncture Experiments

In basic experimental acupuncture research, rats are commonly used as laboratory animals. However, it is difficult for them to maintain a fixed posture. ...

improved-suspension-fixation-using-rat-jacket-rat-acupuncture

Research

JoVE Journal

Medicine

1.2K Views.  Hubei University of Chinese Medicine. Here, we present an improved suspension fixation method using a rat jacket to effectively immobilize rats during acupuncture experiments. Furthermore, the structure and application of this fixation apparatus, as well as instructions and evaluation for its utilization in electroacupuncture therapy, is elucidated. 

Video: Author Spotlight: Efficient Rat Clothing for Improved Rat Fixation During Electroacupuncture 

Hyperinsulinemic-Euglycemic Clamp in the Conscious Rat

Type 2 diabetes (T2D) is rapidly rising in prevalence. Characterized by either inadequate insulin production or the inability to utilize insulin produced, T2D results in elevated blood glucose levels. The "gold-standard" in assessing insulin sensitivity is a hyperinsulinemic-euglycemic clamp or insulin clamp. In this procedure, insulin is infused at a constant rate resulting in a drop in blood glucose. To maintain blood glucose at a constant level, exogenous glucose (D50) is infused into the venous circulation. The amount of glucose infused to maintain homeostasis is indicative of insulin sensitivity. Here, we show the basic clamp procedure in the chronically catheterized, unrestrained, conscious rat. This model allows blood to be collected with minimal stress to the animal. Following the induction of anesthesia, a midline incision is made and the left common carotid artery and right jugular vein are catheterized. Inserted catheters are flushed with heparinized saline, then exteriorized and secured. Animals are allowed to recover for 4-5 days prior to experiments, with weight gain monitored daily. Only those animals who regain weight to pre-surgery levels are used for experiments. On the day of the experiment, rats are fasted and connected to pumps containing insulin and D50. Baseline glucose is assessed from the arterial line and used a benchmark throughout the experiment (euglycemia). Following this, insulin is infused at a constant rate into the venous circulation. To match the drop in blood glucose, D50 is infused. If the rate of D50 infusion is greater than the rate of uptake, a rise in glucose will occur. Similarly, if the rate is insufficient to match whole body glucose uptake, a drop will occur. Titration of glucose continues until stable glucose readings are achieved. Glucose levels and glucose infusion rates during this stable period are recorded and reported. Results provide an index of whole body insulin sensitivity. The technique can be refined to meet specific experimental requirements. It is further enhanced by the use of radioactive tracers that can determine tissue specific insulin-stimulated glucose uptake as well as whole body glucose turnover.

The hyperinsulinemic-euglycemic clamp is the "gold standard" for the assessment of insulin action. Insulin is infused at a constant rate stimulating glucose uptake. The amount of exogenous glucose infused to counter this drop is indicative of insulin sensitivity. Here the procedure is performed on a conscious, unrestrained rat.

Type 2 diabetes (T2D) is rapidly rising in prevalence. Characterized by either inadequate insulin production or the inability to utilize insulin produced, ...

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

Angiogenesis in the Ischemic Rat Lung

The adult lung is perfused by both the systemic bronchial artery and the entire venous return flowing through the pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that responds to a need for enhanced lung perfusion and shows robust neovascularization. Pulmonary vascular ischemia induced by pulmonary artery obstruction has been shown to result in rapid systemic arterial angiogenesis in man as well as in several animal models. Although the histologic assessment of the time course of bronchial artery proliferation in rats was carefully described by Weibel 1, mechanisms responsible for this organized growth of new vessels are not clear. We provide surgical details of inducing left pulmonary artery ischemia in the rat that leads to bronchial neovascularization. Quantification of the extent of angiogenesis presents an additional challenge due to the presence of the two vascular beds within the lung. Methods to determine functional angiogenesis based on labeled microsphere injections are provided.

The lung is perfused by both the systemic bronchial artery and pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that shows robust neovascularization. Cessation of pulmonary blood flow promotes brisk bronchial angiogenesis. We provide surgical details of inducing left pulmonary artery ischemia that promotes bronchial neovascularization.

The  adult lung is perfused by both the systemic bronchial artery and the entire  venous return flowing through the pulmonary arteries. In most lung ...

