Name: using herbal-cake-separated moxibustion for the treatment of rats with chronic renal failure
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We thank Associated Professor Huang and Miss Cui for their strict guidance and support for this paper. The study was supported by: 1. National Natural Science Foundation of China (NSFC), 82160938, &#34;Exploration of mechanism research on renal interstitial fibrosis with cake-separated moxibustion from the interactive effect of&#160; ERK and Smad signal pathways mediated by TGF-&#946;1&#34;; 2. The Health Commission of Yunnan Province &#34;2020 High-level TCM Reserve Talents&#34;Incubation Project (Yunwei TCM Development [2021] No.1); 3. Yunnan Provincial Science and Technology Department-Applied Basic Research Joint Special Funds of Chinese Medicine (No.2019FF002-022, No.202101AZ70001-286, No.202301AZ070001-030); 4.&#160;Yunnan Province Innovation Team Preventing and Treating cerebropathy with Acupuncture and Tuina (2023).

The plan follows the Animal Care Guidelines of the Yunnan University of Chinese Medicine and has been approved by the Animal Ethics Committee of Yunnan University of Chinese Medicine. The experimental animal welfare and ethics review number is R-062021G098.
1. Moxibustion experimental platform device (Figure 1)
NOTE: We designed and constructed the rat moxibustion experimental platform device based on the distance between the hind and front limbs of rats and their behavioral and physiological characteristics.
<ol>
	<li>The size of the moxibustion device
	<ol>
		<li>Fabricate the device using anti-beaten board material and a wooden board; the overall size is 52 cm L x 23.5 cm W x 64 cm H, and the bottom area of the device is 1222&#160;cm2. 
		NOTE: The moxibustion operation is carried out on the platform at this height, and the rats can be kept calm.</li>
	</ol>
	</li>
	<li>The main frame of the moxibustion device
	<ol>
		<li>Make the base of the whole device with anti-beaten board material: The base plate is 52 cm L x 23.5 cm W with a white surface. Ensure that the bottom accounts for a large proportion of the device&#39;s weight to stabilize the whole device.</li>
		<li>Make the elevated platform of the device with solid wood, 60&#160;cm high. 
		NOTE: This is the main frame of the whole instrument, which stabilizes the whole device and supports the placement of the rats in a standing position.</li>
		<li>Put five placement slots above the elevated platform, with a size of 10 cm L x 6 cm W x 10 cm H. Use&#160;interval boards (10 cm L x 1.3 cm W x 10 cm H) to separate the placement slots, corresponding to the distance between the front and rear limbs of the rat in standing position.</li>
	</ol>
	</li>
	<li>The components of the moxibustion device
	<ol>
		<li>Fix two stainless steel strips on top of the placement slots. Each fixed strip is a right angle equal edge steel bar 40 cm L x 1.4 cm W (both sides have the same width), with 15 holes. Three holes at the fixed strip correspond to each slot, used to fix the plum handle screw and the moving hose. The distance between adjacent holes is 7&#160;cm.</li>
		<li>Place the adjustable plum blossom handle screw at both ends of the heat insulation network made of 304 stainless steel and connect to the fixed heat insulation network and the fixed strip.</li>
		<li>Connect the upper end of each placement slot with a liftable heat insulation network surrounded by a galvanized plate frame. Connect this frame to the plum blossom handle screw at both ends. Place 304 stainless steel circular mesh material in the middle of the heat insulation network&#160;to prevent the ash from falling and scalding the rats and to fix the rats to facilitate moxibustion.</li>
		<li>Affix the bottom end of the stainless steel movable hose (34 cm long) with a stainless steel fixed strip and the upper end with the pipe clamp connection plate. 
		NOTE: The movable hose is convenient for adjusting the position of the moxa stick during the suspension moxibustion operation.</li>
		<li>Use the tube clamp made of aluminum alloy to connect the stainless steel movable hose and the moxa clip.</li>
		<li>Connect the stainless steel moxa clip to the pipe clamp connection plate, and fix 8-10 moxa strips simultaneously for moxibustion. 
		NOTE: The movable hose, tube clamp connecting plate, and moxa clip can be used for suspension moxibustion operation in rats.</li>
	</ol>
	</li>
</ol>
2. Establish a rat model of chronic renal failure
<ol>
	<li>Acclimate 24 specific pathogen-free (SPF) SD male rats (aged 3-4 weeks, weighing 180-200 g) for 1 week. Keep the feeding temperature at 18-24 &#176;C; relative humidity at 40-70%; and a 12 h light and 12 h dark cycle. House six animals in every cage and provide free access to water and common feed.</li>
	<li>Establish the rat model of CRF. According to the random number table and following this protocol, select six rats as the control group and the remaining 18 as the model group. Treat the control group (n = 6) with distilled water (10 mL&#8729;kg-1&#8729;day-1) and normal feed and the model group (n = 18) with adenine suspension (150 mg kg-1&#8729;day-1) and high-protein feed for 21 days.</li>
	<li>Observe and record the body weight, mental status, and diet daily. After the end of 21 days, ensure that all the animals are fasted for 12 h before orbital blood sampling.</li>
	<li>Collect the orbit blood samples.
	<ol>
		<li>Anesthetize the rats briefly with isoflurane. Grab the rat&#39;s back with the non-dominant hand, hold the rat&#39;s head and face symmetrically between the thumb and forefinger, fully expose one side of the rat&#39;s canthus, and hold this position.</li>
		<li>With the dominant hand, hold 3 cm long melting point capillaries between the thumb and forefinger and slowly twist the capillary, entering the rat&#39;s inner canthus venous plexus at 45&#176; until the blood slowly flows out of the tube.</li>
		<li>Collect 150-200 &#181;L of blood samples in 0.5 mL blood collection anticoagulant tubes, gently pull out the capillaries, and press the orbit with cotton.</li>
	</ol>
	</li>
	<li>Shake the anticoagulant tube quickly and thoroughly. Measure plasma serum creatinine (Scr) and blood urea nitrogen (BUN) using an automatic biochemical analyzer. 
	NOTE: The increase in Scr and BUN indicates the successful establishment of the model of CRF.</li>
	<li>Divide the rats into four groups: A: six control rats, no treatment; 18 model rats divided into groups B: (model group: no medication or moxibustion), C: (losartan potassium group: administered 2.33 mg&#8729;kg-1&#8729;day-1 by gavage needle 12 G), and D: (herbal-cake-separated-moxibustion group: herbal-cake-separated-moxibustion applied to bilateral Shenshu [B23] and bilateral Pishu [B20]).</li>
</ol>
3. Treatment phase
NOTE: Groups A and B are not given any treatment, while groups C and D will be treated once a day for 24 days, with two days off after 12 days of treatment.
<ol>
	<li>Preparations before treatment
	<ol>
		<li>Prepare 300 g of dried Radix astragali, 300 g of dried Radix Codonopsis, 200 g of dried Radix Rehmanniae Praeparata, 100 g of roasted Shorthorned Epimedium Herb and 100 g of Cornus officinalis. Mix these powders.</li>
