We would like to express our sincere gratitude to Bogazici University for providing the laboratory facilities and technical support necessary for this research. We also thank Dr. Demet Sezgin Mansuroglu, Dr. Eda Karadogan, and Dr. Mustafa Enes Ozden for their valuable assistance in data collection and analysis. The research was funded by the authors. No external financial support was obtained.

1. Sample procurement
<ol>
	<li>Procure 80 acrylic blocks with an internal diameter of 1.5 mm, a radius of curvature of 5 mm, a 55&#176; curvature, and a working length of 16 mm.</li>
</ol>
2. Cleaning and shaping procedure
<ol>
	<li>Set the endomotor to a torque of 2.0 Ncm and a speed of 300 rpm.
	<ol>
		<li>Attach a 10/.04 taper file to the motor and use it in a back-and-forth motion until the working length (16 mm) is reached, ensuring that it does not bind.</li>
		<li>Irrigate the canals with 5.25% NaOCl.</li>
		<li>Attach a 15/.04 tapered file to the motor and use it in a back-and-forth motion until the working length (16 mm) is reached, ensuring that it does not bind.</li>
		<li>Repeat steps 2.1.2 and 2.1.3 with 20/.04, 25/.04, 30/.04, and 35/.04 taper files, used sequentially at the working length (16 mm).</li>
		<li>Dry the canals with paper points.</li>
	</ol>
	</li>
</ol>
3. Obturation
<ol>
	<li>Check the fit of a guttapercha cone to the canal.</li>
	<li>Inject the bioceramic canal sealer into the canal and fill it with bioceramic sealer.</li>
	<li>Insert the appropriate gutta-percha cone into the sealer-filled canal. Cut the gutta-percha 2 mm below the canal orifice using a heat tool.</li>
	<li>Take a periapical radiograph to verify the canal fillings (see Figure 1).</li>
	<li>Store the specimens in an incubator at 37 &#176;C and 100% humidity for 2 weeks.</li>
</ol>
4. Retreatment procedure
NOTE: A total of 24 new Remover files (23 mm) were used in the present study. The files were randomized into three groups of eight samples each. In determining the number of samples and files used in this research, the quota sampling method was used, considering the budget and the sample sizes of other reports in the literature27.
<ol>
	<li>Operate the files at 600 rpm and 2.5 Ncm torque according to the manufacturer&#39;s instructions. Use the files with a back-and-forth motion without applying apical pressure until they are 3 mm short of the working length.</li>
	<li>Remove the file from the canal when resistance is felt and irrigate with 5.25% NaOCl solution.</li>
	<li>Repeat steps 4.1 and 4.2 until the desired length is reached.</li>
	<li>Clean and sterilize the instruments in an autoclave for 18 min at 134 &#176;C before shaping the specimen. 
	NOTE: The files in the first group were used for retreatment in eight curved canals. The files in the second group were used for retreatment 3x each, and the files in the third group were used for retreatment 6x each. The procedures were repeated in group 2 and group 3 according to the number of uses.</li>
</ol>
5. SEM analysis
<ol>
	<li>Sample preparation and loading 
	NOTE: Take the necessary precautions to avoid contamination when handling the sample (e.g., wear gloves). Do not place the sample in a gold sputtering system as the surface is nickel-titanium.
	<ol>
		<li>Mount the sample on an SEM stub using conductive double-sided carbon tape.</li>
		<li>Attach the stub to the stage and tighten the side screw (see Figure 2).</li>
	</ol>
	</li>
	<li>SEM operation
	<ol>
		<li>Open the SEM sample chamber and remove the stage.</li>
		<li>Place the sample stub on the stage and secure it in place.</li>
		<li>Insert the sample stage into the sample chamber and close the chamber.</li>
		<li>Switch on the pumps and wait for the system notification of the vacuum.</li>
		<li>Open the SEM software and select the required operating voltage between 1 kV and 30 kV.</li>
	</ol>
	</li>
	<li>Image analysis
	<ol>
		<li>Have a trained investigator take images of the 4 mm distal end, which is the active part (area of interest), at standard magnifications of 100x, 250x, and 500x. Use an unused Remover file as a reference to evaluate the surface characteristics of the samples (see Figure 3).</li>
		<li>To commence the Auto Focus function, select the key icon within the SEM software. The resulting focused image of the sample is the desired endpoint.</li>
		<li>Set the magnification to the minimum zoom level of 50x.</li>
		<li>Enable the fast scan mode for efficient image acquisition.</li>
		<li>Adjust the focus using the coarse focus mode until a preliminary focus is achieved.</li>
		<li>Gradually increase the magnification to observe the desired feature. Use the coarse focus knob to achieve a rough focus, followed by the fine focus knob for precise focusing. Repeat this step for each magnification increase.</li>
		<li>Increase the magnification until the desired feature is observed. Adjust the coarse focus knob to roughly focus the image at this magnification. Then, use the fine focusing knob to improve the focus to obtain a focused image at the desired magnification. Repeat this step each time the magnification level is increased.</li>
		<li>Once the desired magnification is reached, refine the focus using the fine focus knob for optimal clarity.</li>
		<li>For enhanced image clarity, further increase the magnification to a near-maximum level and adjust the focus using the fine focus knob. If clarity is still not sufficient, adjust the stigmation in both the x and y axes. Continue fine-tuning the focus and stigmation until the clearest image is obtained at the higher magnification.</li>
		<li>After achieving a high-quality image of the sample, return to the desired magnification level. Capture the image by pressing the photo button. Choose either slow photo mode for higher quality and resolution, or fast photo mode for quicker capture.</li>
		<li>Repeat these steps for each sample.</li>
		<li>Download the images to the computer.</li>
		<li>Have two calibrated examiners analyze all SEM images by reviewing the images on a computer screen and recording the presence and type of deformations that occur in the files. The deformations include tip deformation, microcracks, fracture, unwinding, surface pitting, and blade disruption (Figure 4, Figure 5, Figure 6, Figure 7, and Figure 8).</li>
		<li>Have the same examiners analyze the collected data twice at 1 week intervals. 
		NOTE: Differences of opinion in the interpretation of SEM images of the samples between the observers are to be discussed until a consensus is reached.</li>
	</ol>
	</li>
</ol>
6. Statistical analysis
<ol>
	<li>Present descriptive statistics as counts and percentages.</li>
	<li>Perform analyses using statistical analysis software. Evaluate the differences between groups using the Fisher-Freeman-Halton Exact test. Set a type 1 error rate of 0.05 (two-tailed), and consider p &#60; 0.005 statistically significant.</li>
</ol>

