This work was supported by the 2020 Science and Technology Development Plan in Jinan City (Grant Number 202019059), the Traditional Chinese Medicine Science and Technology Project of Shandong Province (Grant Number 2021Q080), and the Qilu School of Traditional Chinese Medicine Inherit Project (Grant Number [2022]93).

This study was approved by the Ethics Committee of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine (Number: AWE-2022-023).
1. Experimental animals
<ol>
	<li>Twenty male Sprague-Dawley (SD) rats (7 weeks old, 250-280 g) were housed&#160;in standard conditions (room temperature [RT] 20-24 &#176;C, humidity 40%-60 %, and a 12 h/12 h light/dark cycle).</li>
</ol>
2. Grouping method
<ol>
	<li>Group the SD rats into control group (C), FS model control group (M), FS model Tuina treatment group (MT), and FS model oral treatment group (MO), each consisting of 5 rats. Keep 5 rats per cage (same group).</li>
	<li>After 7 days of acclimation, immobilize one shoulder of the rats in the M, MT, and MO groups using plaster cast immobilization for 3 weeks to mimic FS as described in the next section.</li>
	<li>Administer Tuina therapy to the rats in the MT group for 2 weeks, as described in section 4 (Figure 1).</li>
	<li>Calculate the required dosage of dexamethasone for each kilogram of rats (0.0675 mg/day) based on the adult dosage (0.75 mg/day) and the ratio of rat to human body surface area (0.018).</li>
	<li>Administer daily intragastric dexamethasone solution to rats in the MO group at 0.067 mg/ kg/day at 7:00 AM for 2 weeks. 
	&#8203;NOTE: Use this grouping method to confirm the effect of the Tuina protocol in this study. Perform the grouping method according to experimental purposes in different studies.</li>
</ol>
3. Development of the FS model
<ol>
	<li>Anesthetize rats using tribromoethanol (250 mg/kg, by intraperitoneal injection)11. 
	NOTE: In accordance with the requirements of the institution&#39;s ethics committee, a stock solution composed of&#160;tribomoethanol (10 g) and&#160;tert-amyl alcohol (10 mL)&#160;was stored at 4&#160;&#176;C.&#160;Before use, it&#160;was diluted to 2% with distilled water.</li>
	<li>Apply plaster-soaked bandages to the right shoulder and chest of the rats, keeping the right forelimb at 90&#176; of internal rotation of the shoulder joint for 3 weeks (Figure 2)12. 
	NOTE: Monitor rats to ensure they can perform normal physiological activities such as walking, eating, and drinking. Refix the plaster bandage if the rats can not perform normal physiological activities.</li>
	<li>Confirm the successful establishment of the FS model by observing the development of symptoms such as stiffness in the right shoulder joint, contraction of the right upper limb, muscular atrophy, and limping in rats13.</li>
</ol>
4. Tuina method
NOTE: Throughout the procedure, the investigator must wear personal protective equipment. Only a single professional Tuina doctor must perform all manipulations (Figure 3, Figure 4, and Figure 5).
<ol>
	<li>Train by the Intelligent-massage technique parameter determination system, which includes a mechanoreceptor and a computer (Figure 3A).
	<ol>
		<li>Perform manipulations on the mechanoreceptor and force parameters in three directions displayed through software (Figure 3B).</li>
		<li>Use the thumb finger to perform the rotatory-kneading method in a rotatory movement at a strength of 0.5 kg and a frequency of 100-120 times/min (Figure 3C).</li>
		<li>Use the thumb fingertip to perform the point-pressing method at a strength of 0.5 kg (Figure 3D).</li>
		<li>Perform Tuina on the rats by maintaining the mechanical display mentioned in steps 4.1.2 and 4.1.3 for 1 min.</li>
	</ol>
	</li>
	<li>Hold the rat until it calms down (~2 min). Then, perform the manipulation. Place the rat in the lateral recumbent position, but the position may change based on different manipulation methods.</li>
	<li>Use the right index finger and middle finger to clamp the right forelimb of the rat and flex and extend it several times to determine the positions of the rat&#39;s shoulder joint, elbow joint, and humerus.</li>
	<li>Knead the right shoulder, forelimb, and back of the rat by clockwise rotation with the thumb pulp at a strength of 0.5 kg and a frequency of 100-120 times/min for 3 min (Figure 4A-C).
	<ol>
		<li>Manipulate the forelimb muscles in the lateral recumbent position.</li>
		<li>Manipulate the shoulder and back muscles in the prone position.</li>
	</ol>
	</li>
	<li>Press acupoint LI15 (Jianyu), SI11 (Tianzong), HT01 (Jiquan,) and LI11 (Quchi) vertically with the thumb fingertip 30 times per acupoint at a strength of 0.5 kg (Figure 4D-G).
	<ol>
		<li>Use the rat-acupoint atlas to define the location of each acupoint (Figure 5)14,15.</li>
		<li>Press LI15, located in the depression anterior-inferior to the acromial end, in the prone position.</li>