		<li>Put 500 g of Artemisiae Argyi Folium into the traditional Chinese medicine crusher (speed of 34,000 rotations/min) for 5 min, transfer the ground powder onto a plate, and then screen with a 600 mesh sieve. After two screening rounds, eliminate the coarse moxa and transfer the fine moxa to a bag for use (Figure 2).</li>
		<li>Prepare the moxa cone by filling the fine moxa into the mold to make a small moxa cone of 0.5 cm diameter and 0.5 cm height. Alternatively, malaxate the moxa between the thumbs and index fingers of both hands to form moxa cones (Figure 3A).</li>
		<li>Prepare the herbal cake mold by cutting a 5 mL syringe into four 0.5 cm thick circular plastic rings and gluing the four rings on the same horizontal plane (on the corners of a square plane with a side length of 26&#160;mm, Figure 3D).</li>
		<li>Make medicine cakes by mixing the powder and water in a 1:1 ratio and filling the mixture into the herbal cake mold (Figure 3E). Leave the medicine cake in the mold and store it in a dry place.</li>
	</ol>
	</li>
	<li>Marking the acupoints
	<ol>
		<li>Locate the acupoints as follows: Shenshu (B23), both sides of the second lumbar vertebra, one acupoint on each side; Pishu (B20), the intercostal of the 11th thoracic vertebra on both sides, one acupoint on each side (Figure 4). 
		NOTE: For the location of the acupoints, refer to the Manual of Acupuncture for Experimental Animals20.</li>
		<li>Wear a thick rubber glove on the non-dominant hand to gently cover the rat&#39;s head, locate the acupoints on the rat&#39;s back with the dominant hand, and then mark the acupoints with a color marker.</li>
	</ol>
	</li>
	<li>Placing the rats
	<ol>
		<li>Wear thick rubber gloves. Use the thumb and the other four fingers of the dominant hand to slightly pinch the skin on the back and the neck of the rat. Use the thumb and index finger of the non-dominant hand to pinch the rat&#39;s tail gently.</li>
		<li>Lift the rat, align the head of the rat into the placement&#160;slot of the special moxibustion platform, and move the rat slowly forward. Ensure that the front paw of the rat is close to the edge of the placement&#160;slot and that the rat climbs into the slot. 
		NOTE: Disinfect the entire moxibustion platform before starting. Before catching the rat, touch the back of the rat gently or talk to it to calm it. Adjust the height of the insulation network from a low position (when not in use) to the highest, which is convenient for rats to enter.</li>
	</ol>
	</li>
	<li>Rat fixation
	<ol>
		<li>Place the thumb and index fingers of both hands at the screw of the plum blossom screw at both ends of the heat insulation network. Unscrew the plum blossom screw to lower the heat insulation net to the appropriate position, close to the back of the rat, but not causing compression, and then tighten the screws.</li>
	</ol>
	</li>
	<li>Placing the moxa cone and medicated cake.
	<ol>
		<li>Place the prepared mold containing the medicine cake on the heat insulation net corresponding to the acupoints of both Pishu (B20) and Shenshu (B23) according to the marks of acupoints on the rats. There are four acupoints, each of which corresponds to a medicine cake.</li>
		<li>Place the moxa cone on the medicated cake smoothly and steadily.</li>
	</ol>
	</li>
	<li>Moxibustion
	<ol>
		<li>Light the moxa cone with a lighter and start moxibustion till the moxa cone burns out and the residual heat dissipates naturally (the wet medicated cake dries up).</li>
		<li>Replace the burnt-out moxa cone with a new moxa cone and medicated cake on the same acupoint and continue moxibustion till the moxa cone burns out and the medicated cake dries. Repeat this for a total of 5x at each acupoint. 
		NOTE: Clean and disinfect the liftable heat insulation network before replacing it with a new moxa cone and medicated cake.</li>
		<li>Extinguish the moxa cones and the medicated cakes in a water-containing waste tank after burning five moxa cones at each acupoint. 
		NOTE: Each acupoint receives five cones of moxibustion; each moxa cone can be replaced after burning out.</li>
	</ol>
	</li>
	<li>Returning the rat to the cage
	<ol>
		<li>Put the empty rearing cage in front of the rat, close to the lower end of the placement slot of the moxibustion platform, and keep still. Pat lightly the rat&#39;s buttock so that the rat can move from the placement slot to the feeding cage. After the rat enters, put down the cage. Use the dominant hand to grasp the fur on the back of the rat, hold the rat firmly, and place it on the ground for further observation.</li>
		<li>Observe whether the rats have shortness of breath, whether the mood is stable, and whether the skin surface is scalded. If there is no abnormality, put it back in the cage. If it is scalded, immediately apply the scald ointment on the skin surface and place it in a single cage.</li>
	</ol>
	</li>
</ol>
4. Sampling phase
<ol>
	<li>After 24 days of treatment, prepare&#160;the rats for 12&#160;hours of fasting before sampling.</li>
	<li>Pour isoflurane into the gas anesthesia machine and anesthetize the rats with 2-3% isoflurane. After 2-3 min, ensure that reflexes are absent (pinch the tail with surgical tweezers to check that they do not have any reflex) and continued&#160;existence&#160;of a heartbeat and switch to maintenance anesthesia with 1-1.5% isoflurane.</li>
	<li>Collect 5 mL of blood from the abdominal aorta by inserting the needle into the quasi-abdominal aorta. Let the blood stand for 2 h, centrifuge at 2490&#160;&#215; g for 10 min at 4 &#176;C, and transfer the serum supernatant to a 1.5 mL microcentrifuge tube for further analysis.</li>
	<li>Deeply anesthetize the rats for a long period of time and take blood from the abdominal aorta under maintenance anesthesia. Further, euthanize the rats by cervical dislocation.</li>
	<li>Harvest tissues from both kidneys by stripping the bilateral renal fascia and fat with surgical scissors and tweezers and rinsing with 0.9% normal saline. Weigh the left and right kidneys and record the weight and morphology.</li>
	<li>Preserve the renal tissue by cutting the left kidney into six parts and putting them into a 50 mL centrifuge tube containing 4% paraformaldehyde for soaking, fixation, and preservation. Cut the right kidney into six pieces using a knife in the longitudinal direction and two knives in the transverse direction. Place these pieces into six 2 mL Cryotubes and immediately put them in liquid nitrogen; transfer them to a -80 &#176;C freezer for storage.</li>
</ol>
5. Detection phase
<ol>
	<li>Use the right kidney tissue of appropriate size for protein extraction, gel electrophoresis, membrane transfer, blocking, primary antibody incubation, secondary antibody, chemiluminescence, development, fixation, and film scanning.</li>
	<li>Use a gel image data analysis system (e.g., Image-Pro Plus 6.0) to analyze the band&#39;s gray value and calculate each protein&#39;s content.</li>
	<li>Subject the left kidney tissues to dehydration, clearing, embedding in paraffin, sectioning (thickness of 4 &#181;m), staining with hematoxylin-eosin and Masson&#39;s trichrome stain, and observe the pathological changes21 in the renal tissue under a light microscope at 200x magnification.</li>
</ol>