Surface Defects in NiTi Files After Single and Multiple Uses in Endodontic Retreatment

The authors have no conflicts of interest to disclose.

This study evaluated the presence and types of microscopic defects on the external surfaces of Remover files after single, triple, and six-times use in acrylic blocks simulating curved canals. Ideally, human teeth are recommended for use in studies evaluating the fracture resistance of files to better simulate clinical use28. In their study, Peters and Barbakow29 found an increase in fracture initiation and propagation rates in instruments used in blocks compared to extract...

Endodontic retreatment is a procedure performed when a previously treated tooth fails to heal or develops new pathologies, such as persistent infection, reinfection, or missed anatomy. The procedure involves the removal of the existing root canal filling material, thorough cleaning and disinfection of the canal system and subsequent refilling1,2.
Nickel-titanium (NiTi) instruments are of great importance in improving and facilitating endodontic procedures due to their flexibility and high cutting efficiency3,4. The superelasticity of NiTi instruments permits them to better adapt to canal curvature, exhibit less wear, and have a higher resistance to fracture5,6. However, one of the major concerns with NiTi files is that they can fracture without visible deformation3.
The most common cause of fracture in NiTi rotary instruments is cyclic fatigue7. Cyclic fatigue occurs due to alternating tensile and compressive stresses on opposing surfaces of the instrument as it rotates continuously in a curved root canal without binding8,9. Fracture due to cyclic fatigue results from metal exhaustion10. Several factors influence the occurrence of fracture due to cyclic fatigue, including the physical properties of the instrument11,12, the root canal morphology13, repeated clinical use, and the sterilization process14,15. Therefore, to improve the fatigue resistance of NiTi rotary files, various modifications in the manufacturing method and core diameter, as well as changes in the cutting-edge and cross-sectional designs, have been attempted16. The Remover file is a new generation file produced by thermal treatment and a special electropolishing process called C-wire. Its design features are claimed to increase fatigue resistance. The file has a 30/100 mm non-cutting (inactive) tip and a minimally invasive core diameter. It is manufactured with a variable triple helix cross section that is symmetrical for the first 3 mm and then becomes asymmetrical towards the shaft. In addition, it is designed to preserve periradicular dentin by having a 7% taper in the first 10 mm, followed by a 0% taper towards the shaft17.
Cyclic fatigue fractures in NiTi rotary files typically occur without any visible plastic deformation18,19,20. As a result, these fractures cannot be evaluated clinically, and structural changes must be examined under high magnification using tools such as a stereomicroscope or scanning electron microscope (SEM)21. Due to the impracticality of performing such examinations on a routine basis, manufacturers recommend that files be used only once22,23. However, due to the high cost of NiTi files, many clinicians choose to reuse them24. Therefore, it is important to investigate the effects of clinical reuse on these files. One clinical study showed that rotary instruments can be safely reused up to 4x25. However, other studies have evaluated much higher reuse rates and there is no consensus on how many times a file can be safely reused24,26.