		<li>Press SI11, located in the depression to the infraspinatus fossa at the midpoint of the scapular spine, in the prone position.</li>
		<li>Press HT01, located at the axilla center, in the supine position.</li>
		<li>Press LI11 located in the depression medial to the extensor carpi radialis at the lateral end of the cubital crease in the lateral recumbent position.</li>
	</ol>
	</li>
	<li>Hold the shoulder joint with the left thumb and middle finger, and stretch the forelimb in the adduction, abduction, anterior extension, and posterior extension positions for 10 s (Figure 4H-K). 
	NOTE: This stretching method must be carried out without resistance in rats.</li>
	<li>Pause the Tuina procedure if the rat becomes agitated. Stroke the rat for 10 s to calm it down, and then proceed with the trial.&#160;</li>
	<li>Perform the procedure daily for 2 weeks.</li>
</ol>
5. Measurement of glenohumeral ROM
NOTE: It is important to complete the measurement process as quickly as possible to prevent degeneration of the joint capsule tissue.
<ol>
	<li>Remove the scapula and proximal two-thirds of the humerus en bloc after sacrificing the rat with an excessive dosage of tribromoethanol (3x initial dose, by intraperitoneal injection), exposing the lower edge of the scapula.</li>
	<li>Insert an injection needle (1.2 cm x 0.45 mm) along the humeral shaft into the humeral head.</li>
	<li>Insert two injection needles vertically into the upper and lower corners of the scapula on plastic foam wrapped with a sterile surgical sheet.</li>
	<li>Attach a thin thread to the injection needle on the humeral shaft and pull it at the other end with a 5 g force to make it parallel to the humeral shaft. Measure the angle between the lower edge of the scapula and the humeral shaft (Figure 6). 
	NOTE: To ensure reliable results, have a separate investigator conduct the measurements.</li>
	<li>Report data as means &#177;standard deviation (SD) using a statistical analysis software application. 
	NOTE: SPSS software (SPSS, version 25.0) was used here.</li>
	<li>Analyze the differences between groups using one-way analysis of variance (ANOVA).</li>
	<li>Obtain bar graphics using appropriate software. 
	NOTE: GraphPad Prism 8 was used here.</li>
	<li>Assess capsule pathology using H&#38;E and Masson staining after measurement.</li>
</ol>
6. Section preparation
<ol>
	<li>After evaluating glenohumeral ROM, fix whole samples in 4% PFA for 3 days, followed by decalcification in EDTA (pH 7.2) solution for an additional 2 months.</li>
	<li>After dehydration, slice embedded tissue blocks containing the samples into 5 &#181;m slices16.</li>
	<li>Dry the slice at 65 &#176;C for 60 min.</li>
	<li>Dewax the slice.</li>
	<li>Soak the slice in xylene I, xylene II, and xylene III for 7 min, followed by a descending ethanol series (anhydrous ethanol, 5 min; 95% ethanol, 2 min; 80% ethanol, 2 min, and 70% ethanol, 2 min), and finally in ultrapure water for 2 min.</li>
</ol>
7. H&#38;E staining
<ol>
	<li>Stain the sections using hematoxylin for 5 min, rinse with 1% hydrochloric acid ethanol for 3 s, and wash with running water for 5 min.</li>
	<li>Stain the section with eosin for 3 min and wash with tap water.</li>
	<li>Soak the section in an ethanol series (95% ethanol I, 3 s; 95% ethanol II, 3 s; anhydrous ethanol I, 3 s, and anhydrous ethanol II, 1 min) and then immerse in a xylene series (xylene I, 1 min; xylene II, 1 min).</li>
	<li>Place a drop of neutral gum sealant on each sample. Seal each sample with a cover glass.</li>
	<li>Collect images using an inverted fluorescence microscope (Scale bar = 100 &#181;m).</li>
</ol>
8. Masson staining
<ol>
	<li>Using an immunohistochemical pen, draw a circle around the sections and then incubate the sections in Bouin&#39;s solution for 2 h at 37 &#176;C to mordant. Subsequently, wash the sections with water until the yellow color disappears.</li>
	<li>Treat the samples with lapis lazuli blue dye for 3 min and then wash them with distilled water.</li>
	<li>After staining the sections with hematoxylin (Mayer) for 2 min, treat the sections for 3 s in the acidic ethanol differentiation solution. Then, wash the sections in running water for 10 min.</li>
	<li>Stain the sections with ponceau magenta dye solution for 10 min and subsequently wash them with water.</li>
	<li>Immerse the sections in the phosphomolybdic acid solution for 10 min.</li>
	<li>Add aniline blue staining solution to the sections for 5 min, and then wash them with a weak acid working solution for 2 min.</li>
	<li>Dehydrate and make the sections transparent as described in step 7.3.</li>
	<li>Place a drop of neutral gum sealing agent on each section and cover it with a cover glass. Leave the sections in a fume hood to dry.</li>
	<li>Collect images as described in step 7.5.</li>
</ol>

