This work was supported by the National Natural Science Foundation of China (No.82074523,82104996).

All animal experiments were reviewed and approved by the Animal Ethics Committee of Beijing University of Chinese Medicine (No. BUCM-4-2022010101-1097). In this protocol, 24 6-week-old male New Zealand rabbits were housed under a specific condition, namely, 20-25 &#176;C, 50-60% humidity, and a 12 h light/12 h dark circadian cycle, with free access to a regular chow diet. The rabbits were anesthetized and sacrificed by combining deep anesthesia and air embolization. Pain is one of the typical pathological features of KOA and is also one of the key indicators used to evaluate animal models of KOA and intervention methods, so analgesics are not used during model preparation.
1. The KOA rabbit model
<ol>
	<li>Anesthetize rabbits with 3% pentobarbital sodium (30 mg/kg) intravenously at the ear margin. To confirm the appropriate level of anesthesia, look for a significantly weakened or absent corneal reflex and the absence of pain upon clamping the skin with hemostatic forceps. During anesthesia, add 2-3 drops of lubricant to the rabbits&#39; eyes every 15 min to prevent the rabbits&#39; eyes from drying out.</li>
	<li>After anesthesia, fix each rabbit in the supine position, pulling the left hindlimb into a fully extended position.</li>
	<li>Fix the left hind limb of each rabbit in the extended position.
	<ol>
		<li>As the first layer, use medical tape to cover the rabbit&#39;s skin from the groin to the ankle joint.</li>
		<li>As the second layer, wrap 36 mm-wide, double-sided foam tape over the medical tape and then wrap a polymer bandage from the groin to the ankle joint. Ensure that the knee joint is 180&#176; straight and the ankle joint is dorsiflexed by 60&#176;.</li>
		<li>As the third layer, immobilize the joints with small splints at the front and back of the knee and ankle joints, and wrap steel mesh around the outermost layer to protect against bites. Expose the rabbits&#39; toes to observe if the blood circulation is normal.</li>
	</ol>
	</li>
	<li>Immobilize the animals for 6 weeks to establish the KOA model (Figure 1). 
	NOTE: 1) During model preparation, inspect the molds every other day. If any molds are loose or detached, anesthetize the rabbits, and re-immobilize the left hind limbs in an extended position. 2) Lay protective mats on the bottom of the cages to prevent the rabbits&#39; limbs from getting stuck and causing injury.</li>
</ol>
2. Acupotomy intervention
NOTE: Before the start of acupotomy intervention, anesthetize the rabbits with 3% pentobarbital sodium (30 mg/kg) by ear margin intravenous injection.
<ol>
	<li>Determine the points of treatment.
	<ol>
		<li>Shave off the fur of the knee joint of the rabbit&#39;s left hind limb.</li>
		<li>Palpate the rabbit knee joint medial femoral muscle tendon insertion, rectus femoral tendon insertion, biceps femoris tendon insertion, and goosefoot bursa. Mark pathological indurations of local muscles with a sterile skin marker. Disinfect the knee joint three times with alternating rounds of medical iodophor and 75% medical alcohol.</li>
	</ol>
	</li>
	<li>Operation of acupotomy
	<ol>
		<li>Keep the acupotomy blade parallel to the direction of travel parallel to the tendon and the longitudinal axis of the limb.</li>
		<li>Use the thumb of the left hand to press down on the skin entering the marker point and move laterally so that blood vessels and nerves are separated on the ventral side of the thumb.</li>
		<li>With the acupotomy handle in the right hand, press down quickly with a small force so that the acupotomy blade instantly passes through the skin. Slowly advance the acupotomy blade to the local muscle indurations and make longitudinal cuts and swings laterally.</li>
		<li>After the acupotomy operation is complete, disinfect the knee joint again and apply a band-aid.</li>
	</ol>
	</li>
	<li>Perform this operation once a week for 4 weeks (Figure 2). 
	&#8203;NOTE: 1) If there is no induration or cord-like tissue touched at the tendon insertion of the vastus medialis, vastus lateralis, rectus femoris, biceps femoris, or the anserine bursa, the acupotomy needle should be used to release their tendon insertions directly. 2) During acupotomy intervention, do not immobilize the left hind limbs of rabbits in the acupotomy group and the model group in extension position.</li>
</ol>
3. Elastic modulus of quadriceps femoris