A Moxibustion Platform for Herbal Cake-Based Treatment of Rats with Renal Failure

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The authors declare that there are no conflicts of interest. The rat moxibustion experimental platform device&#39;s patent&#160;number is ZL202320880924.X.

Clinically, &#160;acupuncture and moxibustion treatment requires patients to be calm and relaxed, avoiding excessive anger and hunger and emphasizing &#39;peace.&#39; However, the current practice of &#160;acupuncture and moxibustion experiments is still based on the premise of binding animals, which is not only contrary to the original intention of the experiment but also does not conform to clinical practice. Binding animals, making animals frightened and angry, will rapidly increase adrenaline and other hormones in ex...

Moxibustion appeared with the application of fire. Ancient doctors accidentally found that some parts of the fire-burning body could relieve the symptoms of discomfort. After long-term practice, &#34;Artemisiae Argyi Folium&#34; was gradually selected as the main moxibustion material1. Moxibustion is one of the important components of traditional Chinese medicine, and when juxtaposed with traditional Chinese medicine decoction and acupoint acupuncture, these three major traditional Chinese medicine therapies are widely used in many diseases.
Chinese doctors often used moxibustion to treat diseases as early as the period of Huangdi Neijing (Yellow Emperor&#39;s Inner Classic). Artemisiae Argyi Folium is a type of Chinese herbal medicine with the function of warming meridians to stop bleeding, dispelling colds, and relieving pain. It is commonly used in the external treatment of traditional Chinese medicine and can also be used in the treatment of traditional Chinese medicine decoction. Moxibustion is a kind of external therapy2 wherein moxa or other drugs are placed on acupoints on the body surface to burn and use the mild heat of moxibustion fire and the effect of drugs to treat diseases3.
As one of the common methods of treatment, moxibustion has been widely used in clinical practice. With the continuous expansion in moxibustion therapy, a growing number of studies have investigated the mechanism of moxibustion and carried out many animal experiments4,5.
The moxibustion method can be classified as direct moxibustion and indirect moxibustion. The method of placing moxibustion on the body&#39;s skin is called direct moxibustion. Indirect moxibustion separates the surface of the moxa cone and the acupoints with drugs or other materials. Moxibustion can treat various diseases, such as irritable bowel syndrome6, chronic fatigue syndrome7, and knee osteoarthritis8.
This paper mainly discusses the mechanism of herbal-cake-separated-moxibustion. Herbal-cake-separated-moxibustion belongs to the indirect moxibustion method, in which a moxa cone is placed on the medicinal cake to apply moxibustion. Current studies have shown that the infrared and warming effects produced by moxa cone burning, the chemical composition of moxa leaf tar, and acupoint stimulation can achieve disease prevention and treatment9 by increasing the penetration of drugs into acupoints. Therefore, herbal-cake-separated-moxibustion mainly produces a &#34;comprehensive effect&#34; through moxibustion, drugs, and acupoints10.
Previous studies show that moxibustion is an effective method for treating CRF11,12,13,14,15, but the underlying mechanism is still undefined. We have been working on clinical and animal research on CRF. At present, we are conducting a moxibustion experimental study on CRF rats based on herbal-cake-separated-moxibustion-mediated protein kinase RNA-like endoplasmic reticulum kinase (PERK) signaling pathway of endoplasmic reticulum stress. Through this animal experiment, we aim to preliminarily explain the potential mechanism of moxibustion in treating chronic kidney disease. In this experiment, the treatment of herbal-cake-separated moxibustion of rats&#160;is a key operation.
In the previous experiment16, the lid of the rearing cage was used to bind and fix the rats. To protect the operator, the rats need to be anesthetized before binding. However, Sprague-Dawley (SD) rats are easy to fear and startle. The binding operation can easily cause restlessness in the rats. Problems such as poor posture fixation of rats, deviation of acupoint positioning, increased experiment time, and operator injury often occurred.
In addition, if the binding is too tight, it can easily cause breathing difficulties in rats, which is not conducive to animal welfare. If the binding is too loose, the rats are prone to struggle and move their bodies, causing moxibustion cones to fall and burn their skin, which affects the experimental results. Based on the literature, animal experiments still involve binding16,17,18, and most experimental devices can only be fixed, hindering the implementation of other techniques such as moxibustion19. Therefore, achieving the integration of fixation and moxibustion in the experimental device while ensuring animal welfare and experimental specifications and efficiency has become one of the focus areas for investigators.