Tuina Therapy for Frozen Shoulders in a Rat Model

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L., Fang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuina+for+periarthritis+of+shoulder:+A+systematic+review+protocol.">Tuina for periarthritis of shoulder: A systematic review protocol.</a> Medicine. 99 (11), e19332 (2020).</li><li>Norlin, R., Hoe-Hansen, C., Oquist, G., Hildebrand, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shoulder+region+of+the+rat:+anatomy+and+fiber+composition+of+some+suprascapular+nerve+branches.">Shoulder region of the rat: anatomy and fiber composition of some suprascapular nerve branches.</a> The Anatomical Record. 239 (3), 332-342 (1994).</li><li>Okajima, S. 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The authors have nothing to disclose.

The first critical step is model selection. Due to the difficulty in implementing the primary FS model, cast immobilization and surgical internal fixation are often used to establish FS rat models9,12. The most severe restriction of shoulder mobility and fibrosis of the capsule were observed in the FS model established by cast immobilization for 3 weeks12,20. In this study, the success rates of the FS model were excellent, with 100% success.
The second critical step is the manipulations used in this protocol. Three manipulations (kneading, pressing, and stretching) were used in this study. The soft tissue kneading manipulation was applied to the shoulder, scapula and upper arm to relax the muscles. Pressing manipulation was performed by applying pressure to acupoints such as LI15, SI11, HT01, and LI11, which are most commonly used in clinical practice for FS5,21. LI15, SI11, and HT01 are located in positions around the shoulder capsule and can be effective in improving ROM and shoulder function22. LI11 is often used for upper extremity motor impairment and is located in the same meridian as LI15. This acupoint matching method helps improve the efficacy of LI1523. Following full relaxation, stretching techniques were used to restore functional activities.
The possible problem in this protocol is that rats exhibit intense resistance during Tuina, which may be caused by fear rather than exceeding the tolerance of the rats. At this point, the manipulations should be stopped until the rats calm down (stroking for 10 s calms down the rats). In addition, the extent of stretching should be adjusted according to the symptoms of the rats. Initially, the limitation of the shoulder joint was obvious, and the stretching amplitude was small. Along with the intervention, the shoulder joint function of the rats gradually recovered and the amplitude of the stretch progressively increased. The standard is that rats can accept the stretching method without resistance. Finally, rats have a certain degree of aggression, and Tuina requires prolonged contact with rats, so it is important to wear personal protective equipment.
The quantitative control of manipulation is the most difficult in Tuina experiments. While a massage manipulation simulator can be used to control the strength and frequency of a single manipulation, this method is limited when multiple manipulations and treatment sites are involved24,25. In clinical practice, Tuina is typically performed directly by practitioners, and in this study, it was difficult to intervene with medical equipment. To control the stimulation, the intelligent massage technique parameter determination system can be used to standardize the training of Tuina. After training, the investigator can apply the same force to each rat to an extent. The major limitation of this protocol is that manipulations cannot be completely controlled.
TCM Tuina therapy has a rich history of use across China, with various doctors in hospitals using different manipulation and treatment site combinations. Therefore, it is important to establish replicable and effective protocols for both animal experiments and clinical studies. In this study, the manipulations and acupoints used were based on a previous study by our team, combining our clinical experience with the characteristics of the FS animal model21. This study demonstrated the effectiveness of the developed Tuina protocol in improving shoulder joint function and reducing capsule fibrosis in FS rats. These findings provide a foundation for further investigations into the mechanisms underlying Tuina treatment. Moreover, the protocol can be useful for researchers interested in exploring the efficacy of alternative medical treatments for FS.
A previous study found that the mechanism of Tuina intervention on fibrosis may be related to the down-regulation of TGF-&#946; and CTGF while regulating the balance of MMP-1/TIMP-1, thereby alleviating the production of extracellular matrix (ECM)26. The effect of Tuina on the fibrosis of the shoulder capsule may be achieved through the regulation of various mechanisms. However, further research is required to fully understand the mechanisms involved in this improvement.

Frozen shoulder (FS), also known as adhesive capsulitis of the shoulder, is a self-limiting disease characterized by shoulder pain and mobility deficits. It typically affects people between the ages of 30 and 70 years old, with a mean age of 50 years, and has a prevalence of around 5% in the Chinese population1. Females are reported to have a 1.6 times higher incidence of FS compared to males2. The prevalence of FS is higher in people with diabetes, glucose and lipid metabolism disorders or other related diseases, ranging between 10% and 36%2,3. Current clinical treatments for FS include physiotherapy, steroid medications, and surgical treatments4.
Tuina, a traditional Chinese medicine (TCM) therapy, has been shown to effectively relieve shoulder pain in FS patients, improving their quality of life5,6. However, the underlying mechanisms of this treatment are not well understood. Thus, using animal models to study the effects and mechanisms of Tuina in treating FS is crucial.
The rat shoulder joint has a complex structure similar to that of the human shoulder and is often used in mechanistic studies of FS7. The FS rat model is characterized by a decline in glenohumeral ROM and capsule fibrosis8. Furthermore, this model allows for the observation of the shoulder capsule and enables pathological research while repairing the injury9. Moreover, oral corticosteroids are often used as the control group in FS treatment research10. This study aims to develop a Tuina protocol for the FS rat model and demonstrates the feasibility of conducting animal experiments in Tuina research by comparing the efficacy of Tuina therapy and oral dexamethasone.

