Implementation of Minimally Invasive Brain Tumor Resection in Rodents for High Viability Tissue Collection
May 9th, 2022
The present protocol describes a standardized resection of brain tumors in rodents through a minimally invasive approach with an integrated tissue preservation system. This technique has implications for accurately mirroring the standard of care in rodent and other animal models.
This is a brain tumor resection paradigm that is highly needed in preclinical research on therapeutics for brain tumors. It allows a standardized resection through a minimally-invasive approach, while at the same time, it's also coupled to an automated tissue preservation system. This protocol allows animal to survive after surgery and thus, aids in understanding the changes in disease state after therapy in the same animal.
This helps in sequential and time-lapse therapeutic applications. Using this technique, we can evaluate the biology of the disease and plan for possible subsequent management. This has significant implications and opportunities in drug discovery and novel therapeutics.
The concepts of standardized resection, a minimally-invasive approach, and automated tissue preservation can be extended to other types of tumors in different organ systems, such as liver tumors and head and neck cancers. The protocol is simple and the system is user-friendly. It is best to read the published protocol, which contains all the necessary steps and troubleshooting.
Begin the experiment by assessing the mouse for sedation by pinching the toe. Apply ophthalmic ointment to the eyes to avoid dryness of the cornea. Then, place the mouse on a stereotactic frame.
Remove the staple from the previous surgery and disinfect the skin with alternating cycles of a chlorhexidine-or betaine-based scrub and alcohol. Then create a one-centimeter longitudinal midline incision along the previous surgical scar using a sterile scalpel. Attach the MIRS hand piece to the stereotactic arm through the stage adapter.
To set up the MIRS machine, insert the power cord set on the rear panel into the power cord receptacle. Turn the power to the system on or off by toggling between zero and one. Insert one end of the nitrogen hose into the male fitting on the rear panel of the console.
Rotate the connection nut clockwise to tighten it. Ensure that the supply pressure does not exceed 100 PSIG and attach the hose to the nitrogen supply. Seal the lid of the vacuum port to avoid any leakage.
Check that the aspiration knob on the front of the console is set at 100, that there is no leakage in the aspiration system, and that the nitrogen input supply pressure is correct. Insert the gray foot pedal connector into its gray receptacle until it clicks. In the same way, insert the blue handpiece connector into its blue receptacle.
Prime each handpiece by aspirating sterile fluid into the aperture through the tubing and handpiece and then into the canister to ensure that the inside of the tubing and handpiece are lubricated. Select the mode for aspiration on the console front panel and initiate using the foot pedal. Insert the 23-G MIRS cannula into the burr hole to a depth of 2.5 millimeters.
Initiate the resection process by pressing the foot pedal connected to the cannula. Perform full cycles of resection using the control knob in the handpiece. After the resection process, withdraw the 23-gauge MIRS cannula and add five milliliters of 1X PBS to flush the tubing and dislodge any residual debris.
Then, close the wound with a stapler and remove the mouse from the stereotactic frame. Return the mouse to the heating pad to recover from anesthesia before placing it back into its cage. After the experiment, purge the cannula with chilled media and air to push all of the resected tissue back to the collection canister.
Remove the collection canister from the system and place a cap. Next, place the distal tip of the cannula into 3%hydrogen peroxide and apply suction to fill the suction line back to the suction collection canister. Let it stand for 60 to 90 seconds and intermittently pulse air and media to flush it.
Immerse the tumor sample in a tissue culture dish containing RBC lysis medium for five minutes at room temperature. Then place a 70-micron filter on a 50-milliliter conical tube and use a syringe plunger to pass the tumor sample through the filter. With a transfer pipette, use the RPMI 1640 media to pass the cells and any tissue mass through the filter.
Centrifuge at 428 times G for five minutes at four degrees Celsius. Discard the supernatant and resuspend each sample in five milliliters of prepared RPMI 1640 medium. Place the samples in a shaker incubator for 20 minutes at 200 RPM at 37 degrees Celsius.
After incubation, centrifuge the samples at 428 times G for five minutes at four degrees Celsius and discard the supernatant. Filter single cells through a 70-micron cell strainer and centrifuge at 274 times G for three minutes at four degrees Celsius. Conduct cell viability analysis with trypan blue and a hemocytometer.
Surgical resection in mice using MIRS caused a significant decrease in the tumor burden as indicated by the reduction in the mean baseline bioluminescent signal for groups with both large and small tumor burden. Compared to the pre-resection MRI images of tumors, the resection cavity can be identified on the post-resection scans as a large, round hypointense area at the tumor inoculation site. In H&E stained sections, a clear circular resection cavity with a rim of blood products, inflammation, and residual tumor cells were observed.
