This work was supported with funds from the National Natural Science Foundation of China (NSFC) Nos. 82374564 (to YJD) and Nos. 82074566 (to YJD) and from Sun Guojie Inheritance Base for TCM Acupuncture-Moxibustion of World Federation of Acupuncture-Moxibustion Societies in Wuhan, China (World Federation of Acupuncture-Moxibustion Societies [2019] No. 26) to YJD and the National Famous TCM Inheritance Studio construction project of the National Administration of Traditional Chinese Medicine, China (the National Administration of Traditional Chinese Medicine [2022] No. 5) to YJD.

All experiments were carried out following the National Institutes of Health - Guide for the Care and Use of Laboratory Animals and were approved by the Hubei University of TCM - Institutional Animal Care and Use Committee. Adult female Sprague-Dawley (SD) rats, weighing 220 &#177; 20 g, were used for the test. All rats were maintained on a 12 h light/dark cycle at 23 &#177; 1 &#176;C and 40%-50% humidity, with free access to food and water.
1. Pre-operative preparation
<ol>
	<li>Group design
	<ol>
		<li>After genotyping 30, 3-month-old female rats, randomly divide them into three groups (n = 10 in each group): the normal group, the model group, and the fire-needle group. Treat the rats in the fire-needle group with fire-needle intervention 1x a day for 6 sessions, with 1 day of rest between sessions. 
		NOTE: The normal group and the model group suspended the rat bound with a secure coat according to the same frequency and the same duration as the fire-needle group, but without the fire-needle treatment.</li>
	</ol>
	</li>
	<li>Preparation of rat osteoporosis model
	<ol>
		<li>For the rats in the model group and the fire-needle group, surgically remove their ovaries to establish an osteoporosis model as described in8,9,10.
		<ol>
			<li>For ovariectomy, administer anesthesia by intraperitoneal injection of 2% pentobarbital sodium (0.2 mL/100 g) before the operation, and consider anesthetization successful when animals are unresponsive in a surgical plane of anesthesia based on lack of response from a toe pinch test. Use vet ointment on eyes to prevent dryness while under anesthesia. Inject buprenorphine (0.05 mg/kg) subcutaneously as an analgesic before the surgery. Maintain aseptic conditions during the operation. 
			NOTE: If the surgery has not been completed and the rat shows signs of resuscitation, intraperitoneal injection of 2% pentobarbital sodium solution may be supplemented temporarily and appropriately, with each supplemental dose not exceeding 20% of the total amount injected.</li>
			<li>After surgery, place the rats on a small animal heating pad and set the temperature to 32 &#176;C. Do not leave rats unattended until they regain consciousness and can crawl freely. Do not return them to the company of other animals until they have fully recovered.</li>
			<li>When the rats are revived, check the sutures for integrity and place the rats back in the cage. Inject all rats intramuscularly with penicillin sodium (50,000 U per day, for 3 days) and inject intraperitoneally with 0.1 mL 0.05% diclofenac sodium solution (per day, for 3 days) to prevent infection and accelerate healing9,10.</li>
		</ol>
		</li>
		<li>Observe the estrous cycle according to the vaginal exfoliated cell smear of female rats as described in11. If the cycle is disordered, the model is established successfully11. 
		NOTE: It takes approximately 8-12 weeks for rats to develop osteoporosis after ovariectomy.</li>
	</ol>
	</li>
	<li>Selection of needle tools
	<ol>
		<li>Purchase fire needles from medical device manufacturers. Ensure that needle tools are made of tungsten-manganese alloy, which is temperature resistant, is non-malleable, not easy to deform and gets uniformly heated. The fire needle is shaped like a filament needle. Ensure the needle is thick, the tip is sharp and straight, and the handle is wrapped with copper wire, which is heat-insulating and does not burn hands.</li>