<table><tbody><tr><td>4% paraformaldehyde</td><td>Solarbio</td><td>P1110</td><td></td></tr><tr><td>Embedding machine</td><td>Changzhou Paisijie Medical Equipment Co., Ltd</td><td>BM450A</td><td></td></tr><tr><td>Ethylene Diamine Tetraacetic Acid (EDTA)</td><td>Solarbio</td><td>E1171</td><td></td></tr><tr><td>Hematoxylin eosin (HE) staining kit</td><td>Sparkjade</td><td>EE0012</td><td></td></tr><tr><td>Intelligent-massage technique parameter determination system</td><td>Shanghai Dukang Intrument Equipment Co. Ltd</td><td>ZTC-&lt;img alt=&quot;Equation 1&quot; src=&quot;/files/ftp_upload/65440/65440eq01.jpg&quot; style=&quot;margin: auto;&quot;/&gt;</td><td></td></tr><tr><td>Microtome</td><td>Leica</td><td>531CM-Y43</td><td></td></tr><tr><td>&lt;br/&gt; Modified Masson Trichrome Staining Solution</td><td>Shanghai yuanye Bio-Technology Co., Ltd</td><td>R20381-8</td><td>Bouin 50 mL;&lt;br/&gt; lapis lazuli blue dye 50 mL;&lt;br/&gt; Hematoxylin (Mayer) 50 mL;&lt;br/&gt; acidic ethanol differentiation solution 50 mL;&lt;br/&gt; ponceau magenta dye solution 50 mL;&lt;br/&gt; phosphomolybdic acid solution 50 mL;&lt;br/&gt; aniline blue staining solution 50 mL;&lt;br/&gt; &amp;nbsp;weak acid 50 mL</td></tr><tr><td>Tribromoethanol</td><td>Macklin</td><td>T903147-5</td><td></td></tr></tbody></table>

