Establishment of Acute Pontine Infarction in Rats by Electrical Stimulation
August 27th, 2020
Presented here is a protocol for establishing acute pontine infarction in a rat model via electrical stimulation with a single pulse.
We provide a protocol to establish infarction in a pons of rat. This pontine infarction rat model was easily performed, easily reproduced, and highly successful model. The electrical stimulation could be used to make infarction in any other brain area like thalamus.
People who want to perform this model should learn the pontine autonomy before the experiment. Before the surgery, weigh the rats and assess their neurological performance according to the behavioral tests as described in the accompanying text. Preheat the heating pad to 37 degrees Celsius immediately before anesthesia and attach the skull drill to the holder on the stereotaxic frame.
Confirm a lack of response to pedal reflex in the first anesthetized rat and mount the rat onto the stereotaxic frame in the prone position on the heating pad. Position the ear bars above the ear canal to secure the head, taking care that the skull is kept horizontal to avoid any skewing of the injection. Apply ointment to the animal's eyes and use a micro shaver to remove the hair from the skull.
Use a marker to draw a three centimeter line along the midline of the skull from the line of the bilateral lateral canthus to 0.5 centimeters behind the posterior fontanelle and use a scalpel to make a midline incision along the marked line. Use a cotton swab to remove any blood and place a piece of surgical tape on each side of the skin flap to expose the skull. Use a new swab soaked with 0.9%sodium chloride to gently remove any connective tissue from the skull and locate the bregma.
Use a fine tip marker pen to indicate the central point of the bregma as the origin point, place the drill at six millimeters anterior posterior, two millimeters medial lateral. Use the drill to carefully perform a one millimeter diameter craniotomy. Next, replaced the drill with a 22 gauge probe with an insulated sheath with the tip placed two millimeters above the proximal end of the sheath.
Insert the sheets seven millimeters into the brain and advance the probe along the sheath until the top of the probe is nine millimeters below the surface of the brain. Connect the electrodes to an electrical stimulator and connect the anode to the probe. Then connect to the cathode to the ear of the rat.
Next, turn on the electrical stimulator and set the single pulse width to 4, 050 milliseconds, the voltage to 50 volts and the current to four milliamps. Leave the probe in position for five minutes after stimulation before removing the device from the brain. Cover the craniotomy with bone cement and allow the cement to dry before using 4.0 polyamide suture filaments to close the wound.
After three or four stitches, tie 2-1-1 standard surgical knots. Then intraperitoneally inject the rat with 250 microliters of penicillin and monitor the rat every 15 minutes until fully awake before returning the animal to its cage. In this analysis, six animals were subjected to the surgery as demonstrated and six animals were treated as controls.
The brain slices in these images were derived from one rat per group. MRI scanning revealed that the infarction was located in the basis of the pons mimicking anterolateral infarctions in human patients. Because an insulated sheath was used, the infarction was not observed in the cortex, cerebellum, or midbrain.
Diffusion weighted images also revealed the acute pontine infarction. TTC staining was used to confirm the infarction 24 hours post-surgery. As expected, the infarction volume was significantly higher in the surgery group compared to the control group.
Behavioral scores were measured before and after surgery. Compared to the control group, rats with a pontine infarction circled to the left. Significant differences were observed in the Longa, Berderson, limb placement, beam balance, and adhesive removal somatosensory test scores between rants with pontine infarction and control group rats.
Any specific brain area are going to be destroyed by the electrical stimulation.
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Overview
This study presents a protocol for establishing acute pontine infarction in a rat model through electrical stimulation with a single pulse. The model is reproducible and successful for investigating pontine infarctions, which mimic anterolateral infarctions in humans.
Key Study Components
Area of Science
- Neuroscience
- Experimental Models
- Electrophysiology
Background
- Pontine infarctions are relevant to understanding stroke mechanisms.
- Existing models may not have effectively mimicked human conditions.
- This protocol allows for targeted investigation of cerebrovascular diseases.