		<li>Choose a single-head fire needle with a size of 0.40 mm x 13 mm because rats are small. Check whether the fire needle has barbs and cracks. 
		NOTE: In clinical practice, most of the fire needles used are those developed by He Puren, a national medical master12. Based on this, Lin Guohua created the Lingnan Long-Headed Fire Needle and Lingnan Three-Headed Fire Needle13. Choose different sizes of fire needles based on the need of the experiment.</li>
	</ol>
	</li>
	<li>Selection and positioning of acupoints
	<ol>
		<li>According to the relevant data and literature, select acupoints for the treatment of postmenopausal osteoporosis. 
		NOTE: The most frequently used acupoints are, in order of frequency, Shenshu (BL 23), Zusanli (ST 36), Pishu (BL 20), Xuanzhong (GB 39), and Dazhu (BL 11)14,15. In basic research on acupuncture intervention for osteoporosis in de-ovulatory model rats, most used acupuncture points are Shenshu (BL 23), Sanyinjiao (SP 6), Guanyuan (CV 4) and Zusanli (ST 36)16,17,18.</li>
		<li>Determine the acupoints selected for this experiment. Referring to the acupuncture points selected in previous studies14,15,16,17,18 and the pathogenesis of osteoporosis, select four acupuncture points, namely, Shenshu (BL 23), Pishu (BL 20), Zusanli (ST 36) and Sanyinjiao (SP 6), for this study. These correspond to the bladder meridian, the Du meridian, the spleen meridian, and the kidney meridian. 
		NOTE: It is forbidden to use fire needles on parts with large vessels and nerve trunks.</li>
		<li>Shave the acupuncture site with a pet electric clipper before positioning to expose the acupuncture point.</li>
		<li>Location of acupoints refers to names and location of commonly used acupoints in19. Locate the acupoints as follows: the Shenshu (BL 23) is located on the lower two sides of the second lumbar vertebra, 6 mm from the mid-dorsal line; the Pishu (BL 20) is located between the ribs on the lower two sides of the 12th thoracic vertebra, 6 mm from the mid-dorsal line; the Zusanli (ST 36) is located on the posterior and lateral side of the knee, about 3 mm below the small head of the fibula; and Sanyinjiao (SP 6) is located about 10 mm straight up from the tip of the inner ankle of the hind limb.</li>
		<li>Mark them with an oil-based marker after locating the points.</li>
	</ol>
	</li>
	<li>Rat restraint method 
	NOTE: In order to reduce stress during immobilization and treatment of rats, the suspended rats bound with a secure coat20,21 was chosen as the immobilization method. The rat coat suspension method has the following advantages: Firstly, it makes full use of the rat&#39;s habit of preferring a dark environment and reduces the rat&#39;s stress reaction during fixation. Secondly, the suspended rats are quieter and more cooperative during the whole process of fire-needling treatment due to the lack of force points on their limbs and toes. Thirdly, the acupoints on the back and limbs of the rats were sufficiently exposed to facilitate the treatment. A single treatment requires restraining the rat for 5-10 min. When secure in the coat, rats do not struggle in the restraint and are calm.
	<ol>
		<li>Prepare the rat coat. Sew the cleaned fabric and make it into a 50 cm x 13 cm tube shaped rat coat. Cut holes for each of the four limbs and back of the rat coat and sew ties at the four corners.</li>
		<li>Put on gloves, place the rat on a table that is not slippery, hold the rat&#39;s tail with the left hand and gently pull back. Open the port of the rat coat with the right hand. Using the rat&#39;s habit of darkness and burrowing, place it at the mouth of the rat coat, the rat would burrow into it on its own.</li>
		<li>When the rat burrows into the middle of the rat coat, use the left hand to hold both sides of the rat&#39;s shoulder blades, and pull out the rat&#39;s two forelimbs with the right hand.</li>