tuina in a frozen shoulder rat model: an efficient and reproducible protocol

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

The physical activity of rats was observed to evaluate the success or failure of the FS model. A previous study showed that cast immobilization significantly reduced the distance traveled and walking speed compared to normal rats17. Another research suggested that FS did not affect the distance traveled, and limping was the most common presenting symptom13. This study showed stiffness in the right shoulder joint, contraction of the right upper limb, muscular atrophy and limping in rats after modeling. These lesions in the MT and MO groups were resolved completely by 2 weeks of intervention. But there was no significant change in the M group.
The primary criterion for evaluating the effectiveness of Tuina in FS is the measurement of glenohumeral ROM18. We observed that the average values of glenohumeral ROM were 149.3&#176; &#177; 5.9&#176; in the C group, 111.1&#176; &#177; 3.9&#176; in the M group, 128.5&#176; &#177; 2.8&#176; in the MT group, and 119.56&#176; &#177; 2.9&#176; in the MO group. As depicted in Figure 7, the glenohumeral ROM of rats in the M group was significantly lower than that in the C group (P &#60; 0.0001). Moreover, the ROM in the MT group and MO group was significantly higher than that in the M group (P &#60; 0.05, P &#60; 0.0001). However, the ROM in the MO group was significantly lower than that in the MT group (P &#60; 0.0001). This finding suggests that Tuina can significantly improve shoulder joint function in FS rats.
Additionally, H&#38;E staining and Masson staining can further demonstrate the effects of Tuina in preserving the structure and reducing fibrosis in capsule. To facilitate observation, the capsule of the glenohumeral joint was used for histological findings. The shoulder joint capsule comprises synovial and fibrous layers19. H&#38;E staining revealed synoviocyte proliferation, flattened synovial folds, erythrocyte stasis, and vascular proliferation in the M group, which are typical features of FS (Figure 8A,B). These features diminished to some extent after Tuina and oral dexamethasone therapy (Figure 8C,D). Compared to the MT group, the MO group also showed much synovial cells. Masson staining showed the arrangement of fiber bundles in each group (yellow arrows). The capsule comprises a loose network of reticular fibers with fiber bundles arranged in a neat direction (Figure 8E). In the M group, the fiber bundles were arranged disorderly, indicating capsule fibrosis (Figure 8F). The capsules of rats in the MT group showed that the fiber bundles are neatly and clearly stratified but remain slightly disordered in the MO group (Figure 8G,H).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65440/65440fig01.jpg" /> 
Figure 1: Protocol for establishing the FS model and Tuina intervention. The rats were on adaptive feeding for 7 days, FS model establishment for 21 days, and Tuina therapy was performed daily for 14 days. On day 36, all rats were sacrificed. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65440/65440fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65440/65440fig02.jpg" /> 
Figure 2: Cast immobilization for establishing a rat model of FS. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65440/65440fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65440/65440fig03.jpg" /> 
Figure 3: Quantitative control of manipulation. (A) Intelligent massage technique parameter determination system. (B) Three forces can be measured as parallel force along the X direction, longitudinal force along the Y direction, and vertical force along the Z direction. (C) Strength of the rotatory-kneading method. The red curve represents the stabilized vertical force (0.5 kg). The orange curve represents the regular parallel force. The white curve represents the regular longitudinal force. (D) The strength of the point-pressing method. The red curve represents the vertical force (0.5 kg). Orange and white curves represent nonparallel and longitudinal forces. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65440/65440fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65440/65440fig04.jpg" /> 
Figure 4: Manipulation used in Tuina therapy. (A-C) Knead muscles of the right shoulders, forelimbs, and back. (D-G) Point-press LI15, SI11, HT01, and LI11. (H-K) Stretch the forelimb in adduction, abduction, anterior extension, and posterior extension positions. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65440/65440fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65440/65440fig05.jpg" /> 
Figure 5: Anatomical positions of LI15, SI11, HT01, and LI11 in rats. &#9679; Lateral surface, &#9675; Medial surface. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65440/65440fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65440/65440fig06.jpg" /> 
Figure 6: Measurement of glenohumeral ROM. A thin thread is attached to an injection needle inserted into the humeral shaft and pulled at the other end with a 5 g force to make it parallel to the humeral shaft. The angle between the lower edge of the scapula and the humeral shaft is measured as glenohumeral ROM. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65440/65440fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/65440/65440fig07.jpg" /> 
Figure 7: Glenohumeral ROM across three groups of rats. Values are means &#177; S.D., n = 5. Significant differences are indicated by one-way ANOVA (aP &#60; 0.001 and bP &#60;&#160;0.0001). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65440/65440fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/65440/65440fig08.jpg" /> 
Figure 8: Histological findings of the shoulder capsule. (A,E) The control group contains a normal capsule structure (H&#38;E and Masson staining). (B,F) The FS model group illustrates changes in the structure of the capsule as follows: flattened synovial folds, capsule fibrosis, and disturbed fiber bundles (H&#38;E and Masson staining). (C,G). The FS model combined with Tuina group illustrates that the structure of the capsule is close to normal, and fibrosis is not obvious (H&#38;E and Masson staining). (D,H) The FS model combined with oral dexamethasone shows that the structure of the capsule is close to normal, and fibrosis is obvious (H&#38;E and Masson staining). Scale bar = 100 &#181;m. HH: head of the humerus; black arrow: synovial folds; red arrow: erythrocyte stasis and vascular proliferation; yellow arrow: fiber bundles. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65440/65440fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Investigating the Effectiveness of Tuina Therapy for Frozen Shoulder at JoVE.com

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

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

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

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

tuina-frozen-shoulder-rat-model-an-efficient-reproducible

Research

JoVE Journal

Medicine

1.7K Views.  Shandong University of Traditional Chinese Medicine. This study develops an efficient and reproducible Tuina protocol for treating frozen shoulder established in a rat model. This approach will help to study the Tuina therapy treatment method for frozen shoulders.

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

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

The In Ovo Chick Chorioallantoic Membrane (CAM) Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and highly vascularized, making it an ideal system for tumor implantation. Furthermore, the CAM contains extracellular matrix proteins such as fibronectin, laminin, collagen, integrin alpha(v)beta3, and MMP-2, making it an attractive model to study tumor invasion and metastasis. Scientists have long taken advantage of the physiology of the CAM by using it as a model of angiogenesis. More recently, the CAM assay has been modified to work as an in vivo xenograft model system for various cancers that bridges the gap between basic in vitro work and more complex animal cancer models. The CAM assay allows for the study of tumor growth, anti-tumor therapies, and pro-tumor molecular pathways in a biologically relevant system that is both cost- and time-effective. Here, we describe the development of CAM xenograft model of hepatocellular carcinoma (HCC) with embryonic survival rates of up to 93% and reliable tumor take leading to growth of three-dimensional, vascularized tumors.

The In Ovo Chick Chorioallantoic Membrane CAM Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) is immunodeficient and highly vascularized, making it a natural in vivo model of tumor growth and angiogenesis. In this protocol, we describe a reliable method of growing three-dimensional, vascularized hepatocellular carcinoma (HCC) tumors using the CAM assay.

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and ...