NOTE: 1) This experiment used the real-time shear wave&#160;elastography (SWE) ultrasound diagnostic instrument to&#160;measure the elastic modulus of the quadriceps femoris in vivo&#160;in each group of rabbits. 2)&#160;The tester should be an experienced&#160;sonographer in ultrasound detection. When&#160;measuring, the ultrasound probe should be gently&#160;placed on the skin surface of the quadriceps to&#160;avoid local muscle tension. Measurements need to&#160;be taken when the animal is in a quiet state, without&#160;struggling or activity. If the animal is active, wait until&#160;it is calm before conducting the test.
<ol>
	<li>Shave the fur to expose the skin in the quadriceps region of the left hindlimb.</li>
	<li>Use conventional two-dimensional ultrasound to locate the quadriceps musculo-abdomen and determine the region of interest (ROI), set to a depth of 1-2 cm.</li>
	<li>Start the SWE mode for inspection.
	<ol>
		<li>Set the area of interest uniformly to a circular area with a diameter of 2 mm and the area of interest to ~0.5-1 cm deep from the surface of the skin.</li>
		<li>Use the ultrasonic diagnostic instrument to generate an acoustic radiation force impulse to stimulate the muscle tissue and obtain tissue elastography.</li>
		<li>Wait for 2-3 s for the image to stabilize and then freeze the image. Activate the Q-BOX function of the instrument to measure the Young&#39;s modulus of the quadriceps muscle.</li>
		<li>Wait for the system to automatically calculate the maximum, minimum, and average values (unit: KPa) of Young&#39;s modulus of the ROI. Select three ROIs at the same depth for three measurements and take the average value for statistical analysis. 
		NOTE: The tester should be an experienced sonographer in ultrasound detection. When measuring, the ultrasound probe should be gently placed on the skin surface of the quadriceps to avoid local muscle tension. Measurements need to be taken when the animal is in a quiet state, without struggling or activity. If the animal is active, wait until it is calm before conducting the test.</li>
	</ol>
	</li>
</ol>
4. Measuring the contraction force of quadriceps femoris
NOTE: After the measurement of the contraction force of the quadriceps femoris, the rabbits were euthanized by air embolism under anesthesia.
<ol>
	<li>Anesthetize the rabbits with 3% pentobarbital sodium (30 mg/kg) intravenously at the ear margin. To confirm that the appropriate level of anesthesia has been reached, look for a significantly weakened or absent corneal reflex and the absence of pain upon clamping the skin with hemostatic forceps. During anesthesia, add 2-3 drops of lubricant to the rabbits&#39; eyes every 15 min to prevent the rabbits&#39; eyes from drying out.</li>
	<li>Expose the quadriceps muscles and attach the tension transducer.
	<ol>
		<li>Cut the skin below the patella, along the longitudinal axis of the limb upwards to the base of the thigh, and continue to cut the skin upwards by 3-4 cm. Carefully peel off the skin and fascia, and expose the muscle. Cut the patellar ligament and carefully separate the quadriceps from the iliac junction, keeping the quadriceps in connection with the iliacium.</li>
		<li>Ligate the surgical sutures at the tendon junction between the patella and the quadriceps muscle. Stretch the muscle to its full length in its natural state and then attach it to the tension transducer. Keep the ligation line on the muscle in a straight line with the ligation line on the force transducer.</li>
		<li>Secure the tension transducer to the bracket. Connect the signal acquisition line on the tension transducer to the biosignal acquisition system processor.</li>
	</ol>
	</li>
	<li>Measure the contractile performance of the quadriceps muscle.
	<ol>
		<li>Insert the electrodes parallel to the quadriceps abdomen and avoid any contact between the electrodes.</li>
		<li>Press the oscilloscope button. Adjust the position of the force transducer on the bracket to maintain the baseline at&#160;zero. Select the stimulation parameters of the stimulator with a wave width of 5 ms and a delay of 10 ms.</li>
		<li>Use a single stimulus first and gradually adjust the stimulus intensity from zero with an increment of 0.1 V each time. Observe the changes in the muscle contraction curve and contraction amplitude until the maximum single contraction amplitude (Pt) of the quadriceps is determined. Record it for subsequent statistics.</li>
		<li>Use a cluster stimulus, and use the stimulus amplitude that induces the maximum single contraction amplitude as the baseline to continuously stimulate the muscle and gradually increase the stimulus frequency. Observe the changes in the muscle contraction curve until the maximum contraction amplitude (Pt) of the quadriceps is determined. Record it for subsequent statistics. 
		&#8203;NOTE: 1) After each muscle contraction, the muscle should be given 30 s to relax with the muscle buffer solution being dripped continuously on the muscle. 2) During the operation, judge the anesthesia status by monitoring the rabbits&#39; eyelid reflex, respiratory rhythm, muscle relaxation and skin pinching response.</li>