		<li>Tighten the ropes on the front of the coat, cross the ropes and wrap them around the rat&#39;s back. Adjust the elasticity of the ropes and tie the knot on the back of the rat. Finally, fix the back end of the rat body in a similar way so that its bilateral hind limbs are located outside the rat coat.</li>
		<li>For the swing type suspension, take two S-hooks and hook them into the knot on the back of the rat coat and hook the hooks onto the iron frame. At this point, the rat is suspended from the strip of cloth, in a slightly swinging state (Figure 1). 
		NOTE: The height of the strip of cloth from the ground is about 70 cm, for the experimenter sitting on a stool to operate comfortably and effortlessly. Place a thick padding to the flat surface below the rat as a precaution if animal slips.</li>
	</ol>
	</li>
</ol>
2. Acupuncture treatment
<ol>
	<li>Disinfection
	<ol>
		<li>Sterilize the local area with 5% iodophor disinfecting solution 3x before acupuncturing. Add alcohol rub after each iodophor scrub.</li>
	</ol>
	</li>
	<li>Burning needles 
	NOTE: Safety precautions should be taken to prevent burns and injuries.
	<ol>
		<li>Take the hemostatic forceps to pick up the cotton ball, soaked with 95% alcohol, with the left hand and light it with a lighter.</li>
		<li>Hold the handle of the needle with the thumb, forefinger, and middle finger of the right hand in a slightly curved position, as if holding a pen.</li>
		<li>Put the needle tip and needle body on the alcohol cotton balls and heat it; first heat the needle body, and then heat the needle tip. Burn the needle until it is bright white or red, depending on the purpose of treatment (Figure 2). 
		NOTE: The needle body should be fully burned bright white or red, otherwise it will affect the efficacy of acupuncture or cause postoperative infection and other issues.</li>
	</ol>
	</li>
	<li>Acupuncture 
	NOTE: According to the speed, depth, range, needle retention time and treatment purpose, Lingnan Fire-Needle can be divided into various operating techniques such as point acupuncture, scatter acupuncture, dense acupuncture, fast acupuncture, and slow acupuncture22. The key to operating with Lingnan Fire-Needle is red, accurate, and fast.
	<ol>
		<li>Wait until the needle burns red, move the needle away from the flame and hold it above the acupoint.</li>
		<li>Point the needle tip vertically at the acupoint, quickly puncture and pull out. Ensure that the whole process is completed within 1 s.</li>
		<li>The depth of acupoints penetration is about 1-2 mm, only pierce the red-hot needle tip. 
		NOTE: The depth of acupuncture should be determined according to the condition, constitution, age, muscle thickness of the acupoint, the depth of blood vessels, and the distribution of nerves.</li>
		<li>Puncture each acupoint 3x-5x. Repeat this for all acupoints (Figure 3). 
		NOTE: When performing the operation, be fully focused and agile. In the same procedure on a rat, the same fire needle should be used, with the needle being re-sterilized by flaming between every two punctures; the needle should not be placed down or become unsterile.</li>
	</ol>
	</li>
</ol>
3. Post-acupuncture management
<ol>
	<li>Lift the fire needle away from the skin and hold a sterile dry cotton ball to press the needle hole to prevent bleeding. Following the fire-needle therapy, the rats displayed unimpaired mobility and no detectable complications. 
	NOTE: If bleeding after fire needling occurs, there is no need to stop the bleeding, just wipe with a dry cotton ball after it stops naturally.</li>
</ol>
4. Data collection and analysis
<ol>
	<li>After 6 courses of fire-needling intervention, euthanize the rats by high dose intraperitoneal injection of pentobarbital sodium (150-200 mg/kg). Collect femur specimens and perform micro-CT scans23.</li>
	<li>Analyze the data statistically, using analysis software. Show measurements as mean and analyze using one-way ANOVA, with p &#60;0.05 being considered as statistically significant.</li>
</ol>

