Jove-Journal of Visualized Experiments最新文献

Combined antiretroviral therapy (cART) has dramatically improved the quality of life for people living with HIV (PLWH). However, over 4 million PLWH are over the age of fifty and experience accompanying HIV-associated neurocognitive disorders (HAND). To understand how HIV impacts the central nervous system, a reliable and feasible model of HIV is necessary. Previously, a novel biological system using chimeric HIV (EcoHIV) inoculation was developed in a rat model to investigate neurocognitive impairments and synaptic dysfunction. Nevertheless, a significant challenge remains in clarifying EcoHIV's neuroanatomical distribution, particularly its differential expression in various cell types in the brain. In the current study, EcoHIV with mScarlet fluorescence labeling was modified and retro-orbitally injected into Tmem119-EGFP knock-in mice (which express enhanced green fluorescence protein primarily in microglia) to determine if microglia are the major cell type responsible for viral expression and reservoirs of HIV in the brain. The current data show that: (1) in vitro, EcoHIV-mScarlet fluorescence signals were predominantly localized in microglia-like cells among primary rodent brain cells; (2) in vivo, injection of EcoHIV-mScarlet into Tmem119-EGFP mice induced significant HIV expression in the mouse brain. The co-localization of mScarlet and EGFP signals suggests that microglia are the main cell type harboring HIV in the brain. Overall, EcoHIV in rodents offers a valuable biological system to study microglial alterations, viral reservoirs in the brain, and the neurological mechanisms of HIV-associated neurocognitive disorders.

Laparoscopic partial splenectomy (LPS) is gradually becoming the preferred method for treating benign splenic lesions. However, due to the abundant blood supply and its soft, fragile tissue texture, especially when the lesion is located near the splenic hilum or is particularly large, performing partial splenectomy (PS) in clinical practice is extremely challenging. Therefore, we have been continuously exploring and optimizing hemorrhage control methods during PS, and we here propose a method to perform LPS with complete spleen blood flow occlusion. This study describes an optimized approach to control intraoperative hemorrhage during LPS. First, it involves the thorough dissection of the splenic ligaments and careful separation of the pancreatic tail from the spleen. With complete exposure to the splenic hilum, we temporarily occlude the entire blood supply of the spleen using a laparoscopic bulldog clip. Subsequently, we employ intraoperative ultrasound to identify the boundary of the lesion and resect the corresponding portion of the spleen under complete blood flow control. This approach embodies the essence of 'spleen preservation' through effective hemorrhage control and precise resection. It is particularly suitable for laparoscopic surgery and deserves further clinical promotion.

Live imaging methods allow the analysis of dynamic cellular processes in detail and in real-time. The Drosophila ovary represents an excellent model to explore the dynamics of a myriad of developmental processes, such as cell division, stemness, differentiation, migration, apoptosis, autophagy, cellular adhesion, etc., over time. Recently, we have implemented an extended ex vivo culture and live imaging of the female Drosophila GSC niche. Using a Drosophila line harboring a GFP::Par-1 transgene as an example, this method allows the visualization of the GSCs' asymmetric division within their niche and the description of the changes in the spectrosome morphology along the cell cycle. Here, we present a detailed protocol for the ex vivo culture of Drosophila germaria, enabling prolonged visualization of the female GSC niche. Importantly, this protocol is broadly applicable to live imaging GSCs with multiple fluorescently tagged proteins of interest that are available in stock centers and/or in the Drosophila research community.

Ischemia-reperfusion injuries are known to cause a range of retinal pathologies, including diabetic retinopathy, glaucoma, retinal vascular occlusions, and other vaso-occlusive conditions. This manuscript presents a method for inducing ischemia-reperfusion injury in a mouse model. The method utilized anterior chamber cannulation attached to a saline reservoir, generating hydrostatic pressure to raise the intraocular pressure to 90-100 mmHg. This method effectively caused constriction of retinal capillaries to induce retinal ischemia. At the end of the ischemic period (60 min), the intraocular pressure was normalized (≤20 mmHg) before removing the cannula from the anterior chamber to initiate reperfusion. Days after the ischemia/reperfusion procedure, the eyes were collected and sectioned for histological staining. The histopathology of the retinal sections was scored by evaluating eight parameters of retinal injury: folds, hemorrhage, deformation, cell loss in the ganglion cell, inner nuclear, outer nuclear, and photoreceptor layers, and damage to retinal pigment epithelial cells. This method provided a reproducible model to study the mechanisms and pathology of retinal ischemia/reperfusion injury. In addition, this model can facilitate the discovery of potential therapeutic targets to treat retinal ischemia/reperfusion injury, advancing the study of retinal pathologies and improving patient outcomes.