	</ol>
	</li>
</ol>
5. The mechanical performance of the quadriceps tendon
<ol>
	<li>Preprocessing: On the day of testing, measure the length, width, and thickness of the quadriceps tendon with a vernier caliper, and install a special anti-slip clamp in the fatigue testing machine. Repeat loading and unloading 15x for preprocessing.</li>
	<li>Stress relaxation test: Use the sensor ranging from 0 N to 100 N, stretch it at a speed of 5 mm/min until it reaches the required length, and then start collecting data. Set the computer data acquisition time from t (0), collecting data every 0.1 s, lasting for 1,800 s. After reaching the set time, record data and curves.</li>
	<li>Tensile test: Use the sensor ranging from 0 N to 100 N and stretch it at a speed of 5 mm/min to the maximum load until the specimen is pulled apart. After the test, calculate the maximum displacement, ultimate load, and stiffness of the specimen.</li>
</ol>
6. Joint contact surface pressure and pressure per unit area of the cartilage
<ol>
	<li>Fix the femur and tibia specimens on both sides in a straight position on the fixture and perform a preload test. Measure the approximate range of the knee joint, cut the pressure-sensitive paper into the same shape, and seal it with plastic wrap.</li>
	<li>Place the sealed pressure-sensitive paper between the tibia and femur joints, and conduct a pressure test on the knee joint with a pressure of 5 mm/min and a maximum pressure of 50 N. Maintain the pressure for 2 min until it reaches 50 N when the pressure-sensitive paper is stably colored.</li>
	<li>After 2 min, remove the pressure-sensitive paper, fix the colored surface on an A4 size sheet of paper, and acquire images with the scale set aside.</li>
	<li>Upload the image to the computer. Use the referenced software for area measurement and multi-segment measurement for irregular figures. Measure the pressure on the inside and outside of the joints of the femur and tibia and record the results.</li>
</ol>
7. Safranin O/Fast Green staining of the knee joint cartilage
<ol>
	<li>After the end of the acupotomy intervention, take the cartilage-subchondral bone complex tissues and embed them in paraffin. Slice the prepared tissue wax blocks and prepare slides. Deparaffinize the prepared tissue slides with environmental dewaxing solution(I) and environmental dewaxing solution (II) for 15 min each; then, dip them successively in xylene and anhydrous ethanol (1:1), anhydrous ethanol (I), 95% ethanol, 85% ethanol, and 75% ethanol, 2-5 min each step; and finally, soak them in distilled water for 15 min.</li>
	<li>Perform staining.
	<ol>
		<li>Stain the slides with Fast Green solution for 1&#160;min. During this process, take the slides out of the solution and observe them under the microscope until the tissue is stained dark green.</li>
		<li>Color separation: Rinse the excess Fast Green solution with ultrapure water. Soak the slides quickly in 1% acetic acid solution for 5 - 10 seconds.&#160; Again, rinse the slide with ultrapure water.</li>
		<li>Stain the slides in Safranine O solution for 10-15 min until the cartilage is stained red.</li>
	</ol>
	</li>
	<li>Dehydrate and clarify the tissue, seal the glass slides, and observe them under the microscope.
	<ol>
		<li>Soak the slides in 75% ethanol, 85% ethanol, 95% ethanol, and 100% ethanol for 3 - 5 seconds successively.</li>
		<li>Dip the slides in environmental dewaxing solution(I) and environmental dewaxing solution (II) for 10 min successively. Take out the slides and drop the neutral resinous medium on the front of the slides, avoiding the tissue. Place the edge of the coverglass on the slide and then, slowly put it down to cover the neutral balsam. Remove the air and avoid air bubbles. Wipe off the extra xylene and neutral balsam, and let it stand overnight at room temperature.</li>
		<li>Observe the slides under the microscope and acquire images. For each group, select six samples of rabbit knee cartilage and randomly select four different viewing fields for each sample for evaluation. Score the cartilage histology of each group according to the Mankin method (Table 1).</li>
	</ol>
	</li>
</ol>
8. Statistical analysis
<ol>
	<li>Express data as mean &#177; standard deviation (<img alt="Equation 1" src="/files/ftp_upload/65584/65584eq01.jpg" style="margin: auto;" /> &#177; s).</li>
	<li>Perform one-way analysis of variance (ANOVA) and LSD&#39;s test for determining the statistical significance of multiple group comparisons.</li>
	<li>Consider differences statistically significant when P &#60; 0.05.</li>
</ol>