In previous studies that have evaluated the reuse of NiTi files, the primary focus has been on the effects of root canal widening and shaping on the fracture resistance of the files. A review of the literature, therefore, reveals that there is only one study that specifically evaluates the repeated use of retreatment file systems27. The aim of this study is to evaluate the impact of repeated use on the surface characteristics of the Remover file using scanning electron microscopy (SEM). It is hypothesized that increased clinical use will result in an increase in surface defects, thereby elevating the risk of fatigue fractures. The specific objective is to analyze the changes in surface defects of the Remover file after single and multiple uses, and to discuss the implications of these changes for clinical practice.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acrylic block</td>
			<td>ArdaDent Medical,Ankara,Turkey</td>
			<td></td>
			<td>for obturation</td>
		</tr>
		<tr>
			<td>DiaRoot Biosealer</td>
			<td>DiaDent, South Korea</td>
			<td>BS23101161</td>
			<td>for obturation</td>
		</tr>
		<tr>
			<td>DualMove Endomotor</td>
			<td>MicroMega, Coltene, France</td>
			<td>52002023</td>
			<td>for preparation</td>
		</tr>
		<tr>
			<td>&#160;EndoArt&#160; Smart Gold&#160;</td>
			<td>EndoArt, Inci Dental, Turkey</td>
			<td>SGK10114</td>
			<td>&#160;for initial preparation</td>
		</tr>
		<tr>
			<td>&#160;Gutta Percha</td>
			<td>EndoArt, Inci Dental, Turkey</td>
			<td>GD23080701</td>
			<td>for obturation</td>
		</tr>
		<tr>
			<td>Quattro ESEM&#160;</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td></td>
			<td>SEM analysis</td>
		</tr>
		<tr>
			<td>Paper Points</td>
			<td>Dentsply Maillefer, Ballaigues, Switzerland&#160;</td>
			<td>1I0305</td>
			<td>for dry to root canal</td>
		</tr>
		<tr>
			<td>Remover File</td>
			<td>MicroMega, Besan&#231;on, France</td>
			<td>891144/873757/</td>
			<td>for retreatment procedure</td>
		</tr>
		<tr>
			<td>Sodium Hypochlorite&#160;</td>
			<td>Saba Chemical&#38;Medical, Turkey</td>
			<td>3010225</td>
			<td>for irrigation</td>
		</tr>
		<tr>
			<td>SPSS v29&#160;</td>
			<td>IBM SPSS Corp, Armonk, New York, USA</td>
			<td></td>
			<td>Statistical analysis</td>
		</tr>
	</tbody>
</table>

scanning electron microscopic evaluation of surface defects of remover retreatment file after single and multiple uses

This study aimed to evaluate surface defects of Remover rotary Nickel-Titanium (NiTi) files after single and multiple uses in conventional endodontic retreatment procedures using scanning electron microscopy (SEM). Eighty acrylic blocks, simulating root canals with a 1.5 mm internal diameter, a 5 mm radius of curvature, and a 55&#176; curvature, were utilized. After chemomechanical preparation and obturation, 24 new Remover files (N30, 7%, L23) were randomly assigned to three groups: single use, triple use, and six uses. The files were operated at 600 rpm with a torque of 2.5 Ncm, cleaned, and sterilized after each use.
SEM analysis at magnifications of 100x, 250x, and 500x revealed surface defects, including tip deformation, microcracks, fracture, unwinding, surface pitting, and blade disruption. Deformation was observed in 75% of the files after a single use and in 100% of the files after three and six uses. Microcracks were absent after single use but appeared in 25% and 87.5% of files after three and six uses, respectively, showing a statistically significant increase (p &#60; 0.001). Surface pitting also significantly increased among groups (p = 0.004).
No fractures were observed in any group. The most common defects were tip deformation (91.7%) and surface pitting (70.8%). The findings suggest that repeated use of NiTi files significantly increases surface defects, elevating the risk of fatigue fractures. Thus, the results recommend limiting the reuse of Remover files to a maximum of 3x. Further research is needed to correlate defect types with anatomical factors and to assess file effectiveness in retreatment scenarios.