Intravenous (IV) injection is widely recognized as the most effective and commonly utilized method for achieving systemic delivery of substances in mammalian research models. However, its application in adult zebrafish for drug delivery, stem cell transplantation, and regenerative and cancer studies has been limited due to the challenges posed by their small body size and intricate blood vessels. To overcome these limitations, alternative injection techniques such as intracardiac and retro-orbital (RO) injection have been explored in the past for stem cell transplantation in adult zebrafish. However, these techniques have their drawbacks, including the need for meticulous injection techniques or increased risk of mortality. In this study, we have developed a refined and optimized IV injection procedure specifically tailored to adult zebrafish, addressing the challenges associated with their unique anatomy. To demonstrate the effectiveness of this technique, we performed successful IV injections of whole kidney marrow cells from Tg(mpo: EGFP) fish and FITC-dextran dye into adult Casper fish. The subsequent visualization of injected cells and dyes using a fluorescence microscope confirmed their successful delivery and engraftment within the zebrafish. Furthermore, we demonstrated that compared with the intracardiac and RO injections, the IV injection resulted in improved survival rates and engraftment efficiency in treated zebrafish. This approach enables precise delivery and localization of substances and holds great potential for large-scale drug and chemical screening using adult zebrafish. Additionally, the ability to visually track the injected cells and dyes provides invaluable insights into their engraftment, migration, and interactions with host tissues, enabling a more comprehensive evaluation of therapeutic effects and biological processes in zebrafish models.

Patient-derived xenografts (PDXs) provide a clinically relevant method for recapitulating tumor-involved cell types and the tumor microenvironment, which is essential for advancing knowledge of breast cancer (BC). Additionally, PDX models enable the study of BC systemic effects, which is not possible using in vitro models. Traditional methods for implanting BC xenografts typically involve anesthesia and sterile surgical procedures, which are time-consuming, invasive, and limit the scalability of PDX models in BC research. This protocol describes a simple and scalable method for the orthotopic implantation of BC PDXs in mice. The immunodeficient mouse strain NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ (NSG) was used for PDX engraftment. Human BC samples obtained from IRB-consented patients were mechanically and enzymatically dissociated, then resuspended in a solution of basement membrane extract (BME) and RPMI 1640. Animals were restrained by scruffing, and depilatory cream was applied to remove hair from the fat pads at the fourth inguinal nipple, followed by injection. Approximately 2 million cells in a 100 µL suspension were bilaterally injected orthotopically into the mammary fat pads using a 26 G needle. Notably, no anesthetic was required, and the total procedure time was under 5 min, from cell preparation to injection. After a growth period of several months, tumors were excised and processed for authentication. Validation included receptor status assessment using immunohistochemistry with specific antibodies for traditional BC receptors (i.e., ER, PR, HER2). Tumor morphology was confirmed with hematoxylin and eosin (H&E) staining, which was interpreted by a pathologist. Genetic similarity to the patient sample was verified through bulk RNA sequencing and short tandem repeat (STR) analysis. This approach to PDX engraftment and validation supports the rigorous development of models and high-throughput tumor implantation, enabling well-powered studies across various BC subtypes.

Acupuncture Point Embedding Therapy is a comprehensive treatment method that combines modern biomaterials with acupuncture techniques. Acupoint catgut embedding (ACE) not only avoids the adverse reactions associated with Western medicine but also incorporates the benefits of traditional acupuncture, extending the duration of its therapeutic effects. Allergic rhinitis (AR) is a condition known for its challenging treatment and tendency to persist. This randomized controlled trial included 128 AR patients to evaluate the effectiveness of ACE for AR management. Data were collected at baseline, four weeks after treatment, and eight weeks after treatment. Baseline analysis was performed, and the visual analog scale (VAS) was used as a clinical observation index for this method. The rhinoconjunctivitis quality of life questionnaire (RQLQ) served as a quality of life index. Specific IgE (sIgE), IL-4, IL-10, and IL-12 were used as laboratory observation indices. The results of the experiment validate the clinical effectiveness of ACE in treating AR. Although this technique is efficient and commonly used in clinics, its invasive nature poses risks of hospital-acquired infections. Based on this experiment, this protocol refines traditional techniques, providing detailed instructions on patient preparation, operation techniques, and postoperative care to ensure the safe and effective administration of therapy. By standardizing this therapy, ACE is anticipated to become an important non-drug treatment option for alleviating symptoms in AR patients, thereby significantly enhancing their quality of life.