Osteoporosis Management in Rats Through Lingnan Fire-Needle Therapy

The authors declare that they have nothing to disclose.

Lingnan fire-needle therapy has a profound historical background. Records on fire needles and needling methods can be traced back to Huangdi Neijing. Modern acupuncturists Situ Bell and Zhang Jiawei combined Lingnan&#39;s climate and the physical characteristics of its people to create Lingnan fire-needle therapy3. Lin Guohua summarized Lingnan Fire-Needle therapy, as warming, dredging, tonifying, clearing, and eliminating, with warming as the essence24.
<p class="jove_...

Chinese medicine believes that factors such as climate and environment can affect a person&#39;s physique, which in turn affects the occurrence and development of diseases. The Lingnan region of China, mainly including Guangdong, Guangxi, and Hainan Island, is located in the tropical monsoon climate region. As a result, people living in this region for a long time are prone to a form of spleen deficiency. In some cases, it spreads to the kidney and liver, leading to the prevalence of osteoporosis1. The spleen and stomach function in transporting and absorbing water and grains, which are the source of qi and blood biochemistry, while the liver helps maintain the marrows. So, bone nutrition relies on the supply of the spleen, stomach, and liver. In Chinese medicine, the kidney is capable of generating marrow and strengthening bones, and its vitality directly affects the strength and function of bones. Therefore, kidney deficiency is directly related to osteoporosis. According to TCM, liver (Gan)-kidney (Shen) deficiency and lack of essence (Jing) are the underlying causes. In some cases, malnutrition may occur due to a spleen (Pi) and stomach (Wei) deficiency or an imbalance of the two. That is, the deficiency of the liver (Gan), kidneys (Shen), spleen (Pi), and stomach (Wei) are the root causes, while external qi (Xie qi) blocks meridians and damages bones and muscles. According to the World Health Organization (WHO), OP is a medical condition characterized by low bone mass and tissue deterioration2, which is a serious concern for the elderly. The selection of appropriate interventions is crucial for the prevention and treatment of osteoporosis.
Modern acupuncturists have developed a unique Lingnan characteristic of fire-needling therapy by combining the climate of Lingnan and the physical characteristics of Lingnan people3. Fire needle therapy is a kind of treatment method in which special needles are burned red and quickly pierced into specific parts or acupoints of the human body to treat various ailments. This method is widely used in skin diseases, musculoskeletal and joint diseases, neurological diseases, and other systemic diseases and shows good efficacy4. The treatment of osteoporosis with fire needles has been used in clinics for a long time, and it has a better curative effect than simple acupuncture or oral administration of traditional Chinese medicine5,6,7. The Lingnan fire-needle therapy integrates acupuncture, three-edged needles, and scarring moxibustion. It can improve the qi and blood circulation in the meridians while warming the kidney. This treatment can address both blood stasis and kidney (Shen) deficiency, which contribute to the pathogenesis of OP in TCM. What&#39;s more, it has the advantages of being low cost, having quick effects, a short treatment period, and being safe. However, there is a lack of research on its mechanism. This paper introduces a specific procedure of Lingnan Fire-Needle in osteoporosis model rats and provides a methodological basis for conducting related basic research.

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	<tbody>
		<tr>
			<td>75% Alcohol</td>
			<td>Shandong Lircon medicaltechnology co., ltd</td>
			<td></td>
			<td>Disinfection</td>
		</tr>
		<tr>
			<td>95% Alcohol</td>
			<td>Shandong Lircon medicaltechnology co., ltd</td>
			<td></td>
			<td>Burn needles</td>
		</tr>
		<tr>
			<td>Adult female Sprague-Dawley (SD) rats</td>
			<td>Liaoning Changsheng biotechnology co., Ltd.</td>
			<td>SCXK 2020-0001</td>
			<td>Modeling of osteoporosis</td>
		</tr>
		<tr>
			<td>Deluxe thermostat heat mat</td>
			<td>Zhenhua Instruments</td>
			<td></td>
			<td>For maintaining the body temperature of rats.</td>
		</tr>
		<tr>
			<td>Diclofenac sodium injection</td>
			<td>Shandong Province Jianmu Bio-pharmaceutical Co.</td>
			<td>150182913</td>
			<td>Antipyretic and analgesic</td>
		</tr>
		<tr>
			<td>Elevated fixation rack</td>
			<td>Taizhou Shi Ai Scientific and Educational Instruments and Equipment Co.</td>
			<td></td>
			<td>Fix rats</td>
		</tr>
		<tr>
			<td>Fire needles</td>
			<td>Beijing Luoya Shanchuan Medical Equipment Co., Ltd.</td>
			<td>20190240</td>
			<td>A tool of needling</td>
		</tr>
		<tr>
			<td>Hemostatic forceps</td>
			<td>Qingdao Schultz Biotechnology Co.</td>
			<td></td>
			<td>Fix rats</td>
		</tr>
		<tr>
			<td>Liguter</td>
			<td>Shanghai Machi Trading Co.</td>
			<td>H5</td>
			<td>Ignite alcohol</td>
		</tr>
		<tr>
			<td>Lodophor disinfecting solution</td>
			<td>Qingdao Hai&#39;s Hainuo Inovation Disinfection Technology Co., Ltd.</td>
			<td>2016-0604</td>
			<td>Disinfection</td>
		</tr>
		<tr>
			<td>Medical cotton ball</td>
			<td>Jiangsu Misawa Medical Supplies Co., Ltd.</td>
			<td>20172142357</td>
			<td>Disinfection</td>
		</tr>
		<tr>
			<td>pentobarbital sodium</td>
			<td>Beijing Jiehui Biotechnology Co.</td>
			<td>P3761</td>
			<td>anesthesia</td>
		</tr>
		<tr>
			<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>
		</tr>
		<tr>
			<td>Self-made rat coat</td>
			<td></td>
			<td></td>
			<td>Fix rats</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Qingdao Schultz Biotechnology Co.</td>
			<td></td>
			<td>Pinch cotton balls</td>
		</tr>
	</tbody>
</table>