Establishment of the KOA Model in Rabbits Using the Modified Videman Method

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The authors have no conflicts of interest to disclose.

An appropriate animal model is one of the key factors to achieve experimental objectives and clarify a specific scientific question. This study was based on the theories of &#34;Zongjin controlling bones and lubricating joints&#34; and &#34;mechanical imbalance&#34; in acupotomology, aiming to explain the scientific connotation behind the treatment of KOA by &#34;modulating muscles and tendons to treat bone disorders&#34; in acupotomy therapy. In other words, acupotomy improves the abnormal mechanical environment of the ...

Knee osteoarthritis (KOA) is the most frequent form of osteoarthritis, often recognized as a whole-joint disease characterized by articular cartilage degeneration, which manifests clinically as pain, swelling, and limited movement of the affected joints1. According to recent epidemiological statistics, KOA is reported to have affected 654.1 million individuals globally who were 40 years of age or older by 2020. The prevalence and incidence of KOA rise with age, are the highest in middle-aged and older adults, and affect more women than men2. The prevalence of KOA is likely to increase due to the aging population and obesity epidemic worldwide, posing a growing threat to global public health. Age, sex, obesity, trauma, and other complicated risk factors associated with KOA all directly impact knee instability, making a biomechanical imbalance in knee joints one of the primary causes of KOA3.
Under normal physiological conditions, the knee joint is in a state of mechanical balance, ensuring that the mechanical loads in the joint are evenly distributed on the cartilage. Any mechanical imbalance in the knee joint can lead to abnormal stress in cartilage, resulting in cartilage degeneration and the onset of KOA4. The muscle-tendon system is the main dynamic system that maintains the mechanical balance of the knee joint. The coordinated movement of the extensor and flexor muscle-tendon system can evenly distribute the load generated by the movement on the cartilage surface, avoiding the metabolic imbalance of local cartilage stresses beyond its physiological load that results in cartilage loss5. Decreased muscle strength is the main cause of intramuscular movement disorder and cartilage damage, which may occur before symptomatic KOA. 
KOA can also induce arthrogenous muscle inhibition (AMI), manifesting as muscle weakness and decreased muscle strength around the knee6. Among these muscles, the quadriceps femoris group functions as the only knee extensor, an important structure in maintaining knee joint stability. Studies have shown that a decrease in quadriceps cross-sectional area and muscle strength is significantly and positively correlated with KOA progression7. The decline in quadriceps strength affects the gait pattern, knee stability, movement patterns, and many other functions. Moreover, the decline in muscle strength impairs tendon function, manifested as a decrease in tendon stiffness, elastic modulus, and other biomechanical properties8. In long-term strain repair, changes such as adhesion and contracture may occur in the muscles and tendons of the knee joint, damaging their mechanical properties, causing joint instability, and ultimately forming a vicious cycle of pathological changes of KOA. It is, therefore, crucial for KOA treatment to improve the mechanical properties of the muscle-tendon system and restore the joint mechanical balance.
Among the causes of KOA, biomechanical imbalance is the main inducing factor for knee pain, dysfunction, inflammatory lesions, and cartilage degeneration9. Therefore, the key to treating KOA is to restore the biomechanical balance of the knee joint. Acupotomology believes that the etiology and pathogenesis of KOA are "mechanical imbalance." When the mechanical characteristics of the soft tissues around the knee change abnormally, the knee joint loses its mechanical balance, and the abnormal mechanical stress environment of the joint accelerates degeneration, causing inflammatory stimulation to further aggravate the soft tissue adhesions, contractures, and further decline in joint stability. This vicious cycle eventually develops into KOA. By loosening soft tissue adhesions and contractures, as well as reducing stress concentration in the muscles and tendons, acupotomy in conjunction with the theory of "Modulating Muscles and Tendons to Treat Bone Disorders" improves the soft tissue mechanics and "modulates muscles and tendons," which balances the mechanical stress of the joint, effectively alleviating cartilage degeneration and "treating bone disorders"10. In terms of animal model selection, based on the purpose of this study, we prepared the KOA model by the modified Videman method of left hindlimb extension immobilization.
This paper details the establishment of the KOA model using the modified Videman method of left hind limb extension immobilization and the method of operation and precautions of acupotomy. We demonstrate the effectiveness of acupotomy by testing the mechanical properties of quadriceps femoris and tendon and detecting changes in articular cartilage stress and morphology.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acupotomy</td>
			<td>Beijing Zhuoyue Huayou Medical Devices Co., Ltd.</td>
			<td></td>
			<td>0.4 x 40 mm</td>
		</tr>
		<tr>
			<td>Connect Cast Orthopedic Casting Tape</td>
			<td>Suzhou Connect Medical Technology Co.,Ltd.</td>
			<td>KCP06</td>
			<td>15.0 cm x 360 cm</td>
		</tr>
		<tr>
			<td>Double-sided Foam Tape</td>
			<td>Deli Group Co.,Ltd.</td>
			<td>NO.30416</td>
			<td>36 mm x 5 yard x 2.5 mm</td>
		</tr>
		<tr>
			<td>Environmental Dewaxing Solution</td>
			<td>Wuhan Servicebio Technology Co.,Ltd.</td>
			<td>G1128</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>Beijing Hengkangda Medicine Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fast Green solution</td>
			<td>Wuhan Servicebio Technology Co.,Ltd.</td>
			<td>G1031</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast grenn FCF</td>
			<td>Sigma,America</td>
			<td>2353-45-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Fatigue testing machine</td>
			<td>BOSE, America</td>
			<td></td>
			<td>Bose Electro Force 3300</td>
		</tr>
		<tr>
			<td>Four-channel physiological recorder</td>
			<td>Chengdu Instrumeny Frctory</td>
			<td></td>
			<td>RM-6420</td>
		</tr>
		<tr>
			<td>FPD-305E</td>
			<td>Fuji, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FPD-306E</td>
			<td>Fuji, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin solution</td>
			<td>Wuhan Servicebio Technology Co.,Ltd.</td>
			<td>G1005</td>
			<td></td>
		</tr>
		<tr>
			<td>Medical iodophor disinfectant</td>
			<td>Shan Dong Lircon Medical Technology Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Medical Tape</td>
			<td>Shandong Rongjian Sanitary Products Co., Ltd.</td>
			<td>200402</td>
			<td>1.5 x 500 cm</td>
		</tr>
		<tr>
			<td>Muscle tension transducer&#160;</td>
			<td>Chengdu Instrumeny Frctory</td>
			<td></td>
			<td>JH-2204005, 50 g</td>
		</tr>
		<tr>
			<td>Prescale</td>
			<td>Fuji, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Real-time SWE ultrasound diagnostic instrument</td>
			<td>SuperSonic Imagine SA,France</td>
			<td></td>
			<td>SuperSonic Imagine AixPlorer</td>
		</tr>
		<tr>
			<td>Rhamsan gum</td>
			<td>Wuhan Servicebio Technology Co.,Ltd.</td>
			<td>WG10004160</td>
			<td></td>
		</tr>
		<tr>
			<td>Safranine O</td>
			<td>Sigma,America</td>
			<td>477-73-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Safranine O solution</td>
			<td>Wuhan Servicebio Technology Co.,Ltd.</td>
			<td>G1015</td>
			<td></td>
		</tr>
		<tr>
			<td>Statistical Package for the Social Sciences (SPSS)</td>
			<td>IBM, America</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