Deformation was observed in 75% of files after single use and in 100% of files after three and six uses, but the differences between the groups were not statistically significant (Table 1). The evaluation of deformation types among groups is shown in Table 2. When the types of deformation were evaluated separately, no microcracks were observed after a single use, while microcracks were observed in 25% of the files after three uses and in 87.5% of the files after six uses; this difference...

Scanning Electron Microscopic Evaluation of Surface Defects of Remover Retreatment File After Single and Multiple Uses

Here, we present a protocol for evaluating the surface characteristics of endodontic retreatment files after repeated use in retreatment procedures, utilizing scanning electron microscopy to identify and analyze potential surface defects.

This study aimed to evaluate surface defects of Remover rotary Nickel-Titanium (NiTi) files after single and multiple uses in conventional endodontic ...

scanning-electron-microscopic-evaluation-surface-defects-remover

Research

JoVE Journal

Medicine

332 Views.  Marmara University. Here, we present a protocol for evaluating the surface characteristics of endodontic retreatment files after repeated use in retreatment procedures, utilizing scanning electron microscopy to identify and analyze potential surface defects.

Video: Scanning Electron Microscopic Evaluation of Surface Defects of Remover Retreatment File After Single and Multiple Uses

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher frequency of fractures 1. As these fractures often occur in regions that have lost normal sensory function, the patient is at a greater risk of fracture-dependent pathologies, including death. SCI-dependent loss in both bone mineral density (BMD, grams/cm2) and bone mineral content (BMC, grams) has been attributed to mechanical disuse 2, aberrant neuronal signaling 3 and hormonal changes 4. The use of rodent models of SCI-induced osteoporosis can provide invaluable information regarding the mechanisms underlying the development of osteoporosis following SCI as well as a test environment for the generation of new therapies 5-7 (and reviewed in 8). Mouse models of SCI are of great interest as they permit a reductionist approach to mechanism-based assessment through the use of null and transgenic mice. While such models have provided important data, there is still a need for minimally-invasive, reliable, reproducible, and quantifiable methods in determining the extent of bone loss following SCI, particularly over time and within the same cohort of experimental animals, to improve diagnosis, treatment methods, and/or prevention of SCI-induced osteoporosis.
An ideal method for measuring bone density in rodents would allow multiple, sequential (over time) exposures to low-levels of X-ray radiation. This study describes the use of a new whole-animal scanner, the IVIS Lumina XR (Caliper Instruments) that can be used to provide low-energy (1-3 milligray (mGy)) high-resolution, high-magnification X-ray images of mouse hind limb bones over time following SCI. Significant bone density loss was seen in the tibiae of mice by 10 days post-spinal transection when compared to uninjured, age-matched control (na&iuml;ve) mice (13% decrease, p&lt;0.0005). Loss of bone density in the distal femur was also detectable by day 10 post-SCI, while a loss of density in the proximal femur was not detectable until 40 days post injury (7% decrease, p&lt;0.05). SCI-dependent loss of mouse femur density was confirmed post-mortem through the use of Dual-energy X-ray Absorptiometry (DXA), the current "gold standard" for bone density measurements. We detect a 12% loss of BMC in the femurs of mice at 40 days post-SCI using the IVIS Lumina XR. This compares favorably with a previously reported BMC loss of 13.5% by Picard and colleagues who used DXA analysis on mouse femurs post-mortem 30 days post-SCI 9. Our results suggest that the IVIS Lumina XR provides a novel, high-resolution/high-magnification method for performing long-term, longitudinal measurements of hind limb bone density in the mouse following SCI.

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

A longitudinal examination of bone loss in the femurs and tibiae of adult mice was performed following spinal cord injury using sequential low-dose X-ray scans. Tibia bone loss was detected throughout the study, while bone loss in the femur was not detected until 40 days post injury.

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher ...

Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem cells&#8221; (CSCs) responsible for tumor initiation, maintenance and relapse following conventional chemotherapies. Identification of ovarian CSCs is therefore an important goal. A commonly used method to assess CSC potential in vitro is the spheres assay in which cells are plated under non-adherent culture conditions in serum-free medium supplemented with growth factors and sphere formation is scored after a few days. Here, we review currently available protocols for human ovarian cancer spheres assays and perform a side-by-side analysis between commonly used multi cell-based assays and a more accurate system based on single cell plating. Our results indicate that both multi cell-based as well as single cell-based spheres assays can be used to investigate sphere formation in vitro. The more laborious and expensive single cell-based assays are more suitable for functional assessment of individual cells and lead to overall more accurate results while multi cell-based assays can be strongly influenced by the density of plated cells and require titration experiments upfront. Methylcellulose supplementation to multi cell-based assays can be effectively used to reduce mechanical artifacts.

In vitro spheres assays are commonly used to identify cancer stem cells. Here we compare single with multi cell-based spheres assays. The more laborious single cell-based assays or methylcellulose supplementation give more accurate results while multi cell-based assays performed in liquid medium can be highly influenced by cell density.

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem ...

Evaluation of Patients' Posture and Gait Profile After Lumbar Fusion Surgery by Video Rasterstereography and Treadmill Gait Analysis

This protocol provides guidance on how to perform high resolution video rasterstereography and treadmill gait analysis on patients after lumbar fusion surgery to obtain results about altered variables of gait and posture. These observed changes can then be correlated with the patient-reported outcome measure of pain relief. The rasterstereographic device projects lines of parallel light onto the surface of the tested subject&#39;s back. The deformation of these lines is recognized by the device. From these data, a special software then generates a 3-D profile based on the principle of triangulation. With an inaccuracy of only 0.2 mm it can measure changes in posture at very high precision. Gait and stance parameters are recorded using a treadmill equipped with an electric sensor mat that contains 10,200 miniature force sensors in the registering zone under the belt. Initial walking speed on the treadmill is 0.5 km/h. Speed is then gradually increased by increments of 0.1 km/h until each subject reaches his or her individual maximum well tolerable walking speed. At this speed, parameters are recorded during a 20 s measurement interval. Subjects are tested barefoot and without holding a handrail. Among various other parameters, stride width, step length, stance phase and foot rotation are measured. Both methods used reportedly have a high intra- and inter-observer reliability. The advantage of these highly accurate techniques is that they offer an objective and very detailed perspective on changes in the patient&#39;s posture and gait. Due to the amount of data generated, these techniques are, however, not so much suitable for everyday routine use, but rather interesting to scientifically evaluate long term alterations in posture and gait in patients like for example after lumbar fusion surgery.

Here, we present a protocol to analyze the posture and the gait of patients after lumbar fusion surgery by means of high-resolution video rasterstereography and a treadmill equipped with an integrated sensor mat. Allowing critical functional postoperative evaluation on a less subjective level may enhance accuracy and reliability of indication for surgery.

This protocol provides guidance on how to perform high resolution video rasterstereography and treadmill gait analysis on patients after lumbar fusion ...

Evaluation of Coronary Flow Reserve After Myocardial Ischemia Reperfusion in Rats

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via recanalization of the coronary artery is the most effective strategy for reducing the size of ischemic myocardium. The coronary microvasculature cannot be visualized and imaged&#160;in vivo, but there are several invasive and noninvasive techniques that can be used to assess parameters which depend directly on coronary microvascular function. The endothelial function after ischemia reperfusion can be assessed also at the level of the coronary circulation via the coronary flow reserve (CFR).&#160;In this study, peak velocity of left anterior descending (LAD) coronary arteries was measured in rats in vivo via Transthoracic Doppler Echocardiography during resting and stress challenge (induced by Dobutamine). A normal heart can increase its coronary blood flow up to four times above the resting values during stress induction. Following ischemia reperfusion, we found a significantly diminished CFR, which can be used as a marker of coronary microvascular dysfunction. CFR has opened a window on the importance of microvascular dysfunction and has been shown to predict cardiovascular risk independent of whether the severe obstructive disease is present.

Coronary flow reserve (CFR), is defined as the ratio of maximal coronary blood flow to the resting coronary blood flow. We present a protocol for evaluating CFR in rats via ultrasound, which offers the opportunity to predict cardiovascular risk factors in the absence of obstructive coronary disease.