operation procedure and precautions of lingnan fire-needle therapy in osteoporosis model rats

Compared to filiform needle therapy, fire-needle therapy has both the stimulation of needles and the warming effect of heat, making it have unexpected effects on some chronic diseases and incurable diseases. Osteoporosis (OP) has a high incidence in postmenopausal women and middle-aged and elderly men, and the treatment cycle is long. According to Traditional Chinese Medicine (TCM), Lingnan fire-needle therapy has shown potential in treating osteoporosis. However, there is still a long way to go before it can be widely used. This article focuses on the application of Lingnan fire-needle therapy in the intervention of OP in rats. It covers the selection of needle tools, acupuncture point selection, positioning of rats' bodies, and fixation methods. We also outline the steps and precautions to be taken during and after needling with fire needles. The experiment was done with three groups: a normal group, a model group, and a fire-needle group, each containing 10 rats. The rats in the fire-needle group were treated with fire-needle intervention for six sessions. After the intervention period, we collected femoral specimens and performed micro-CT scans. The results suggest that fire needling can enhance bone morphology and mineral density in OP rats. This information can serve as a methodological basis for conducting basic research on fire-needle therapy.

The data in Table 1 shows that compared with the normal group, the thickness of trabecular bone (Tb.Th), the number of trabecular bone (Tb.N) and the static parameter bone volume fraction of trabecular bone (BV/TV) in the model group were all significantly reduced (p &#60;0.05), and the separation of trabecular bone (Tb.Sp) was elevated (p &#60;0.05); indicating that the bone structure of trabecular bone in trabecular bone-enriched areas of the femoral epiphysis of the rat was significantly disrupted aft...

Read this Scientific Article Protocol about Author Spotlight: Exploring the Impact of Lingnan Fire-Needle Therapy in Osteoporosis Intervention at JoVE.com

Author Spotlight: Exploring the Impact of Lingnan Fire-Needle Therapy in Osteoporosis Intervention

Here, we present a protocol for the application of Lingnan Fire-Needle therapy in the intervention of osteoporosis model rats.

Operation Procedure and Precautions of Lingnan Fire-Needle Therapy in Osteoporosis Model Rats

Compared to filiform needle therapy, fire-needle therapy has both the stimulation of needles and the warming effect of heat, making it have unexpected ...

operation-procedure-precautions-lingnan-fire-needle-therapy

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461 Views.  Hubei University of Chinese Medicine. Here, we present a protocol for the application of Lingnan Fire-Needle therapy in the intervention of osteoporosis model rats.