application of acupotomy in a knee osteoarthritis model in rabbit

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department, which seriously reduces the quality of life of people with KOA. Among several pathogenic factors, the biomechanical imbalance of the knee joint is one of the main causes of KOA. Acupotomology believes that restoring the mechanical balance of the knee joint is the key to treating KOA. Clinical studies have shown that acupotomy can effectively reduce pain and improve knee mobility by reducing adhesion, contracture of soft tissues, and stress concentration points in muscles and tendons around the knee joint. 
In this protocol, we used the modified Videman method to establish a KOA model by immobilizing the left hindlimb in a straight position. We have outlined the method of operation and the precautions related to acupotomy in detail and evaluated the efficacy of acupotomy in conjunction with the theory of "Modulating Muscles and Tendons to Treat Bone Disorders" through the detection of the mechanical properties of quadriceps femoris and tendon, as well as cartilage mechanics and morphology. The results show that acupotomy has a protective effect on cartilage by adjusting the mechanical properties of the soft tissues around the knee joint, improving the cartilage stress environment, and delaying cartilage degeneration.

Experimental results of mechanical properties of quadriceps femoris and tendon 
To evaluate the effect of acupotomology on the mechanical properties of quadriceps femoris in rabbits with KOA, we used real-time shear wave elastic ultrasound imaging and a muscle tension transducer, respectively. Compared with the control group, Young&#39;s modulus of the quadriceps femoris in the KOA group was decreased (P &#60; 0.05). Compared with the KOA group, Young&#39;s modulus of the acupotomy group wa...

Watch this Scientific Journal Video about Author Spotlight: Investigating the Mechanism of Action of Acupotomy in Treating Knee Osteoarthritis at JoVE.com

Author Spotlight: Investigating the Mechanism of Action of Acupotomy in Treating Knee Osteoarthritis

In this protocol, a knee osteoarthritis model was prepared using the modified Videman method, and the operation procedures and precautions of acupotomy are detailed. The effectiveness of acupotomy has been demonstrated by testing the mechanical properties of quadriceps femoris and tendon and the mechanical and morphological properties of cartilage.