- It emphasizes the importance of pre-surgical assessments for animal welfare.
Purpose of Study
- To develop a reliable rat model for studying pontine infarctions.
- To assess neurological performance before and after inducing infarction.
- To validate the effectiveness of electrical stimulation in inducing localized brain damage.
Methods Used
- Involves a rat model and electrical stimulation to create an infarction.
- Neurological assessments are performed pre- and post-surgery.
- Critical steps include anaesthesia, craniotomy, and precise electrode placement.
- Post-surgical care includes monitoring and antibiotic administration.
Main Results
- The protocol successfully produced an acute pontine infarction.
- Behavioral tests indicated impaired performance in rats with infarction.
- Significant differences in neurological scores were noted compared to controls.
- MRI and TTC staining confirmed the presence and extent of infarction.
Conclusions
- The study establishes a valuable model for investigating pontine strokes.
- It facilitates further research into interventions for cerebrovascular diseases.
- The findings underscore the relevance of localized infarction models in neuroscience.
Frequently Asked Questions
Executive Industry Relevance
Establishing reliable rodent models of stroke subtypes is critical for preclinical target validation and mechanistic de-risking in cerebrovascular drug development. This protocol enables reproducible induction of acute pontine infarction, a common posterior circulation stroke, supporting hypothesis testing in neuroprotective and thrombolytic research. The model facilitates predictive confidence by mimicking human anterolateral pontine lesions and enabling quantitative behavioral and imaging readouts.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses in pontine stroke pathways through localized electrical lesioning.
- Operational Value: Provides a highly reproducible model for consistent target engagement and pathway modulation studies.
Screening & Assay Development
- Scientific Value: Generates quantifiable neurological and sensorimotor deficits for assay standardization and compound screening readiness.
- Operational Value: Supports development of scalable behavioral readouts (Longa, Berderson, beam balance, adhesive removal, limb placement) for high-throughput functional assessment.
Translational & Preclinical Research
- Scientific Value: Mirrors human anterolateral pontine infarction, enhancing translational relevance for cerebrovascular intervention studies.
- Operational Value: Permits longitudinal evaluation via MRI and TTC staining to correlate structural damage with functional outcomes.
Pipeline & Workflow Integration
The model fits within the discovery continuum from target validation to preclinical efficacy testing, enabling mechanistic de-risking of stroke therapeutics.
- Discovery Biology: Supports pathway clarification and target validation in pontine circuits via precise lesion induction.
- Screening: Delivers standardized neurological and proprioceptive outputs for reliable compound effect measurement.
- Analytics: Provides multimodal validation (behavioral scores, MRI, TTC) to enable cross-functional data comparison and threshold setting.
- Translational Research: Aligns with human stroke pathology to inform preclinical go/no-go decisions based on infarct location and severity.
- Enterprise Reuse: The electrical stimulation approach can be adapted to other brain regions, increasing platform utility across neuroscience discovery programs.
Operational & Enterprise Impact
- Scientific Value: Reduces mechanistic ambiguity in stroke modeling through anatomically precise, electrically induced infarction.
- Operational Value: Ensures reproducibility and standardization via stereotaxic guidance and defined stimulation parameters.
- Strategic Value: Improves risk-adjusted advancement by enabling early functional and structural assessment of lesion models.
- Portfolio Impact: Supports data-driven prioritization of cerebrovascular candidates using validated infarct volume and behavioral deficit correlations.
Implementation Considerations
- Requires expertise in stereotaxic surgery, neuroanatomy, and electrophysiology for accurate probe placement.
- Dependent on precision instrumentation including stereotaxic frame, electrical stimulator, and MRI/histology validation tools.
- Necessitates cross-team standardization of behavioral testing protocols and blinding for unbiased outcome assessment.
- Adaptation to other brain regions requires recalibration of coordinates and stimulation parameters to avoid off-target effects.
- Practical limitations include variability in skull thickness and bregma identification, which may affect targeting accuracy.