Video: Author Spotlight: Exploring the Impact of Lingnan Fire-Needle Therapy in Osteoporosis Intervention

Roux-en-Y Gastric Bypass Operation in Rats

Currently, the most effective therapy for the treatment of morbid obesity to induce significant and maintained body weight loss with a proven mortality benefit is bariatric surgery1,2. Consequently, there has been a steady rise in the number of bariatric operations done worldwide in recent years with the Roux-en-Y gastric bypass (gastric bypass) being the most commonly performed operation3. Against this background, it is important to understand the physiological mechanisms by which gastric bypass induces and maintains body weight loss. These mechanisms are yet not fully understood, but may include reduced hunger and increased satiation4,5, increased energy expenditure6,7, altered preference for food high in fat and sugar8,9, altered salt and water handling of the kidney10 as well as alterations in gut microbiota11. Such changes seen after gastric bypass may at least partly stem from how the surgery alters the hormonal milieu because gastric bypass increases the postprandial release of peptide-YY (PYY) and glucagon-like-peptide-1 (GLP-1), hormones that are released by the gut in the presence of nutrients and that reduce eating12.
During the last two decades numerous studies using rats have been carried out to further investigate physiological changes after gastric bypass. The gastric bypass rat model has proven to be a valuable experimental tool not least as it closely mimics the time profile and magnitude of human weight loss, but also allows researchers to control and manipulate critical anatomic and physiologic factors including the use of appropriate controls. Consequently, there is a wide array of rat gastric bypass models available in the literature reviewed elsewhere in more detail 13-15. The description of the exact surgical technique of these models varies widely and differs e.g. in terms of pouch size, limb lengths, and the preservation of the vagal nerve. If reported, mortality rates seem to range from 0 to 35%15. Furthermore, surgery has been carried out almost exclusively in male rats of different strains and ages. Pre- and postoperative diets also varied significantly.
Technical and experimental variations in published gastric bypass rat models complicate the comparison and identification of potential physiological mechanisms involved in gastric bypass. There is no clear evidence that any of these models is superior, but there is an emerging need for standardization of the procedure to achieve consistent and comparable data. This article therefore aims to summarize and discuss technical and experimental details of our previously validated and published gastric bypass rat model.

Numerous studies using gastric bypass rat models have been recently conducted to uncover the underlying physiological mechanisms of Roux-en-Y gastric bypass operations. This article aims to demonstrate and discuss the technical and experimental details of our published gastric bypass rat model to understand advantages and limitations of this experimental tool.

Currently, the most effective therapy for the treatment of morbid obesity to induce significant and maintained body weight loss with a proven mortality ...

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

A Contusion Model of Severe Spinal Cord Injury in Rats

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is described to obtain a consistent model of severe SCI. Use of a stereotactic frame and computer controlled impactor allows for creation of reproducible injury. Hypothermia and urinary tract infection pose significant challenges in the post-operative period. Careful monitoring of animals with daily weight recording and bladder expression allows for early detection of post-operative complications. The functional results of this contusion model are equivalent to transection models. The contusion model can be utilized to evaluate the efficacy of both neuroprotective and neuroregenerative approaches.

A contusion model of severe spinal cord injury is described. Detailed pre-operative, operative and post-operative steps are described to obtain a consistent model.

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is ...

Transarterial Administration of Oncolytic Viruses for Locoregional Therapy of Orthotopic HCC in Rats

Hepatocellular carcinoma (HCC) is a disease with limited treatment options and poor prognosis. In recent years, oncolytic virotherapies have proven themselves to be potentially powerful tools to fight malignancy. Due to the unique dual blood supply in the liver, it is possible to apply therapies locally to orthotopic liver tumors, which are predominantly fed by arterial blood flow. We have previously demonstrated that hepatic arterial delivery of oncolytic viruses results in safe and efficient transduction efficiency of multifocal HCC lesions, resulting in significant prolongation of survival in immune competent rats. This procedure closely mimics the application of transarterial embolization in patients, which is the standard palliative care provided to many HCC patients. The ability to administer tumor therapies through the hepatic artery in rats allows for a highly sophisticated preclinical model for evaluating novel viral vectors under development. Here we describe the detailed protocol for microdissection of the hepatic artery for infusion of oncolytic virus vectors to treat orthotopic HCC.

Oncolytic virotherapies are under development as novel therapeutics for the treatment of hepatocellular carcinoma (HCC). Here we describe a method for locoregional therapy of HCC via hepatic arterial administration of oncolytic virus.