Rat Model of Adhesive Capsulitis of the Shoulder

This proposal aims to create an in vivo rat model of adhesive capsulitis for researching potential treatment options for this condition and other etiologies of comparable arthrofibrosis. The model includes extra-articular fixation of the shoulder in rats via scapular to humeral suturing, resulting in a secondary contracture without invading the intra-articular space and resulting in decreased rotational ROM and increased joint stiffness.
We used 10 Sprague-Dawley rats for the purpose of this study. Baseline ROM measurements were taken before glenohumeral immobilization. The rats were subjected to 8 weeks of immobilization before the fixation sutures were removed and changes in ROM and joint stiffness were evaluated. To evaluate whether immobilization resulted in a significant reduction in ROM, changes in kinematics were calculated. ROM was measured at each time point in the follow-up period and was compared to the baseline internal and external ROM measurements. In order to evaluate the stiffness, joint kinetics were calculated by determining the differences in torque (text and tint ) needed to reach the initial external rotation of 60&#176; and initial internal rotation of 80&#176;.
After the removal of the extra-articular suture fixation on follow-up day 0, we found a 63% decrease in total ROM compared to baseline. We observed continuous improvement until week 5 of follow-up, with the progress slowing down around a 19% restriction. On week 8 of follow-up, there was still an 18% restriction of ROM. Additionally, on follow-up day 0, we found the torque increased by 13.3 Nmm when compared to baseline. On week 8, the total torque was measured to be 1.4 &#177; 0.2 Nmm higher than initial measurements. This work introduces a rat model of shoulder adhesive capsulitis with lasting reduced ROM and increased stiffness.

This protocol presents an in vivo rat model of adhesive capsulitis. The model includes an internal fixation of the glenohumeral joint with extra-articular suture fixation for an extended time, resulting in a decreased rotational range of motion (ROM) and increased joint stiffness.

This proposal aims to create an in vivo rat model of adhesive capsulitis for researching potential treatment options for this condition and other ...

Bioengineering

Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

Glaucoma is a disease of the central nervous system affecting retinal ganglion cells (RGCs). RGC axons making up the optic nerve carry visual input to the brain for visual perception. Damage to RGCs and their axons leads to vision loss and/or blindness. Although the specific cause of glaucoma is unknown, the primary risk factor for the disease is an elevated intraocular pressure. Glaucoma-inducing procedures in animal models are a valuable tool to researchers studying the mechanism of RGC death. Such information can lead to the development of effective neuroprotective treatments that could aid in the prevention of vision loss. The protocol in this paper describes a method of inducing glaucoma - like conditions in an in vivo rat model where 50 &#181;l of 2 M hypertonic saline is injected into the episcleral venous plexus. Blanching of the vessels indicates successful injection. This procedure causes loss of RGCs to simulate glaucoma. One month following injection, animals are sacrificed and eyes are removed. Next, the cornea, lens, and vitreous are removed to make an eyecup. The retina is then peeled from the back of the eye and pinned onto sylgard dishes using cactus needles. At this point, neurons in the retina can be stained for analysis. Results from this lab show that approximately 25% of RGCs are lost within one month of the procedure when compared to internal controls. This procedure allows for quantitative analysis of retinal ganglion cell death in an in vivo rat glaucoma model.

Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

Glaucoma is characterized by damage to retinal ganglion cells. Inducing glaucoma in animal models can provide insight into the study of this disease. Here, we outline a procedure that induces loss of RGCs in an in vivo rat model and demonstrates the preparation of whole-mount retinas for analysis.

Glaucoma is a disease of the central nervous system affecting retinal ganglion cells (RGCs). RGC axons making up the optic nerve carry visual input to the ...

Reproducible Arterial Denudation Injury by Infrarenal Abdominal Aortic Clamping in a Murine Model

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These complications can be attributed to impairments in re-endothelialization within the wound margins. Yet, the cellular and molecular mechanisms of re-endothelialization remain to be defined. While several animal models to study re-endothelialization after arterial denudation are available, few are performed in the mouse because of surgical limitations. This undermines the opportunity to exploit transgenic mouse lines and investigate the contribution of specific genes to the process of re-endothelialization. Here, we present a step-by-step protocol for creating a highly reproducible murine model of arterial denudation injury in the infrarenal abdominal aorta using external vascular clamping. Immunocytochemical staining of injured aortas for fibrinogen and &#946;-catenin demonstrate the exposure of a pro-thrombotic surface and the border of intact endothelium, respectively. The method presented here has the advantages of speed, excellent overall survival rate, and relative technical ease, creating a uniquely practical tool for imposing arterial denudation injury in transgenic mouse models. Using this method, investigators may elucidate the mechanisms of re-endothelialization under normal or pathological conditions.

Understanding the cellular and molecular mechanisms of re-endothelialization following arterial denudation injury is of paramount importance in preventing thrombosis and restenosis of arteries. Here we describe a protocol for reproducible arterial denudation injury of the infrarenal abdominal aorta. The procedure was developed to investigate the underlying mechanisms that regulate endothelial regeneration using mouse models.

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These ...