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via ...

3D Planning and Printing of Patient Specific Implants for Reconstruction of Bony Defects

We are in the midst of the 3D era in most aspects of life, and especially in medicine. The surgical discipline is one of the major players in the medical field using the constantly developing 3D planning and printing capabilities. Computer-assisted design (CAD) and computer assisted manufacturing (CAM) are used to describe the 3D planning and manufacturing of the product. The planning and manufacturing of 3D surgical guides and reconstruction implants is performed almost exclusively by engineers. As technology advances and software interfaces become more user-friendly, it raises a question regarding the possibility of transferring the planning and manufacturing to the clinician. The reasons for such a shift are clear: the surgeon has the idea of what he wants to design, and he also knows what is feasible and could be used in the operating room. It allows him to be prepared for any scenario/unexpected results during the operation and allows the surgeon to be creative and express his new ideas using the CAD software. The purpose of this method is to provide clinicians with the ability to create their own surgical guides and reconstruction implants. In this manuscript, a detailed protocol will provide a simple method for segmentation using segmentation software and implant planning using a 3D design software. Following the segmentation and stl file production using segmentation software, the clinician could create a simple patient specific reconstruction plate or a more complex plate with a cradle for bone graft positioning. Surgical guides can be created for accurate resection, hole preparation for proper reconstruction plate positioning or for bone graft harvesting and re-contouring. A case of lower jaw reconstruction following plate fracture and nonunion healing of a trauma sustained injury is detailed.

This protocol describes the use of 3D planning and printing for reconstruction of bony defects. We use segmentation tools to create 3D models followed by 3D design software to create patient specific implants for reconstruction purposes concomitant to ablative surgery or as a second stage.

We are in the midst of the 3D era in most aspects of life, and especially in medicine. The surgical discipline is one of the major players in the medical ...

Microscopic Observation of Lymphocyte Dynamics in Rat Peyer's Patches

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

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

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

Visualizing Membrane Ruffle Formation using Scanning Electron Microscopy

Membrane ruffling is the formation of motile plasma membrane protrusions containing a meshwork of newly polymerized actin filaments. Membrane ruffles may form spontaneously or in response to growth factors, inflammatory cytokines, and phorbol esters. Some of the membrane protrusions may reorganize into circular membrane ruffles that fuse at their distal margins and form cups that close and separate into the cytoplasm as large, heterogeneous vacuoles called macropinosomes. During the process, ruffles trap extracellular fluid and solutes that internalize within macropinosomes. High-resolution scanning electron microscopy (SEM) is a commonly used imaging technique to visualize and quantify membrane ruffle formation, circular protrusions, and closed macropinocytic cups on the cell surface. The following protocol describes the cell culture conditions, stimulation of the membrane ruffle formation in vitro, and how to fix, dehydrate, and prepare cells for imaging using SEM. Quantification of membrane ruffling, data normalization, and stimulators and inhibitors of membrane ruffle formation are also described. This method can help answer key questions about the role of macropinocytosis in physiological and pathological processes, investigate new targets that regulate membrane ruffle formation, and identify yet uncharacterized physiological stimulators as well as novel pharmacological inhibitors of macropinocytosis.

Macropinocytosis is a highly conserved endocytic process initiated by the formation of F-actin-rich sheet-like membrane projections, also known as membrane ruffles. Increased rate of macropinocytotic solute internalization has been implicated in various pathological conditions. This protocol presents a method to quantify membrane ruffle formation in vitro using scanning electron microscopy.

Membrane ruffling is the formation of motile plasma membrane protrusions containing a meshwork of newly polymerized actin filaments. Membrane ruffles may ...

Repetitive Blood Sampling from the Subclavian Vein of Conscious Rat

Rats are widely used in pharmacokinetics (PK) and toxicokinetic (TK) studies that need to collect a certain amount of blood at specific time points to detect drug exposure. The rat blood sampling method determines the quality of the plasma and further affects the precision of the test results. The subclavian vein blood collection method described in this protocol collects blood samples repeatedly in the conscious state of animals to meet the needs of PK and TK tests. The skills of restraint handling and appropriate procedure of needle incision ensure the success rate of blood collection. It is easy to operate while ensuring the quality of plasma and at the same time catering to animal welfare. However, this method requires skilled operation, and an improper one may cause animal weakness, pain, lameness, and even mortality. The current method has been used in the testing facility for a 4-week oral toxicity study in Sprague Dawley (SD) rats with TK. The maximum amount of blood collected within 24 h did not exceed 20% of the animal's total blood. The animals' body weight was more than 200 g for males and females. The data showed that the animals' bodyweight increased steadily every week, and the clinical observation was normal after repetitive sample collection.