Hepatocellular carcinoma (HCC) is a disease with limited treatment options and poor prognosis.  In recent years, oncolytic virotherapies have proven ...

A Model of Cardiac Remodeling Through Constriction of the Abdominal Aorta in Rats

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and fluid retention. Changes in myocardial structure, electrical conduction, and energy metabolism develop with heart failure, leading to contractile dysfunction, increased risk of arrhythmias, and sudden death. Hypertensive heart disease is one of the key contributing factors of cardiac remodeling associated with heart failure. The most commonly-used animal model mimicking hypertensive heart disease is created via surgical interventions, such as by narrowing the aorta. Abdominal aortic constriction is a useful experimental technique to induce a pressure overload, which leads to heart failure. The surgery can be easily performed, without the need for chest opening or mechanical ventilation. Abdominal aortic constriction-induced cardiac pathology progresses gradually, making this model relevant to clinical hypertensive heart failure. Cardiac injury and remodeling can be observed 10 weeks after the surgery. The method described here provides a simple and effective approach to produce a hypertensive heart disease animal model that is suitable for studying disease mechanisms and for testing novel therapeutics.

A rat model of abdominal aortic constriction that induces cardiac hypertrophy and remodeling is described. An efficient, highly-reproducible, and minimally-invasive method is used to provide a simple yet useful platform for research in myocardial hypertrophy and dysfunction.

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and ...

A Method for Quantifying Upper Limb Performance in Daily Life Using Accelerometers

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. It has become important to measure a person's activities in daily life, and not just measure their capacity for activity in the structured environment of a clinic or laboratory. A wearable sensor that is now enabling measurement of daily movement is the accelerometer. Accelerometers are commercially-available devices resembling large wrist watches that can be worn throughout the day. Data from accelerometers can quantify how the limbs are engaged to perform activities in peoples' homes and communities. This report describes a methodology to collect accelerometry data and turn it into clinically-relevant information. First, data are collected by having the participant wear two accelerometers (one on each wrist) for 24 h or longer. The accelerometry data are then downloaded and processed to produce four different variables that describe key aspects of upper limb activity in daily life: hours of use, use ratio, magnitude ratio, and the bilateral magnitude. Density plots can be constructed that visually represent the data from the 24 h wearing period. The variables and their resultant density plots are highly consistent in neurologically-intact, community-dwelling adults. This striking consistency makes them a useful tool for determining if upper limb daily performance is different from normal. This methodology is appropriate for research studies investigating upper limb dysfunction and interventions designed to improve upper limb performance in daily life in people with stroke and other patient populations. Because of its relative simplicity, it may not be long before it is also incorporated in clinical neurorehabilitation practice.

This protocol describes a method to quantify upper limb performance in daily life using wrist-worn accelerometers.

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

A Model of Glaucoma Induced by Circumlimbal Suture in Rats and Mice

The circumlimbal suture is a technique for inducing experimental glaucoma in rodents by chronically elevating intraocular pressure (IOP), a well-known risk factor for glaucoma. This protocol demonstrates a step-by-step guide on this technique in Long Evans rats and C57BL/6 mice. Under general anesthesia, a "purse-string" suture is applied on the conjunctiva, around the equator and behind the limbus of the eye. The fellow eye serves as an untreated control. Over the duration of our study, which was a period of 8 weeks for rats and 12 weeks for mice, IOP remained elevated, as measured regularly by rebound tonometry in conscious animals without topical anesthesia. In both species, the sutured eyes showed electroretinogram features consistent with preferential inner retinal dysfunction. Optical coherence tomography showed selective thinning of the retinal nerve fiber layer. Histology of the rat retina in cross-section found reduced cell density in the ganglion cell layer, but no change in other cellular layers. Staining of flat-mounted mouse retinae with a ganglion cell specific marker (RBPMS) confirmed ganglion cell loss. The circumlimbal suture is a simple, minimally invasive and cost-effective way to induce ocular hypertension that leads to ganglion cell injury in both rats and mice.

Chronic ocular hypertension is induced by applying a circumlimbal suture in rats and mice, leading to functional and structural deterioration of the retinal ganglion cells consistent with glaucoma.