Reproducible Motor Deficit Following Aortic Occlusion in a Rat Model Of Spinal Cord Ischemia

Spinal cord ischemia is a fatal complication following thoracoabdominal aortic aneurysm surgery. Researchers can investigate the strategies for preventing and treating this complication using experimental models of spinal cord ischemia. The model described here demonstrates varying degrees of paraplegia that relate to the length of occlusion following thoracic aortic occlusion in a rat spinal cord ischemia model.
A 2-Fr. balloon-tipped catheter was advanced through the femoral artery into the descending thoracic aorta until the catheter tip was placed at the left subclavian artery in anesthetized male Sprague-Dawley rats. Spinal cord ischemia was induced by inflating the catheter balloon. After a set period of occlusion (9, 10, or 11 min), the balloon was deflated. Neurologic assessment was performed using the motor deficit index at 24 h after surgery, and the spinal cord was harvested for histopathological examination.
Rats that underwent 9 min of aortic occlusion showed mild and reversible motor impairment in the hind limb. Rats subjected to 10 min of aortic occlusion presented with moderate but reversible motor impairment. Rats subjected to 11 min of aortic occlusion displayed complete and persistent paralysis. The motor neurons in the spinal cord sections were more preserved in rats subjected to shorter duration of aortic occlusion.
Researchers can achieve a reproducible hind limb motor deficit following thoracic aortic occlusion using this spinal cord ischemia model.

This study demonstrates the technique to make a minimally invasive and easily reproducible model of spinal cord ischemia in rats. Various degrees of hind limb motor deficit can be produced by controlling the aortic occlusion time.

Spinal cord ischemia is a fatal complication following thoracoabdominal aortic aneurysm surgery. Researchers can investigate the strategies for preventing ...

Standardized Tuina Manipulation for Knee Osteoarthritis in Rats

Tuina Manipulation to Reduce Inflammation and Cartilage Loss in Knee Osteoarthritis Rats

Clinical trials suggest that Tuina manipulation is effective in treating knee osteoarthritis (KOA), while further studies are required to discover its mechanism. Therefore, the manipulation of animal models of knee osteoarthritis is critical. This protocol provides a standard process for Tuina manipulation on KOA rats and a preliminary exploration of the mechanism of Tuina for KOA. The press and kneading manipulation method (a kind of Tuina manipulation that refers to pressing and kneading the specific area of the body surface) is applied on 5 acupoints around the knee joint of rats. The force and frequency of the manipulation were standardized by finger pressure recordings, and the position of the rat during manipulation is described in detail in the protocol. The effect of manipulation can be measured by pain behavior tests and microscopic findings in synovial and cartilage. KOA rats showed significant improvement in pain behavior. The synovial tissue inflammatory infiltration was reduced in the Tuina group, and the expression of tumor necrosis factor (TNF)-&#945; was significantly lower. Compared to the control group, chondrocyte apoptosis was less in the Tuina group. This study provides a standardized protocol for Tuina manipulation on KOA rats and preliminary proof that the therapeutic effects of Tuina may be related to reducing synovial inflammation and delayed chondrocyte apoptosis.

Author Spotlight: Understanding the Mechanism of Tuina Manipulation in Rats 

We present a protocol for Tuina manipulation performed on plaster-immobilized-induced knee osteoarthritis rats. Based on the preliminary results, it suggested that the efficacy of the method relied on the reduction of inflammation and cartilage loss.

Clinical trials suggest that Tuina manipulation is effective in treating knee osteoarthritis (KOA), while further studies are required to discover its ...

Standardized Tuina Protocol for Rat DRG Compression Model

Analgesic Effect of Tuina on Rat Models with Compression of the Dorsal Root Ganglion Pain

Neuropathic pain is a prevalent condition that affects 6.9%-10% of the population and results from nerve damage due to various etiologies, such as lumbar disc herniation, spinal canal stenosis, and intervertebral foramen stenosis. Although Tuina, a traditional Chinese manual therapy, has shown analgesic effects in clinical practice for the treatment of neuropathic pain, its underlying neurobiological mechanisms remain unclear. Animal models are essential for elucidating the basic principles of Tuina. In this study, we propose a standardized Tuina protocol for rats with compression of the dorsal root ganglion (DRG), which involves inducing DRG compression by inserting a stainless steel rod into the intervertebral foramen, performing Tuina manipulation with specific parameters of location, intensity, and frequency in a controlled environment, and assessing the behavioral and histopathological outcomes of Tuina treatment. This article also discusses the potential clinical implications and limitations of the study and suggests directions for future research on Tuina.

Author Spotlight: Analgesic Effect of Tuina on Rat Models with Compression of the Dorsal Root Ganglion Pain  

This article presents a manipulation for treating chronic compression of the dorsal root ganglion in rats using Tuina therapy, along with a method for evaluating its effectiveness based on pain behavior and histopathological results.

Neuropathic pain is a prevalent condition that affects 6.9%-10% of the population and results from nerve damage due to various etiologies, such as lumbar ...