A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the luminal surface area in arteries thereby reducing adequate blood flow to organs and distal tissues. Clinically, revascularization procedures such as balloon angioplasty with or without stent placement aim to restore blood flow. Although these procedures reestablish blood flow by reducing plaque burden, they damage the vessel wall, which initiates the arterial healing response. The prolonged healing response causes arterial restenosis, or re-narrowing, ultimately limiting the long-term success of these revascularization procedures. Therefore, preclinical animal models are integral for analyzing the pathophysiological mechanisms driving restenosis, and provide the opportunity to test novel therapeutic strategies. Murine models are cheaper and easier to operate on than large animal models. Balloon or wire injury are the two commonly accepted injury modalities used in murine models. Balloon injury models in particular mimic the clinical angioplasty procedure and cause adequate damage to the artery for the development of restenosis. Herein we describe the surgical details for performing and histologically analyzing the modified, pressure-controlled rat carotid artery balloon injury model. Additionally, this protocol highlights how local periadventitial application of therapeutics can be used to inhibit neointimal hyperplasia. Lastly, we present light sheet fluorescence microscopy as a novel approach for imaging and visualizing the arterial injury in three-dimensions.

The rat carotid artery balloon injury mimics the clinical angioplasty procedure performed to restore blood flow in atherosclerotic vessels. This model induces the arterial injury response by distending the arterial wall, and denuding the intimal layer of endothelial cells, ultimately causing remodeling and an intimal hyperplastic response.

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the ...

Evaluation of Cerebral Blood Flow Autoregulation in the Rat Using Laser Doppler Flowmetry

When investigating the body&#39;s mechanisms for regulating cerebral blood flow, a relative measurement of microcirculatory blood flow can be obtained using laser Doppler flowmetry (LDF). This paper demonstrates a closed skull preparation that allows cerebral blood flow to be assessed without penetrating the skull or installing a chamber or cerebral window. To evaluate autoregulatory mechanisms, a model of controlled blood pressure reduction via graded hemorrhage can be utilized while simultaneously employing LDF. This enables the real time tracking of the relative changes in the blood flow in response to reductions in arterial blood pressure produced by the withdrawal of circulating blood volume. This paradigm is a valuable approach to study cerebral blood flow autoregulation during reductions in arterial blood pressure and, with minor modifications in the protocol, is also valuable as an experimental model of hemorrhagic shock. In addition to evaluating autoregulatory responses, LDF can be used to monitor the cortical blood flow when investigating metabolic, myogenic, endothelial, humoral, or neural mechanisms that regulate cerebral blood flow and the impact of various experimental interventions and pathological conditions on cerebral blood flow.

This article demonstrates the use of laser Doppler flowmetry to evaluate the ability of the cerebral circulation to autoregulate its blood flow during reductions in arterial blood pressure.

When investigating the body&#39;s mechanisms for regulating cerebral blood flow, a relative measurement of microcirculatory blood flow can be obtained ...

Microscopic Observation of Lymphocyte Dynamics in Rat Peyer's Patches

Na&#239;ve lymphocytes recirculate from the blood to the lymphoid tissues under physiological condition and it is commonly recognized as an important phenomenon in the gut immunity. The stroma of secondary lymphoid organs, such as Peyer&#8217;s patches (PPs) or mesenteric lymph nodes, are where na&#239;ve lymphocytes sense antigens. Na&#239;ve lymphocytes circulate through the bloodstream to reach high endothelial venules, the portal of entry into PPs. Some immunomodulators are estimated to influence lymphocyte migration, but the precise evaluation of microcirculation dynamics is very difficult, and establishing a method to observe lymphocyte migration in vivo can contribute to the clarification of the precise mechanisms. We refined the method of collecting lymphocytes from the lymph duct and observing the detailed dynamics of gut-tropic lymphocytes in rat PPs. We chose confocal laser scanning microscopy to observe rat PPs in vivo and recorded it using time-lapse photography. We can now obtain clear images that can contribute to the analysis of lymphocyte dynamics.

Here, we describe a precise method for collecting thoracic duct lymphocytes and observing the migration of gut-tropic lymphocytes in rat Peyer&#8217;s patches for 3 hours using time-lapse photography. This technique can clarify how the dynamics of lymphocytes are affected under inflammatory conditions.

Na&#239;ve lymphocytes recirculate from the blood to the lymphoid tissues under physiological condition and it is commonly recognized as an important ...

Acupuncture in a Rat Model of Asthma

A high platform can fix rats without restriction and completely expose the acupoints on the back during acupuncture manipulation. This article describes methods for the fabrication of the high platform, establishes a rat model of asthma and measures changes in respiratory function using a noninvasive and real-time whole-body plethysmography (WBP) system.