Application of Acupotomy in a Knee Osteoarthritis Model in Rabbit

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department, which seriously reduces the quality of life of ...

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954 Views.  Beijing University of Chinese Medicine. In this protocol, a knee osteoarthritis model was prepared using the modified Videman method, and the operation procedures and precautions of acupotomy are detailed. The effectiveness of acupotomy has been demonstrated by testing the mechanical properties of quadriceps femoris and tendon and the mechanical and morphological properties of cartilage.

Video: Author Spotlight: Investigating the Mechanism of Action of Acupotomy in Treating Knee Osteoarthritis

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

The Monoiodoacetate Model of Osteoarthritis Pain in the Mouse

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The current treatments for OA pain such as NSAIDS or opiates are neither sufficiently effective nor devoid of detrimental side effects. Animal models of OA are being developed to improve our understanding of OA-related pain mechanisms and define novel pharmacological targets for therapy. Currently available models of OA in rodents include surgical and chemical interventions into one knee joint. The monoiodoacetate (MIA) model has become a standard for modelling joint disruption in OA in both rats and mice. The model, which is easier to perform in the rat, involves injection of MIA into a knee joint that induces rapid pain-like responses in the ipsilateral limb, the level of which can be controlled by injection of different doses. Intra-articular injection of MIA disrupts chondrocyte glycolysis by inhibiting glyceraldehyde-3-phosphatase dehydrogenase and results in chondrocyte death, neovascularization, subchondral bone necrosis and collapse, as well as inflammation. The morphological changes of the articular cartilage and bone disruption are reflective of some aspects of patient pathology. Along with joint damage, MIA injection induces referred mechanical sensitivity in the ipsilateral hind paw and weight bearing deficits that are measurable and quantifiable. These behavioral changes resemble some of the symptoms reported by the patient population, thereby validating the MIA injection in the knee as a useful and relevant pre-clinical model of OA pain.
The aim of this article is to describe the methodology of intra-articular injections of MIA and the behavioral recordings of the associated development of hypersensitivity with a mind to highlight the necessary steps to give consistent and reliable recordings.

Osteoarthritis (OA), or degenerative joint disease, is a debilitating condition associated with pain that remains only partially controlled by available analgesics. Animal models are being developed to improve our understanding of OA-related pain mechanisms. Here we describe the methodology for the monoiodoacetate model of OA pain in the mouse.

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The ...

Preparation, Procedures and Evaluation of Platelet-Rich Plasma Injection in the Treatment of Knee Osteoarthritis

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department. Existing non-surgical treatments have a limited effect on the repair of cartilage and on bone regeneration. Platelet-rich plasma (PRP) is an autologous bioactive substance that can repair cartilage injury and accelerate bone regeneration effectively. However, reporting of PRP preparation protocols in clinical studies is highly inconsistent, with the majority of studies providing insufficient information to allow the protocol to be reproduced. We describe a repeatable method of preparing PRP visually, the treatment of KOA using PRP intra-articular injection, and methods of evaluating the outcome. PRP was prepared using manual double centrifugation. The PRP layer was extracted from peripheral blood and used for knee joint cavity injection. Evaluations included assessments of blood platelet concentrations and clinical outcomes. Preparation of PRP by manual centrifugation requires less apparatus and is less costly than plasma filtration or centrifugation using equipment. The centrifugation time of our double centrifugation method was 6 and 5 minutes for the respective centrifugations at forces of 800 and 1400 x g, respectively, to allow for the consistent preparation of standardized PRP. However, a manual method is susceptible to operator error, and PRP batch preparation is not available. Intra-articular injection of PRP proved to be an effective treatment for knee osteoarthritis. The entire treatment procedure took less than 30 minutes, the blood platelet concentration of PRP could be standardized, and treatment was proven to be effective when evaluated by follow-up.

Knee osteoarthritis is frequently seen in the orthopedic department. We introduce in detail the entire knee osteoarthritis treatment process with platelet-rich plasma injection, including preparation, procedures, and evaluation.

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department. Existing non-surgical treatments have a limited ...