The present protocol describes a simple and efficient method for collecting blood from the subclavian vein in rats. It enables rapid, timely, and easily identifiable sampling without anesthesia and obtains high-quality blood through repetitive sample collection.

Rats are widely used in pharmacokinetics (PK) and toxicokinetic (TK) studies that need to collect a certain amount of blood at specific time points to ...

Development and Evaluation of a Rat Model of Full-Thickness Cartilage Defects

Cartilage defects of the knee joint caused by trauma are a common sports joint injury in the clinic, and these defects result in joint pain, impaired movement, and eventually, knee osteoarthritis (kOA). However, there is little effective treatment for cartilage defects or even kOA. Animal models are important for developing therapeutic drugs, but the existing models for cartilage defects are unsatisfactory. This work established a full-thickness cartilage defects (FTCD) model by drilling holes in the femoral trochlear groove of rats, and the subsequent pain behavior and histopathological changes were used as readout experiments. After surgery, the mechanical withdrawal threshold was decreased, chondrocytes at the injured site were lost, matrix metalloproteinase MMP13 expression was increased, and type II collagen expression decreased, consistent with the pathological changes observed in human cartilage defects. This methodology is easy and simple to perform and enables gross observation immediately after the injury. Furthermore, this model can successfully mimic clinical cartilage defects, thus providing a platform for studying the pathological process of cartilage defects and developing corresponding therapeutic drugs.

This protocol establishes a full-thickness cartilage defects (FTCD) model by drilling holes in the femoral trochlear groove of rats and measuring the subsequent pain behavior and histopathological changes.

Cartilage defects of the knee joint caused by trauma are a common sports joint injury in the clinic, and these defects result in joint pain, impaired ...

Developing an Exercise Program for Rheumatoid Arthritis Patients

Evaluation of Changes in Hydration and Body Cell Mass with Bioelectrical Impedance Analysis after Exercise Program for Rheumatoid Arthritis Patients

Rheumatoid arthritis (RA) is a debilitating disease that can result in complications such as rheumatoid cachexia. While physical exercise has shown benefits for RA patients, its impact on hydration and body cell mass remains uncertain. The presence of pain, inflammation, and joint changes often restrict activity and make traditional body composition assessments unreliable due to altered hydration levels. Bioelectrical impedance is a commonly used method for estimating body composition, but it has limitations since it was primarily developed for the general population and does not consider changes in body composition. On the other hand, bioelectrical impedance vectorial analysis (BIVA) offers a more comprehensive approach. BIVA involves graphically interpreting resistance (R) and reactance (Xc), adjusted for height, to provide valuable information about hydration status and the integrity of the cell mass.
Twelve women with RA were included in this study. At the beginning of the study, hydration and body cell mass measurements were obtained using the BIVA method. Subsequently, the patients participated in a six-month dynamic exercise program encompassing cardiovascular capacity, strength, and coordination training. To evaluate changes in hydration and body cell mass, the differences in the R and Xc parameters, adjusted for height, were compared using BIVA confidence software. The results showed notable changes: resistance decreased after the exercise program, while reactance increased. BIVA, as a classification method, can effectively categorize patients into dehydration, overhydration, normal, athlete, thin, cachectic, and obese categories. This makes it a valuable tool for assessing RA patients, as it provides information independent of body weight or prediction equations. Overall, the implementation of BIVA in this study shed light on the effects of the exercise program on hydration and body cell mass in RA patients. Its advantages lie in its ability to provide comprehensive information and overcome the limitations of traditional body composition assessment methods.

Author Spotlight: Implementation of BIVA for Analyzing Disease Risk Factors in Patients with Low Body Cell Mass 

This protocol assesses alterations in hydration and body cell mass status using bioelectrical impedance vectorial analysis following a dynamic exercise program designed for patients with rheumatoid arthritis. The dynamic exercise program itself is detailed, highlighting its components focused on cardiovascular capacity, strength, and coordination. The protocol details steps, instruments, and limitations.

Rheumatoid arthritis (RA) is a debilitating disease that can result in complications such as rheumatoid cachexia. While physical exercise has shown ...