<table><tbody><tr><td>0.5 mL heparin lithium biochemical tube</td><td>Shijiazhuang Kangweishi Medical Equipment Co., LTD</td><td>220518</td><td>Blood collection</td></tr><tr><td>0.9% normal saline</td><td>Kunming Nanjiang Pharmaceutical Co., LTD</td><td>/</td><td>Rinse</td></tr><tr><td>1.5 mL Eppendorf tube</td><td>Beijing Lanjieke Biotechnology Co., LTD</td><td>12100024</td><td>Subpackage</td></tr><tr><td>2 mL cryopreservation tube</td><td>Baroque Biotechnology Co., LTD</td><td>88-0151</td><td>Packaged kidney&amp;nbsp;</td></tr><tr><td>4 &amp;deg;C refrigerator</td><td>Haier Group Co. LTD</td><td>BCD-236WDGL</td><td>Store</td></tr><tr><td>5 mL syringe</td><td>Sichuan New century Medical polymer products Co., LTD</td><td>20211115</td><td>Gavage&amp;nbsp;</td></tr><tr><td>&amp;nbsp;50 mL centrifuge tube</td><td>Beijing Langeke Biotechnology Co., LTD</td><td>CT-002-50A</td><td>Packaged kidney&amp;nbsp;</td></tr><tr><td>12 G gavage needle</td><td>Beijing Zhongke Hengtian Technology Co., Ltd.</td><td>12#55mm</td><td>Gavage&amp;nbsp;</td></tr><tr><td>96-well plates</td><td>Beijing Lanjieke Biotechnology Co., LTD</td><td>11510</td><td>Determination of protein concentration</td></tr><tr><td>&amp;nbsp;-80 &amp;deg;C freezer</td><td>Thermo Fisher Scientific</td><td>991</td><td>Store</td></tr><tr><td>Adenine</td><td>Beijing Boaotuo Technology Co., LTD</td><td>A0607</td><td>Molding drug</td></tr><tr><td>BCA developer</td><td>Biyuntian Biological Engineering Co., LTD</td><td>P0010</td><td>Determination of protein concentration</td></tr><tr><td>BCL-2 protein antibody reagent</td><td>Abcam Shanghai Trading Co., LTD</td><td>ab196495</td><td>Western Blot</td></tr><tr><td>Caspase-3 protein antibody reagent</td><td>Abcam Shanghai Trading Co., LTD</td><td>ab184787</td><td>Western Blot</td></tr><tr><td>Chemiluminescence imaging system</td><td>Shanghai Qinxiang Scientific Instrument Co., LTD</td><td>100240073</td><td>Protein banding imaging</td></tr><tr><td>Chinese medicine powder</td><td>The Second Affiliated Hospital of Yunnan University of Chinese Medicine</td><td>/</td><td>Cure</td></tr><tr><td>Direct-load Color Prestained Marker</td><td>Beijing Kangrunchengye Biotechnology Co., LTD (GenStar)</td><td>M221</td><td>Western Blot</td></tr><tr><td>Disposable blood collection needle</td><td>Liuyang Sanli Medical science and technology development Co., LTD</td><td>YZB/ Country 0411-2010</td><td>Blood sampling</td></tr><tr><td>Disposable human venous blood sample collection container</td><td>Liuyang Sanli Medical science and technology development Co., LTD</td><td>20210901</td><td>Blood sampling</td></tr><tr><td>DK-30 Automatic snow ice maker</td><td>Henan Brothers instrument equipment Co., LTD</td><td>SHDX0023</td><td>Ice-making</td></tr><tr><td>ECL luminescent substrate kit</td><td>Beijing Lanjieke Biotechnology Co., LTD</td><td>BL520B</td><td>Convert latent images in exposed film into visible images</td></tr><tr><td>electronic scales</td><td>Shanghai Ran Hao Electronics Co., LTD</td><td>JCS-31002C</td><td>The body weight and kidney weight of rats were measured</td></tr><tr><td>Freezing microtome&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>HM525 NX</td><td>Microtome</td></tr><tr><td>fully automatic biochemical analyser</td><td>Tianjin Micro nano core Technology Co., LTD</td><td>PV2112001Z02410305</td><td>Renal function test</td></tr><tr><td>Gas anesthesia system</td><td>Zhongshi Technology Co., Ltd</td><td>ZS-MV-1</td><td>Anesthesia</td></tr><tr><td>Glycine</td><td>Beijing Soleibao Technology Co., Ltd.</td><td>Cat#G8200</td><td>Running buffer, Western transfer buffer</td></tr><tr><td>HE staining reagent</td><td>Beijing Soleibao Technology Co., Ltd.</td><td>G1120&amp;nbsp;</td><td>Staining</td></tr><tr><td>High-protein feed</td><td>Jiangsu Synergetics Biological Engineering Co., LTD</td><td>XT19008</td><td>Feeding</td></tr><tr><td>Intelligent laboratory ultra-pure water machine</td><td>Chongqing huachuag water treatment engineering co.,LTD</td><td>15L</td><td>Filtration</td></tr><tr><td>Internal reference &amp;beta;-actin</td><td>Jiangsu Qinke Biological Research Co., LTD</td><td>AF7018</td><td>Western Blot</td></tr><tr><td>Isoflurane</td><td>Jiangsu Zhongmu Beikang Pharmaceutical Co., Ltd</td><td>20220701</td><td>Anesthesia</td></tr><tr><td>liquid nitrogen</td><td>Kunming Botai dry ice sales Co., LTD</td><td>/</td><td>Renal tissue freezing</td></tr><tr><td>Losartan potassium tablets</td><td>Merck Sharp &amp;amp; Dohme (China) Investment Co., LTD</td><td>J20180054</td><td>Cure</td></tr><tr><td>Low temperature high speed tissue grinder</td><td>Wuhan Servicebio technology CO.,LTD</td><td>SKZ3F20200191</td><td>Tissue grinding</td></tr><tr><td>Maason staining kit</td><td>Beijing Soleibao Technology Co., Ltd.</td><td>G1340</td><td>Staining</td></tr><tr><td>Methanol</td><td>Guangdong Guanghua Sci-Tech Co., Ltd</td><td>20220519</td><td>Western transfer buffer</td></tr><tr><td>Mini-PROTEAN Tetra</td><td>Bole Life Medical Products (Shanghai) Co., Ltd</td><td>552BR 233193</td><td>Electrophoresis</td></tr><tr><td>Moxa</td><td>The Second Affiliated Hospital of Yunnan University of Chinese Medicine</td><td>/</td><td>Cure</td></tr><tr><td>Multiskan Spectrum Microplate Spectrophotometer</td><td>TECAN</td><td>Spark</td><td>The absorbance and concentration of tissue protein were detected</td></tr><tr><td>operating scissors</td><td>Zhangjiagang city Jinfeng deer knife cutting factory</td><td>201118</td><td>Cut the skin</td></tr><tr><td>Paraformaldehyde</td><td>Beijing Lanjieke Biotechnology Co., LTD</td><td>BL539A</td><td>Fixed tissue</td></tr><tr><td>PERK protein antibody reagent</td><td>Shanghai Pumai Biotechnology Co., LTD (CST China Agent)</td><td>3192</td><td>Western Blot</td></tr><tr><td>Positive fluorescence microscope</td><td>Carl Zeiss Suzhou Co.,Ltd.</td><td>5446000456</td><td>Capture picture</td></tr><tr><td>Prefabricated glue</td><td>Derun Cheng (Beijing) Biological Co., LTD (ACE China Agent)</td><td>B230418</td><td>Western Blot</td></tr><tr><td>ProteinFind Goat Anti-Rabbit LgG(H+L), HRP Conjugate</td><td>Shanghai Dinran electronic Technology Co., LTD (TRAN)</td><td>HS101</td><td>Western Blot</td></tr><tr><td>pulverizer</td><td>Hefei Rongzhida small household appliances Co., LTD</td><td>RS-FS1401</td><td>Crushed Chinese medicine</td></tr><tr><td>PVDF membrane</td><td>MerckMillipore Corporation&amp;nbsp;</td><td>IPVH00010&amp;nbsp;</td><td>Western Blot</td></tr><tr><td>Refrigerated centrifuge</td><td>Hettich Precision Technology (Zhuhai) Co., LTD</td><td>MIKRO 220R</td><td>Centrifuge</td></tr><tr><td>RIPA lysate</td><td>Beijing Solaibao Technology Co., LTD (Solarbio)</td><td>R0010</td><td>Lytic tissue</td></tr><tr><td>Sampling capillary</td><td>Jiangsu Kangjianhua Medical Supplies Co., LTD</td><td>20160157</td><td>Blood from inner canthus</td></tr><tr><td>Scald ointment (Zilian ointment)</td><td>Yunnan Provincial Hospital of Traditional Chinese Medicine</td><td>/</td><td>Used for the treatment of scalding from moxibustion</td></tr><tr><td>Skim milk powder</td><td>Guangzhou Saiguo Biotechnology Co., LTD (BIOFROXX)</td><td>1172GR500</td><td>Western blocking buffer</td></tr><tr><td>surgical blade</td><td>Shanghai Medical Equipment Co., LTD</td><td>JOBO10</td><td>Incision kidney tissue</td></tr><tr><td>surgical knife handle</td><td>Shanghai Medical Equipment Co., LTD</td><td>J11010</td><td>Mounting blade</td></tr><tr><td>TS-200 Orbital shaker</td><td>Haimen Qilin Bell Manufacturing Co., Ltd.</td><td>TS-8S</td><td>Gel fixation</td></tr><tr><td>Tween 80</td><td>MedChemExpress</td><td>HY-Y1819</td><td>&amp;nbsp;TBST&amp;nbsp;</td></tr><tr><td>tweezers</td><td>Shanghai Medical Equipment Co., LTD</td><td>JD1050&amp;nbsp;</td><td>Pick up the moxa</td></tr><tr><td>Veterinary drugs*Micro core renal function test disc</td><td>Tianjin Micro nano core Technology Co., LTD</td><td>47031-35.5-0145-0227-62-220750</td><td>Renal function test</td></tr></tbody></table>