Transthoracic Echocardiographic Examination in the Rabbit Model

Large animal models such as the rabbit are valuable for translational preclinical research. Rabbits have a similar cardiac electrophysiology compared to that of humans and that of other large animal models such as dogs and pigs. However, the rabbit model has the additional advantage of lower maintenance costs compared to other large animal models. The longitudinal evaluation of cardiac function using echocardiography, when appropriately implemented, is a useful methodology for preclinical assessment of novel therapies for heart failure with reduced ejection fraction (e.g. cardiac regeneration). The correct use of this non-invasive tool requires the implementation of a standardized examination protocol following international guidelines. Here we describe, step by step, a detailed protocol supervised by veterinary cardiologists for performing echocardiography in the rabbit model, and demonstrate how to correctly obtain the different echocardiographic views and imaging planes, as well as the different imaging modes available in a clinical echocardiography system routinely used in human and veterinary patients.

Here we describe, step by step, a detailed protocol for performing echocardiography in the rabbit model. We show how to correctly obtain the different echocardiographic views and imaging planes, as well as the different imaging modes available in a clinical echocardiography system routinely used in human and veterinary patients.

Large animal models such as the rabbit are valuable for translational preclinical research. Rabbits have a similar cardiac electrophysiology compared to ...

The Lower Body Positive Pressure Treadmill for Knee Osteoarthritis Rehabilitation

Here, based on a clinician&rsquo;s point-of-view, we propose a two-model lower body positive pressure (LBPP) protocol (walking and squatting models) in addition to a clinical, functional assessment methodology, including details for further encouragement of the development of non-drug surgical intervention strategies in knee osteoarthritis patients. However, we only present the effect of LBPP training in improvement of pain and knee function in one patient through three-dimensional gait analysis. The exact, long-term effects of this approach should be explored in future studies.

Here, based on a clinician&#8217;s point-of-view, we propose a two-model lower body positive pressure (LBPP) protocol (walking and squatting models) in addition to a clinical, functional assessment methodology, including details for further encouragement of the development of non-drug surgical intervention strategies in knee osteoarthritis patients. However, we only present the effect of LBPP training in improvement of pain and knee function in one patient through three-dimensional gait analysis. The exact, long-term effects of this approach should be explored in future studies.

Here, based on a clinician&rsquo;s point-of-view, we propose a two-model lower body positive pressure (LBPP) protocol (walking and squatting models) in ...

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

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

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

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

Standardized Histomorphometric Evaluation of Osteoarthritis in a Surgical Mouse Model

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, primarily in the hip and knee joints, which results in significant impacts on patient mobility and quality of life. To date, there are no existing curative therapies for OA able to slow down or inhibit cartilage degeneration. Presently, there is an extensive body of ongoing research to understand OA pathology and discover novel therapeutic approaches or agents that can efficiently slow down, stop, or even reverse OA. Thus, it is crucial to have a quantitative and reproducible approach to accurately evaluate OA-associated pathological changes in the joint cartilage, synovium, and subchondral bone. Currently, OA severity and progression are primarily assessed using the Osteoarthritis Research Society International (OARSI) or Mankin scoring systems. In spite of the importance of these scoring systems, they are semiquantitative and can be influenced by user subjectivity. More importantly, they fail to accurately evaluate subtle, yet important, changes in the cartilage during the early disease states or early treatment phases. The protocol we describe here uses a computerized and semiautomated histomorphometric software system to establish a standardized, rigorous, and reproducible quantitative methodology for the evaluation of joint changes in OA. This protocol presents a powerful addition to the existing systems and allows for more efficient detection of pathological changes in the joint.

The current protocol establishes a rigorous and reproducible method for quantification of morphological joint changes that accompany osteoarthritis. Application of this protocol can be valuable in monitoring disease progression and evaluating therapeutic interventions in osteoarthritis.

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, ...

Application of Lucilia sericata Larvae in Debridement of Pressure Wounds in Outpatient Settings

Biological therapy using Lucilia sericata larvae has numerous advocates worldwide, yet it is still fairly unknown and not commonly applied in daily practice because of the limited awareness and insufficient experience of medical and nursing personnel. There are case reports suggesting that maggot therapy can be applied and supported by lay caregivers, provided they are supervised and informed by physicians/nurses. The foregoing observation suggests that the method should be considered for implementation by a wider group of caregivers if accepted and meticulously supervised by trained and experienced medical staff. The concerns related to the therapeutic use of maggots in certain regions seem understandable, but are not supported by scientific facts. It should be noted that many therapeutic agents (including brood) used in medicine are of natural origin, and are associated with low production costs and high possibilities of implementation in the course of therapy. By analyzing the literature and using our own clinical and research experience, we have come to conclusions related to using larvae therapy, as a quick and safe method providing cleaning and revitalization in the process of treating wounds of various etiologies, especially pressure ulcers. In the current study, medical-grade Lucilia sericata maggots were applied to remove necrotic tissue from deep pressure sores. The treatment is mostly accepted by both caregivers and patients. In most cases, it is conducted by trained and experienced medical personnel in home and outpatient settings. Over the course of the conducted analyzes involving the collected specimens, there were no statistically significant relationships (p&gt; 0.05) confirmed between the wound surface successfully cleared by brood and variables, such as time from wound formation, location, surface size, and the depth of damage to the tissue structure. The lack of statistical dependence may result from the small size of the studied group. Based on the current findings, we have formulated the following conclusions: Maggot Debridement Therapy (MDT) is a fast and effective method enabling the preparation of the wound bed. The use of MDT in outpatient settings is safe and acceptable for patients and their caregivers.