The circumlimbal suture is a technique for inducing experimental glaucoma in rodents by chronically elevating intraocular pressure (IOP), a well-known ...

Preparation of Decellularized Kidney Scaffolds in Rats

Tissue engineering is a cutting-edge discipline in biomedicine. Cell culture techniques can be applied for regeneration of functional tissues and organs to replace diseased or damaged organs. Scaffolds are needed to facilitate the generation of three-dimensional organs or tissues using differentiated stem cells in vivo. In this report, we describe a novel method for developing vascularized scaffolds using decellularized rat kidneys. Eight-week-old Sprague-Dawley rats were used in this study, and heparin was injected into the heart to facilitate flow into the renal vessels, allowing heparin to perfuse into the renal vessels. The abdominal cavity was opened, and the left kidney was collected. The collected kidneys were perfused for 9 h using detergents, such as Triton X-100 and sodium dodecyl sulfate, to decellularize the tissue. Decellularized kidney scaffolds were then gently washed with 1% penicillin/streptomycin and heparin to remove cellular debris and chemical residues. Transplantation of stem cells with the decellularized vascular scaffolds is expected to facilitate the generation of new organs. Thus, the vascularized scaffolds may provide a foundation for tissue engineering of organ grafts in the future.

This protocol introduces a method to develop a scaffold using decellularized rat kidneys. The protocol includes decellularization and recellularization processes to confirm bioavailability. Decellularization is performed using Triton X-100 and sodium dodecyl sulfate.

Tissue engineering is a cutting-edge discipline in biomedicine. Cell culture techniques can be applied for regeneration of functional tissues and organs ...

A Rodent Model of The Ross Operation: Syngeneic Pulmonary Artery Graft Implantation in A Systemic Position

The Ross operation for aortic valve disease has regained new interest due to its outstanding long-term results. Nonetheless, when employed as freestanding root replacement, the possible dilation of the pulmonary autograft and subsequent aortic regurgitation is described. Several animal models have been proposed. However, these are usually limited to ex-vivo models or in-vivo experiments with relatively expensive large animal models. In this study, we sought to establish a rodent model of pulmonary artery graft (PAG) implantation in a systemic position. A total of 39 adult Lewis rats were included. Immediately after euthanasia, the pulmonary root was harvested from a donor animal (n=17). Syngeneic recipient (n=17) and sham-operated (n=5) rats were sedated and ventilated. In the recipient group, the PAG was implanted with an end-to-end anastomosis in infra-renal abdominal aortic position. Sham-operated rats underwent only transection and re-anastomosis of the aorta. Animals were followed with serial ultrasound studies for two months and post-mortem histological analysis. The median PAG diameter in the native position was 3.20 mm (IQR=3.18-3.23). At follow-up, the median diameter of the PAG was 4.03 mm (IQR=3.74-4.13) at 1 week, 4.07 mm (IQR=3.80-4.28) at 1 month, and 4.27 mm (IQR=3.90-4.35) at 2 months (p&lt;0.01). Peak systolic velocity was 220.07 mm/s (IQR=210.43-246.41) at 1 week, 430.88 mm/s (IQR=375.28-495.56) at 1 month, and 373.68 mm/s (IQR=305.78-429.81) at 2 months (p=0.02) and did not differ from the sham-operated group at the end of the experiment (p=0.5). Histological analysis did not show any sign of endothelial thrombosis. This study showed that rodent models may allow for the evaluation of the long-term adaptation of the pulmonary root to a high-pressure system. A systemically placed syngeneic PAG implantation represents a simple and feasible platform for the development and evaluation of novel surgical techniques and drug therapies to further improve the outcomes of the Ross operation.

We demonstrate how to establish a murine model of pulmonary root implantation into the descending aorta to simulate the Ross procedure. This model enables the medium/long-term evaluation of pulmonary autograft remodeling in a systemic position, representing the basis of developing therapeutic strategies to promote its adaptation.

The Ross operation for aortic valve disease has regained new interest due to its outstanding long-term results. Nonetheless, when employed as freestanding ...