using herbal-cake-separated moxibustion for the treatment of rats with chronic renal failure

In the process of moxibustion in clinical practice, subjects need to be in a stable mood and comfortable posture to avoid problems such as moxa ash falling, scalding skin, and poor curative effect. Such problems also exist in the rat moxibustion experiment. To simulate clinical practice, it is necessary to introduce an experimental instrument in animal experiments, that is, a moxibustion device with fixed rats and moxibustion treatment synchronization, which can make experimental rats receive moxibustion treatment quietly and comfortably under non-anesthesia.
Our research group designed a rat moxibustion experimental platform. The device was framed by a wooden board with a supporting base plate, multiple fixed components, and partitioned components. The device can achieve the operation mode of moxibustion in rats without binding, avoiding anesthesia and scalding and simultaneously exposing multiple acupoints on the back. This operation can avoid physical and mental injury to rats and operators, which improves the research efficiency and further promotes the development and research of moxibustion animal experiments.
The device has a simple structure, is easy to operate and popularize, is comprehensively and innovatively designed, reusable, and is suitable for rat experiments mainly based on moxibustion. This article mainly introduces the structure of the experimental platform device for rat moxibustion, the basic procedure of herbal-cake-separated-moxibustion in experimental rats using the device and describes the establishment of a rat model of chronic renal failure (CRF) and representative experimental results.

Kidney morphology 
As can be seen from Figure 5, compared with the other groups, the kidneys&#39; size, color, and texture in group D (herbal-cake-separated-moxibustion group) were closer to those of the normal group. The kidney morphology of the model and losartan potassium groups showed different degrees white fibrosis.
Kidney weight 
Figure 6 shows that group A&#39;s left...

Watch this Scientific Journal Video about Using Herbal-Cake-Separated Moxibustion for the Treatment of Rats with Chronic Renal Failure at JoVE.com

Author Spotlight: Understanding the Effect of Herbal-Cake-Separated Moxibustion in Rats with Renal Faliure

The moxibustion experimental platform can fix the rats without restriction, and the rats can undergo multi-acupoint moxibustion in the awake state, ensuring the welfare of the rats, protecting the safety of the operators, ensuring therapeutic efficacy, and improving the efficiency of the experiment.