Application of Lucilia sericata Larvae in Debridement of Pressure Wounds in Outpatient Settings

Maggots of Lucilia sericata were used in the debridement of deep pressure sores in outpatient settings for the treatment of pressure sores involving the complete skin thickness. The technique presented in the article does not pose risks for the patient.

Biological therapy using Lucilia sericata larvae has numerous advocates worldwide, yet it is still fairly unknown and not commonly applied in daily ...

FSN Therapy for Knee Osteoarthritis Pain Management

Fu's Subcutaneous Needling for Knee Osteoarthritis Pain

Fu's subcutaneous needling (FSN) is a new acupuncture and dry needling technique based on traditional Chinese medicine. It rapidly produces long-lasting effects in soft tissue injuries, particularly in painful musculoskeletal conditions, by providing stimulation primarily in the subcutaneous area. Osteoarthritis (OA) is the most common joint disease in adults worldwide and is often accompanied by a painful syndrome of structural changes in the peripheral joints of the knee. However, the etiology of OA pain is not fully understood, though myofascial trigger points (MTrPs) are commonly found in the lower limb muscles (so-called "tightened muscles") of patients with knee OA. 
FSN has been used in many fields for the treatment of acute pain problems and can relieve muscle contraction from MTrPs, thereby improving the local circulation. This study recruited patients with pain from knee OA into an FSN group or a transcutaneous electrical nerve stimulation (TENS) group with three treatment sessions and a follow-up over the course of 2 weeks. The results showed that FSN was effective in treating soft tissue pain around the knee with OA. This study aimed to establish and visualize three key technical indicators during FSN therapy, including the FSN needle insertion point and layer; the frequency and duration of the swaying movement; and the manipulation of the reperfusion approach. These findings have great potential for future applications in myofascial pain treatment, especially for pain management. Following this protocol could enhance FSN skills.

Author Spotlight: Fu's Subcutaneous Needling for Knee Osteoarthritis Pain  

We present a protocol for using Fu's subcutaneous needling for knee osteoarthritis pain, which combines swaying movement and reperfusion approach techniques. This protocol has great potential for future applications in myofascial pain treatment and could enhance Fu's subcutaneous needling (FSN) manipulation skills.

Fu's subcutaneous needling (FSN) is a new acupuncture and dry needling technique based on traditional Chinese medicine. It rapidly produces long-lasting ...

In Vitro Application of a Wireless Sensor in Flexion-Extension Gap Balance of Unicompartmental Knee Arthroplasty

Unicompartmental knee arthroplasty (UKA) is an effective treatment for end-stage anteromedial osteoarthritis (AMOA). The key to UKA is the flexion-extension gap balance, which is closely related to postoperative complications such as bearing dislocation, bearing wear, and arthritis progression. The traditional gap balance assessment is performed by indirectly sensing the tension of the medial collateral ligament by a gap gauge. It relies on the surgeon's feel and experience, which is imprecise and difficult for beginners. To accurately assess the flexion-extension gap balance of UKA, we developed a wireless sensor combination consisting of a metal base, a pressure sensor, and a cushion block. After osteotomy, the insertion of a wireless sensor combination allows the real-time measurement of intra-articular pressure. It accurately quantifies the flexion-extension gap balance parameters to guide further femur grinding and tibia osteotomy, to improve the accuracy of gap balance. We conducted an in vitro experiment with the wireless sensor combination. the results showed that there was a difference of 11.3 N after applying the traditional method of flexion-extension gap balance performed by an experienced expert.

In Vitro Application of a Wireless Sensor in Flexion-Extension Gap Balance of Unicompartmental Knee Arthroplasty

This protocol presents a cadaveric study of a wireless sensor used in medial unicompartmental knee arthroplasty. The protocol includes the installation of an angle measuring device, standardized Oxford unicompartmental knee arthroplasty osteotomy, preliminary assessment of flexion-extension balance, and application of the sensor to measure flexion-extension gap pressure.