Therapeutic Efficacy of Small Needle Knife Therapy in Treating Frozen Shoulder

Clinical Efficacy of Small Needle Knife Therapy on Stage I-II Frozen Shoulder

Frozen shoulder is a kind of aseptic inflammatory disease of the shoulder caused by strain, trauma, and other reasons, resulting in shoulder joint pain and limited function. The protocol presented here demonstrates the operation of a small needle knife in treating frozen shoulders, including patient management, material preparation, positioning, operation, and postoperative care. The purpose of this protocol is to relieve the pain and functional limitations and improve the living ability of patients with frozen shoulders. In our study, 76 stage I-II frozen shoulder patients who met the inclusion criteria were randomly divided into a control group and a treatment group (n=38). Patients in the control group received functional exercise, while the treatment group received small needle knife therapy with functional exercise. The visual analogue scores (VAS), the Constant and Murley scores (CMS), and the thickness of the coracohumeral ligament (CHL) under ultrasound were evaluated. After small needle knife therapy, the VAS score was significantly lower in the treatment group (5.11 &plusmn; 0.89) than in the control group (5.49 &plusmn; 0.65; t=-2.065, p&lt;0.05); the CMS score was significantly higher in the treatment group (64.72 &plusmn; 4.78) than in the control group (60.97 &plusmn; 6.00; t=2.947, p&lt;0.05); the CHL thickness was significantly decreased in the treatment group (2.38 &plusmn; 0.36) than in the control group (2.57 &plusmn; 0.42; t=-2.117, p&lt;0.05). These results indicate that the small needle knife significantly relieved the pain symptoms, improved the shoulder function, reduced the CHL thickness, and improved the quality of life and, therefore, had significant therapeutic efficacy in stage I-II frozen shoulder patients.

Author Spotlight: Treating Frozen Shoulder with Small Needle Knife Therapy 

The protocol presented here describes the detailed operation method and proves the effectiveness of a small needle knife in the treatment of frozen shoulders.

Frozen shoulder is a kind of aseptic inflammatory disease of the shoulder caused by strain, trauma, and other reasons, resulting in shoulder joint pain ...

Therapeutic Potential of Tuina Massage for Knee Osteoarthritis

Tuina Intervention in Sodium Monoiodoacetate Injection-Induced Rat Model of Knee Osteoarthritis

Knee osteoarthritis (KOA), a common degenerative joint disorder, is characterized by chronic pain and disability, which can progress to irreparable structural damage of the joint. Investigations into the link between articular cartilage, muscles, synovium, and other tissues surrounding the knee joint in KOA are of great importance. Currently, managing KOA includes lifestyle modifications, exercise, medication, and surgical interventions; however, the elucidation of the intricate mechanisms underlying KOA-related pain is still lacking. Consequently, KOA pain remains a key clinical challenge and a therapeutic priority. Tuina has been found to have a regulatory effect on the motor, immune, and endocrine systems, prompting the exploration of whether Tuina could alleviate KOA symptoms, caused by the upregulation of inflammatory factors, and further, if the inflammatory factors in skeletal muscle can augment the progression of KOA.
We randomized 32 male Sprague Dawley (SD) rats (180-220 g) into four groups of eight animals each: antiPD-L1+Tuina (group A), model (group B), Tuina (group C), and sham surgery (group D). For groups A, B, and C, we injected 25 &#181;L of sodium monoiodoacetate (MIA) solution (4 mg MIA diluted in 25 &#181;L of sterile saline solution) into the right knee joint cavity, and for group D, the same amount of sterile physiological saline was injected. All the groups were evaluated using the least to most stressful tests (paw mechanical withdrawal threshold, paw withdrawal thermal latency, swelling of the right knee joint, Lequesne MG score, skin temperature) before injection and 2, 9, and 16 days after injection.

Author Spotlight: Treating Knee Osteoarthritis with Tuina - A New Perspective 

This protocol describes the methods of Tuina intervention in sodium monoiodoacetate injection-induced rat model of knee osteoarthritis (KOA), which provides a reference for the application of Tuina in KOA animal models. This protocol also studies the effective mechanism of Tuina for KOA, and the results will help promote its application.

Knee osteoarthritis (KOA), a common degenerative joint disorder, is characterized by chronic pain and disability, which can progress to irreparable ...

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

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Chapters in this video

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

The In Ovo Chick Chorioallantoic Membrane (CAM) Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

Rat Model of Adhesive Capsulitis of the Shoulder

Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

Reproducible Arterial Denudation Injury by Infrarenal Abdominal Aortic Clamping in a Murine Model

Reproducible Motor Deficit Following Aortic Occlusion in a Rat Model Of Spinal Cord Ischemia

Tuina Manipulation to Reduce Inflammation and Cartilage Loss in Knee Osteoarthritis Rats

Analgesic Effect of Tuina on Rat Models with Compression of the Dorsal Root Ganglion Pain

Clinical Efficacy of Small Needle Knife Therapy on Stage I-II Frozen Shoulder

Tuina Intervention in Sodium Monoiodoacetate Injection-Induced Rat Model of Knee Osteoarthritis