A high platform can fix rats without restriction and completely expose the acupoints on the back during acupuncture manipulation. This article describes ...

Retinal Pathophysiological Evaluation in a Rat Model

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal detachment, breakdown of the blood-retinal barrier (BRB), and retinal angiogenesis. An in vivo rat model is a valuable experimental tool to examine the changes in the structure and function of the retina. We propose three different experimental techniques in the rat model to identify morphological changes of retinal cells, retinal vasculature, and compromised BRB. Retinal histology is used to study the morphology of various retinal cells. Also, quantitative measurement is performed by retinal cell count and thickness measurement of different retinal layers. A BRB breakdown assay is used to determine the leakage of extraocular proteins from the plasma to vitreous tissue due to the breakdown of BRB. Fluorescence angiography is used to study angiogenesis and leakage of blood vessels by visualizing retinal vasculature using FITC-dextran dye.

Diabetic retinopathy is one of the leading causes of blindness. Histology, blood-retinal barrier breakdown assay, and fluorescence angiography are valuable techniques to understand the pathophysiology of the retina, which could further enhance the efficient drug screening against diabetic retinopathy.

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal ...

Improved Rodent Model of Myocardial Ischemia and Reperfusion Injury

Myocardial ischemia and reperfusion injury (MIRI), induced by coronary heart disease (CHD), causes damage to the cardiomyocytes. Furthermore, evidence suggests that thrombolytic therapy or primary percutaneous coronary intervention (PPCI) does not prevent reperfusion injury. There is still no ideal animal model for MIRI. This study aims to improve the MIRI model in rats to make surgery easier and more feasible. A unique method for establishing MIRI is developed by using a soft tube during a key step of the ischemic period. To explore this method, thirty rats were randomly divided into three groups: sham group (n = 10); experimental model group (n = 10); and existing model group (n = 10). Findings of triphenyltetrazolium chloride staining, electrocardiography, and percent survival are compared to determine the accuracies and survival rates of the operations. Based on the study results, it has been concluded that the improved surgery method is associated with a higher survival rate, elevated ST-T segment, and larger infarct size, which is expected to mimic the pathology of MIRI better.

Myocardial ischemia-reperfusion model of rat heart is improved by using a self-made retractor, polyvinyl chloride tube, and a unique knotting method. Electrocardiogram, triphenyltetrazolium chloride and histological staining, and percent survival analysis results showed that the improved model group has higher success and survival rates than the already existing model group.

Myocardial ischemia and reperfusion injury (MIRI), induced by coronary heart disease (CHD), causes damage to the cardiomyocytes. Furthermore, evidence ...

Development of a Uterosacral Ligament Suspension Rat Model

Pelvic organ prolapse (POP) is a common pelvic floor disorder (PFD) with the potential to significantly impact a woman's quality of life. Approximately 10%-20% of women undergo pelvic floor repair surgery to treat prolapse in the United States. PFD cases result in an overall $26.3 billion annual cost in the United States alone. This multifactorial condition has a negative impact on the quality of life and yet the treatment options have only dwindled in the recent past. One common surgical option is uterosacral ligament suspension (USLS), which is typically performed by affixing the vaginal vault to the uterosacral ligament in the pelvis. This repair has a lower incidence of complications compared to those with mesh augmentation, but is notable for a relatively high failure rate of up to 40%. Considering the lack of standard animal models to study pelvic floor dysfunction, there is an urgent clinical need for innovation in this field with a focus on developing cost-effective and accessible animal models. In this manuscript, we describe a rat model of USLS involving a complete hysterectomy followed by fixation of the remaining vaginal vault to the uterosacral ligament. The goal of this model is to mimic the procedure performed on women to be able to use the model to then investigate reparative strategies that improve the mechanical integrity of the ligament attachment. Importantly, we also describe the development of an in situ tensile testing procedure to characterize interface integrity at chosen time points following surgical intervention. Overall, this model will be a useful tool for future studies that investigate treatment options for POP repair via USLS.

Pelvic organ prolapse affects millions of women worldwide and yet some common surgical interventions have failure rates as high as 40%. The lack of standard animal models to investigate this condition impedes progress. We propose the following protocol as a model for uterosacral ligament suspension and in vivo tensile testing.

Pelvic organ prolapse (POP) is a common pelvic floor disorder (PFD) with the potential to significantly impact a woman's quality of life. Approximately ...