Using Herbal-Cake-Separated Moxibustion for the Treatment of Rats with Chronic Renal Failure

In the process of moxibustion in clinical practice, subjects need to be in a stable mood and comfortable posture to avoid problems such as moxa ash ...

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Research

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Medicine

691 Views.  Yunnan University of Chinese Medicine. The moxibustion experimental platform can fix the rats without restriction, and the rats can undergo multi-acupoint moxibustion in the awake state, ensuring the welfare of the rats, protecting the safety of the operators, ensuring therapeutic efficacy, and improving the efficiency of the experiment.

Video: Author Spotlight: Understanding the Effect of Herbal-Cake-Separated Moxibustion in Rats with Renal Faliure

Acute and Chronic Tactile Sensory Testing after Spinal Cord Injury in Rats

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory testing in rat SCI models is needed. However, until recently, no single testing approach had been validated for SCI so that standardized methods have not been implemented across labs. Additionally, available testing methods could not be implemented acutely or when severe motor impairments existed, preventing studies of the development of SCI-induced allodynia3. Here we present two validated sensory testing methods using von Frey Hair (VFH) monofilaments which quantify changes in tactile sensory thresholds after SCI4-5. One test is the well-established Up-Down test which demonstrates high sensitivity and specificity across different SCI severities when tested chronically5. The other test is a newly-developed dorsal VFH test that can be applied acutely after SCI when allodynia develops, prior to motor recovery4-5. Each VFH monofilament applies a calibrated force when touched to the skin of the hind paw until it bends. In the up-down method, alternating VFHs of higher or lower forces are used on the plantar L5 dermatome to delineate flexor withdrawal thresholds. Successively higher forces are applied until withdrawal occurs then lower force VFHs are used until withdrawal ceases. The tactile threshold reflects the force required to elicit withdrawal in 50% of the stimuli. For the new test, each VFH is applied to the dorsal L5 dermatome of the paw while the rat is supported by the examiner. The VFH stimulation occurs in ascending order of force until at least 2 of 3 applications at a given force produces paw withdrawal. Tactile sensory threshold is the lowest force to elicit withdrawal 66% of the time. Acclimation, testing and scoring procedures are described. Aberrant trials that require a retest and typical trials are defined. Animal use was approved by Ohio State University Animal Care and Use Committee.

We describe two tactile sensory testing methods for acute or chronic periods of spinal cord injury in rats. These validated procedures can detect the development and maintenance of allodynia-like sensations.

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory ...

Assessment of Vascular Function in Patients With Chronic Kidney Disease

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this is only partially explained by traditional CVD risk factors. Vascular dysfunction is an important non-traditional risk factor, characterized by vascular endothelial dysfunction (most commonly assessed as impaired endothelium-dependent dilation [EDD]) and stiffening of the large elastic arteries. While various techniques exist to assess EDD and large elastic artery stiffness, the most commonly used are brachial artery flow-mediated dilation (FMDBA) and aortic pulse-wave velocity (aPWV), respectively. Both of these noninvasive measures of vascular dysfunction are independent predictors of future cardiovascular events in patients with and without kidney disease. Patients with CKD demonstrate both impaired FMDBA, and increased aPWV. While the exact mechanisms by which vascular dysfunction develops in CKD are incompletely understood, increased oxidative stress and a subsequent reduction in nitric oxide (NO) bioavailability are important contributors. Cellular changes in oxidative stress can be assessed by collecting vascular endothelial cells from the antecubital vein and measuring protein expression of markers of oxidative stress using immunofluorescence. We provide here a discussion of these methods to measure FMDBA, aPWV, and vascular endothelial cell protein expression.

The degree of vascular dysfunction and contributing physiological mechanisms can be assessed in patients with chronic kidney disease by measuring brachial artery flow-mediated dilation, aortic pulse-wave velocity, and vascular endothelial cell protein expression.

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this ...

Standardized Operation Process of Zhuang Medicine Line Moxibustion in Rats

Medicated Thread Moxibustion Therapy of Zhuang Medicine and its Application in a Rat Model of Cold-Congealing Syndrome of Primary Dysmenorrhea

Zhuang medicine thread moxibustion therapy is one of the national intangible cultural heritages. It is a Zhuang medicine characteristic therapy that involves igniting ramie thread soaked in Zhuang medicine preparation solution and directly moxibustion on a certain acupoint or part of the body surface. This treatment method is characterized by combining drug, acupoints, and moxibustion, stimulating the skin's receptors at the acupoints through the warmth of moxibustion, affecting the biochemical metabolism of histiocyte and the function of the nervous system so that the drug ingredients can be absorbed through the skin. However, the therapeutic effect of Zhuang medicine line moxibustion is influenced by factors such as moxibustion intensity, heat intensity, and moxibustion frequency. Manual operation cannot precisely control each influencing factor. Here, we design a Zhuang medicine line moxibustion simulation instrument to maintain a constant force, heat, and frequency. The frequency and heat can be adjusted according to experimental needs, and it can be applied to the study of analgesic experiments to observe the analgesic effect of Zhuang medicine line moxibustion on pain animal models to solve the standardization problem of Zhuang medicine line moxibustion in the operation process of experimental animals.This study establishes a model of primary dysmenorrhea in rats with cold coagulation syndrome and provides a detailed introduction to the preparation, process, and precautions of Zhuang medicine thread moxibustion on the model of primary dysmenorrhea in rats with cold coagulation syndrome. The intervention of Zhuang medicine thread moxibustion on primary dysmenorrhea in rats with cold coagulation syndrome is evaluated by twisting score and infrared thermal imaging body surface temperature detection, intuitively demonstrating the therapeutic effect of Zhuang medicine thread moxibustion, and a preliminary analysis of its mechanism of action is conducted.