The resection volume significantly increased when two rotations of the cutting aperture were performed, which allowed optimization of resection volume as per tumor burden. Comparison of survival in mice with untreated tumors versus mice undergoing resection of tumors by MIRS showed an increase in survival from 16 to 22 days in the small tumor group. Similarly, in the group with a larger tumor burden, the median survival increased from 12 to 19 days.
Light microscopy images of the samples taken from the tissue preservation system appeared as single cells with the presence of a few small chunks of tissue on day zero. After seven days, the cells harvested with MIRS showed neurosphere formation in suspension cultures, which indicated their tumor initiation potential. It's important to purge and flush the tubing system right after the procedure to avoid a clogging the system.
Following this procedure, efficacy studies of therapeutics and studies on diagnostic and prognostic markers can be used on surviving animals after surgical resection with the minimally-invasive resection system.
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Overview
This protocol outlines a standardized method for resecting brain tumors in rodents using a minimally invasive approach integrated with a tissue preservation system. This technique enhances the accuracy of preclinical research by allowing for the survival of animals post-surgery, facilitating the study of disease progression and therapeutic effects.
Key Study Components
Area of Science
- Neuroscience
- Preclinical Research
- Oncology
Background
- Brain tumor research is critical for developing effective therapies.
- Standardized surgical techniques improve reproducibility in studies.
- Minimally invasive approaches reduce recovery time and complications.
- Tissue preservation is essential for subsequent analyses.
Purpose of Study
- To establish a reliable method for brain tumor resection in rodent models.
- To enable longitudinal studies on tumor biology and treatment responses.
- To support drug discovery efforts through improved experimental design.
Methods Used
- Minimally invasive surgical techniques for tumor resection.
- Automated tissue preservation systems to maintain sample integrity.
- Post-operative monitoring to assess animal recovery.
- Sequential therapeutic applications to evaluate treatment efficacy.
Main Results
- The protocol allows for successful tumor resection with minimal complications.
- Animals demonstrate good recovery and can be used for follow-up studies.
- Facilitates the assessment of therapeutic interventions over time.
- Provides a framework for future research in brain tumor therapeutics.
Conclusions
- This technique represents a significant advancement in preclinical brain tumor research.
- It enhances the ability to study disease dynamics and treatment outcomes.
- Future applications may lead to improved therapeutic strategies for brain tumors.
Frequently Asked Questions
Executive Industry Relevance
Standardized minimally invasive brain tumor resection in rodents enables high-viability tissue collection and post-surgical survival, directly addressing a critical gap in preclinical oncology models. This approach supports longitudinal therapeutic evaluation and reduces experimental variability, enhancing predictive confidence for translational research. The protocol's integration of automated tissue preservation aligns with enterprise needs for reproducibility and scalable workflow integration.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables rigorous interrogation of tumor biology and perioperative therapeutic effects in vivo.
- Reduces mechanistic ambiguity by allowing sequential interventions and disease-state monitoring in the same animal.
- Supports functional target validation through high-viability tissue and cell recovery for downstream analyses.
Screening & Assay Development
- Provides standardized, reproducible tissue samples for assay development and biomarker discovery.
- Facilitates preparation of validated biological systems for compound screening and efficacy studies.
- Improves assay scalability and platform reuse by minimizing operative variability and tissue loss.
Translational & Preclinical Research
- Enables modeling of surgical intervention and perioperative management relevant to clinical oncology.
- Supports continuity from discovery through preclinical validation by allowing survival and longitudinal assessment.
- Aligns with translational biomarker strategies by preserving viable tumor cells for molecular and functional studies.
Pipeline & Workflow Integration
This protocol bridges early discovery, lead identification, and preclinical validation by enabling standardized surgical modeling and tissue collection in rodents.
- Discovery Biology: Supports hypothesis testing and pathway clarification through controlled resection and tissue analysis.
- Screening: Delivers reproducible, high-quality samples for quantitative assay outputs and comparative studies.
- Analytics: Provides viable cells and tissues for downstream molecular, cellular, and viability measurements.
- Translational Research: Models clinical surgical scenarios and supports biomarker alignment for preclinical-to-clinical continuity.
- Enterprise Reuse: Establishes a reusable, scalable platform for diverse tumor models and therapeutic evaluations.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces biological risk in preclinical oncology studies.
- Operational Value: Enhances standardization, reproducibility, and throughput in tissue collection workflows.
- Strategic Value: Improves go/no-go decision-making and capital efficiency by enabling robust, longitudinal data generation.
- Portfolio Impact: Supports risk-adjusted prioritization and advancement of therapeutic candidates.
Implementation Considerations
- Requires surgical expertise in rodent models and familiarity with stereotactic systems.
- Needs access to the minimally invasive resection device and integrated tissue preservation infrastructure.
- Demands cross-team standardization for consistent tissue handling and viability assessment.
- Adaptable to other tumor models with consideration for anatomical and procedural differences.
- Timely system flushing and maintenance are critical to prevent clogging and ensure sample integrity.