Robot-assisted pancreaticobiliary junction resection is a surgical technique employed to treat benign duodenal tumors. The procedure involves several key steps: making a longitudinal incision in the duodenum, excising the tumor at the pancreaticobiliary junction, inserting a biliary stent, connecting the biliary and duodenal mucosa, and suturing the duodenal incision during phase I. The robotic system enhances visibility, facilitates precise operations, minimizes duodenal traction injuries to the duodenum and surgical trauma, ensures accurate suture and fixation of bile duct stents, connects the bile duct and duodenal mucosa and reduces postoperative recovery time. Given the complexity of the operation and the associated risk of postoperative duodenal fistula, a thorough preoperative evaluation and meticulous perioperative preparation are crucial. Prior to the procedure, a comprehensive assessment was conducted, integrating the patient's medical history, family history, serological tests, and imaging studies. Special emphasis was placed on determining the benign or malignant nature of the tumor and evaluating the status of the duodenal artery blood supply network to ascertain the feasibility and efficacy of the surgery. During the operation, efforts were made to minimize duodenal trauma and avoid compromising the duodenal artery blood supply network. Additionally, the use of bile duct stents was considered essential to prevent biliary strictures, facilitate bile discharge, and mitigate biliary complications. Postoperatively, real-time monitoring of amylase and jaundice indicators in drainage fluid informed the timely removal of drainage tubes in accordance with the enhanced recovery after surgery (ERAS) protocol. Subsequent follow-up indicated a successful recovery, characterized by a notable reduction in preoperative abdominal pain, the absence of long-term complications, and no evidence of tumor recurrence. Consequently, robot-assisted pancreaticobiliary junction resection demonstrates a safe and effective surgical approach for the treatment of benign duodenal tumors.

A successful positron emission tomography imaging program involving carbon-11 radiotracers demands fast, efficient, and reliable synthesis methods, requiring an on-site cyclotron and radiochemistry group, as well as clinical staff trained to operate under the unique constraints of the carbon-11 radionuclide. This study examines the merits and advantages of a captive solvent 'loop method' of radiolabeling four tracers with the carbon-11 radionuclide, producing the radioligands [¹¹C]ER-176, [¹¹C]MRB, [¹¹C]mHED, and [¹¹C]PiB. The 'loop method' is compared against the traditional reactor-based method of carbon-11 methylation in the course of synthesizing the same radiotracers on the identical automated platform. Further, a complete overview of the clinical research preparation of the [¹¹C]ER-176 radiotracer is presented. As demonstrated by the production of [¹¹C]ER-176, the captive solvent 'loop method' of heterogeneous alkylation proved to be more efficient, with excellent radiochemical purity (99.6 ± 0.6%, n = 25), higher and more consistent radiochemical yield (end of synthesis (EOS) = 5.4 ± 2.2 GBq, n = 25) compared to the reactor method (EOS = 1.6 ± 0.5 GBq, n = 6), increased molar activity (loop method = 194 ± 66 GBq/µmol, n = 25; reactor method = 132 ± 78 GBq/µmol, n = 6), along with an average 5 min shorter reaction sequence.

Under current minimally invasive treatment regimes, minor tooth preparation and thinner biomimetic ceramic restoration are used to preserve the restored tooth's vitality, aesthetics, and function. New computer-aided design and computer-aided manufacturing (CAD/CAM) ceramic-like material are now available. To guarantee longevity, a dental clinician must know these newly launched product's mechanical strength compared to the relatively brittle glass-matrix ceramic. Furthermore, a tooth substitute has been promoted for laboratory investigation, especially after the pandemic, and more evidentiary support is required for its application. This study developed a laboratory protocol for a monotonic load-to-fracture test to determine the fracture strength of 1 mm-thick CAD/CAM occlusal veneers. Master dies were milled from high-pressure fiberglass laminate, which has similar elastic modulus and bond strength as hydrated dentin. They were mounted into polyvinyl chloride (PVC) end caps with cold-curing epoxy resin. Occlusal veneers, also called tabletop restorations, were milled from lithium disilicate (LD) and resin nanoceramic blocks (RNC) and cemented to prepared master dies using dual-cured adhesive resin cement. They were allowed to cure fully by storing in distilled water for 48 h at 37 °C. All samples were then placed in a universal testing machine and loaded via a non-fixed 5.5 mm stainless-steel ball that allows lateral movement as would occur against the antagonist teeth. Compression was applied at a 1 mm/min rate, and the load-displacement graph was generated. The average maximum load-bearing capacity of restorations in the RNC group (3,212.80 ± 558.67 N) was significantly higher than in the LD group (2727.10 ± 472.41 N) (p < 0.05). No debonding was found during the test. Both CAD/CAM materials may have a similar flaw distribution. Hertzian cone crack was found at the loading site, whereas radial cracks propagating from the cementation surface were found close to the margin in both groups.