Author Spotlight: Demonstrating Therapeutic Effect of Automated Zhuang Medicine Line Moxibustion Simulator

This article demonstrates the standardized operation process of Zhuang medicine line moxibustion in experimental animals. To achieve repeatability and accuracy of Zhuang medicine line moxibustion, we developed a simulator to maintain strength, heat, and frequency. We constructed a dysmenorrhea model for experimental verification in rats with cold coagulation syndrome.

Zhuang medicine thread moxibustion therapy is one of the national intangible cultural heritages. It is a Zhuang medicine characteristic therapy that ...

Behavior

Early Detection of Drug-Induced Renal Hemodynamic Dysfunction Using Sonographic Technology in Rats

The kidney normally functions to maintain hemodynamic homeostasis and is a major site of damage caused by drug toxicity. Drug-induced nephrotoxicity is estimated to contribute to 19- 25% of all clinical cases of acute kidney injury (AKI) in critically ill patients. AKI detection has historically relied on metrics such as serum creatinine (sCr) or blood urea nitrogen (BUN) which are demonstrably inadequate in full assessment of nephrotoxicity in the early phase of renal dysfunction. Currently, there is no robust diagnostic method to accurately detect hemodynamic alteration in the early phase of AKI while such alterations might actually precede the rise in serum biomarker levels. Such early detection can help clinicians make an accurate diagnosis and help in in decision making for therapeutic strategy. Rats were treated with Cisplatin to induce AKI. Nephrotoxicity was assessed for six days using high-frequency sonography, sCr measurement and upon histopathology of kidney. Hemodynamic evaluation using 2D and Color-Doppler images were used to serially study nephrotoxicity in rats, using the sonography. Our data showed successful drug-induced kidney injury in adult rats by histological examination. Color-Doppler based sonographic assessment of AKI indicated that resistive-index (RI) and pulsatile-index (PI) were increased in the treatment group; and peak-systolic velocity (mm/s), end-diastolic velocity (mm/s) and velocity-time integral (VTI, mm) were decreased in renal arteries in the same group. Importantly, these hemodynamic changes evaluated by sonography preceded the rise of sCr levels. Sonography-based indices such as RI or PI can thus be useful predictive markers of declining renal function in rodents. From our sonography-based observations in the kidneys of rats that underwent AKI, we showed that these noninvasive hemodynamic measurements may consider as an accurate, sensitive and robust method in detecting early stage kidney dysfunction. This study also underscores the importance of ethical issues associated with animal use in research.

Early stage hemodynamic dysfunction is critical to the development of kidney disease. Yet, detection methodologies are limited. Recent advances in sonography provide a noninvasive, accurate option for early detection of kidney injury. This study outlines a step-by-step, sonographic methodology for detecting kidney dysfunction using a drug-induced nephrotoxicity rat model.

The kidney normally functions to maintain hemodynamic homeostasis and is a major site of damage caused by drug toxicity. Drug-induced nephrotoxicity is ...

Network Analysis of Foramen Ovale Electrode Recordings in Drug-resistant Temporal Lobe Epilepsy Patients

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control seizures. However, a significant minority of patients continues to exhibit post-operative seizures, even in those cases in which the suspected source of seizures has been correctly localized and resected. The protocol presented here combines a clinical procedure routinely employed during the pre-operative evaluation of temporal lobe epilepsy (TLE) patients with a novel technique for network analysis. The method allows for the evaluation of the temporal evolution of mesial network parameters. The bilateral insertion of foramen ovale electrodes (FOE) into the ambient cistern simultaneously records electrocortical activity at several mesial areas in the temporal lobe. Furthermore, network methodology applied to the recorded time series tracks the temporal evolution of the mesial networks both interictally and during the seizures. In this way, the presented protocol offers a unique way to visualize and quantify measures that considers the relationships between several mesial areas instead of a single area.

This protocol describes a procedure to track the evolution of mesial network measures in temporal lobe epilepsy (TLE) patients. It is based on the combination of intracranial recordings with a novel numerical technique for data analysis. Specifically, we present a protocol for network analyses of foramen ovale recordings.

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control ...

Tachycardia-Induced Cardiomyopathy As a Chronic Heart Failure Model in Swine

A stable and reliable model of chronic heart failure is required for many experiments to understand hemodynamics or to test effects of new treatment methods. Here, we present such a model by tachycardia-induced cardiomyopathy, which can be produced by rapid cardiac pacing in swine.
A single pacing lead is introduced transvenously into fully anaesthetized healthy swine, to the apex of the right ventricle, and fixated. Its other end is then tunneled dorsally to the paravertebral region. There, it is connected to an in-house modified heart pacemaker unit that is then implanted in a subcutaneous pocket. 
After 4 - 8 weeks of rapid ventricular pacing at rates of 200 - 240 beats/min, physical examination revealed signs of severe heart failure - tachypnea, spontaneous sinus tachycardia, and fatigue. Echocardiography and X-ray showed dilation of all heart chambers, effusions, and severe systolic dysfunction. These findings correspond well to decompensated dilated cardiomyopathy and are also preserved after the cessation of pacing.
This model of tachycardia-induced cardiomyopathy can be used for studying the pathophysiology of progressive chronic heart failure, especially hemodynamic changes caused by new treatment modalities like mechanical circulatory supports. This methodology is easy to perform and the results are robust and reproducible.

Here, we present a protocol to produce tachycardia-induced cardiomyopathy in swine. This model represents a potent way to study the hemodynamics of progressive chronic heart failure and the effects of applied treatment.

A stable and reliable model of chronic heart failure is required for many experiments to understand hemodynamics or to test effects of new treatment ...

Establishing a Swine Model of Post-myocardial Infarction Heart Failure for Stem Cell Treatment

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major causes of mortality and morbidity around the world. Cell-based therapies for cardiac repair and improvement of left ventricular function after MI have attracted considerable attention. Accordingly, the safety and efficacy of these cell transplantations should be tested in a preclinical large animal model of HF prior to clinical use. Pigs are widely used for cardiovascular disease research due to their similarity to humans in terms of heart size and coronary anatomy. Therefore, we sought to present an effective protocol for the establishment of a porcine chronic HF model using closed-chest coronary balloon occlusion of the left circumflex artery (LCX), followed by rapid ventricular pacing induced with pacemaker implantation. Eight weeks later, the stem cells were administered by intramyocardial injection in the peri-infarct area. Then the infarct size, cell survival, and left ventricular function (including echocardiography, hemodynamic parameters, and electrophysiology) were evaluated. This study helps establish a stable preclinical large animal HF model for stem cell treatment.

We sought to establish a swine model of heart failure induced by left circumflex artery blockage and rapid pacing to test the effect and safety of intramyocardial administration of stem cells for cell-based therapies.

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major ...

Traditional Medicine and PI3K/AKT Pathway Activation in Membranous Nephropathy

Mechanism of Kemeng Fang's Inhibition of Podocyte Apoptosis in Rats with Membranous Nephropathy through the PI3K/AKT Signaling Pathway

Membranous nephropathy (MN) is a common pathological type of adult nephrotic syndrome. Up to 20% of patients with MN develop end-stage renal disease (ESRD). Podocytes have an important function in maintaining the glomerular filtration barrier and play a crucial role in the occurrence and development of proteinuria and MN. PI3K/AKT signaling pathway is involved in the entire process of podocyte growth, differentiation, and apoptosis. Kemeng Fang (KMF) is a traditional Chinese medicine formula that has been used to delay kidney injury. However, the therapeutic mechanism of KMF in MN is unclear. Here, the MN rat model was established by axillary, inguinal, and tail vein injections of cationized bovine serum albumin (C-BSA), and then KMF and PI3K inhibitor (LY294002) were administered. The data of liver function, kidney function, blood lipid, renal pathology, podocyte function, expression level of PI3K/AKT signaling pathway, and transcriptomics of rats demonstrated that KMF has a protective effect on the podocytes of MN rats by activating the PI3K/AKT signaling pathway, and it can effectively prevent the progression of MN.

The present protocol describes the establishment of a membranous nephropathy (MN) animal model, and how Kemeng Fang's inhibition reduces MN rat podocyte apoptosis by activating the PI3K/AKT signaling pathway.

Membranous nephropathy (MN) is a common pathological type of adult nephrotic syndrome. Up to 20% of patients with MN develop end-stage renal disease ...

Herbal Moxibustion on Shenque Acupoint for Alleviating Primary Dysmenorrhea

Herbs-Partitioned Moxibustion on the Navel in a Rat Model of Primary Dysmenorrhea with Cold Coagulation and Blood Stasis

Primary dysmenorrhea (PDM) refers to the occurrence of spasmodic colicky pain in the lower abdomen without any obvious pelvic pathology, often accompanied by other systemic symptoms, which significantly reduces the quality of life of women with PDM, 45%-95% of menstrual women are affected by it. Despite the high incidence of PDM, it is often not well treated and is ignored by relevant researchers and women themselves. Herbs-partitioned moxibustion (HPM) is a characteristic external therapy in traditional Chinese medicine (TCM), which is widely used to treat diseases in China; it treats diseases by applying a Chinese herbal formula on the patient's acupoints and then igniting moxa sticks on the herbal formula for moxibustion therapy. The primary effectiveness of the herbs utilized in this study is meridian activation and warmth, which encourages qi circulation to relieve pain. Moxibustion's heat stimulation could hasten the infusion of the herbs into the human body. Consequently, HPM, which combines acupoint, herbal, and heat stimulations, is appropriate for treating gynecological disorders, particularly functional diseases such as PDM. This paper provides a detailed description of the procedures and precautions for HPM at the Shenque (CV8) acupoint in female experimental rats, which 0provides an experimental basis for better promoting the application and development of HPM on the navel in PDM.

Here, we describe in detail the operation procedures and precautions to be taken during herbs-partitioned moxibustion on the navel in the treatment of primary dysmenorrhea in rats with cold coagulation and blood stasis, which can significantly improve the writhing reaction of dysmenorrhea rats and alleviate pain.

Primary dysmenorrhea (PDM) refers to the occurrence of spasmodic colicky pain in the lower abdomen without any obvious pelvic pathology, often accompanied ...

Herbal Munziq Ameliorates Myocardial Ischemia-Reperfusion Injury by Inhibiting Inflammation

The objective of this study was to investigate the cardioprotective effects of Munziq on abnormal body fluid myocardial ischemia-reperfusion injury (MIRI) and its underlying mechanism.Normal rats and rats with abnormal body fluid (ABF) were pre-treated with Munziq for 21 days. Following this, MIRI models were established. Histopathological changes and myocardial ultrastructure changes were observed by Hematoxylin and Eosin (HE)staining and transmission electron microscopy to observe pathological manifestations of myocardial injury. Serum CK-MB, cTn-T, and ICAM-1 levels were detected by Enzyme-Linked Immunosorbent Assay (ELISA) to observe myocardial injury-related markers. The levels of IL-1&#946;, IL-6, and TNF-&#945; in serum and myocardial tissue were also detected by ELISA to observe the anti-inflammatory effect. The expression levels of NF-&#954;B signaling pathway-related proteins NIK, IKK&#945;, Pik&#945;, and p65 were detected by Western blot analysis. The results showed that myocardial injury in the ABF MIRI group was more severe compared to the control MIRI group. Munziq pretreatment has the potential to mitigate the pathological changes induced by ischemia-reperfusion injury and could protect cardiac function. Protein levels of the NF-&#954;B pathway and downstream effectors IL-1&#946;, IL-6, and TNF-&#945; were significantly up-regulated in the MIRI group while down-regulated in the Munziq group. Interestingly, there was more activation of the NF-&#954;B signaling pathway and higher levels of downstream inflammatory cytokines in the ABF MIRI group. The results suggest that MIRI was more severe in ABF. Munziq has cardioprotective effects in ischemia and reperfusion injury. This protective effect may be acted by suppressing the NF-&#954;B signaling pathway.

This study investigates the cardioprotective effects of Munziq, a traditional Uyghur herbal preparation, on myocardial ischemia-reperfusion injury (MIRI) in rats with abnormal body fluid. Through comprehensive experimental methods, we demonstrate Munziq's potential to mitigate MIRI by suppressing the NF-&#954;B signaling pathway.

The objective of this study was to investigate the cardioprotective effects of Munziq on abnormal body fluid myocardial ischemia-reperfusion injury (MIRI) ...