Microiontophoresis and Micromanipulation for Intravital Fluorescence Imaging of the Microcirculation
June 10th, 2011
Microiontophoresis entails movement of ions from a micropipette in response to a difference in electrical potential between the inside and outside of the micropipette. Biologically active molecules are thereby delivered in proportion to electrical current. We illustrate acetylcholine microiontophoresis in conjunction with micromanipulation to study endothelium-dependent vasodilation in the microcirculation.
The overall goal of this procedure is to deliver a charged substance such as the positively charged neurotransmitter, a subtle choline to a discrete site within the experimental preparation. This is accomplished by preparing micro pipettes from glass capillary tubes using a horizontal micro pipette puller backfilling the micro pipettes with a subtle choline and securing the micro pipette in a micro manipulator. The micro pipette tip is then positioned adjacent to a site of interest.
While a negative retaining current is set to prevent leakage of a subtle choline, a subtle choline is ejected from the micro pipette by applying a positive current at a defined intensity and duration while recording fluorescence and diameter responses in the microcirculation, the signals measure calcium fluorescence underlying endothelium dependent vasodilation of arterials controlling tissue blood flow. I'm Dr.Steve Siegel. This method can be used to provide insight into the cellular signaling events that underlie blood flow control and arterials.
It can also be applied to neurophysiology to study synaptic transmission and the activation of ion channels. I'm Dr.Pune Badger. Generally, individuals new to this technique may struggle because it takes practice to learn how to prepare effective micro pipettes to perform micro-manipulation and to adjust the retain and injection currents to control the delivery of the drug from the micro pipette.
Begin by using a pipette puller to prepare micro iontophoresis, micro pipettes from bo silicate glass capillary tubes with an internal glass filament. A five millimeter taper is preferred to provide stiffness, whereas longer tapers are more flexible. Next, secure a backfill needle attached to a 0.2 micrometer filter onto a syringe containing one molar acetylcholine chloride dissolved in 18.2 mega ohm water.
Now insert the backfill needle into the back end of the micro pipette and advance it to the tip. Inject the acetylcholine into the micro pipette while withdrawing the backfill needle as the micro pipet is filled. To remove any air bubbles, hold the micro pipette with its tip pointing down and gently flick the micro pipette.
Next, secure the micro pipette in a micro pipette holder. Then secure the micro pipette holder in a three axis micro manipulator, like a compact model mounted to a magnetic base. Connect both the positive and negative wires to the micro iontophoresis programmer Using wires with an alligator clip at the end, a silver wire is secured to the edge of the preparation to serve as the reference electrode.
Using the alligator clip, connect the negative wire coming from the micro iontophoresis programmer to the reference electrode. Then connect the positive wire from the micro iontophoresis programmer to the external pen of the micro pipette holder. With the second delegator clip, the circuit is completed when the tip of the micro pipette is advanced into the physiological saline solution.
Over the preparation, this microscope is outfitted with a custom platform to increase the surface area of the XY stage. The platform is fabricated from ferromagnetic stainless steel and it provides sufficient space to position the micro manipulator around the experimental preparation as desired. Position the experimental preparation on the platform.
In this example, muscle tissue has been prepared using the cream master muscle preparation protocol from an associated video. Now position the micro manipulator relative to the preparation so that the micro pipette tip is located above the site of interest. Now, to avoid dilating, the arterials apply a retaining current using the micro pheresis programmer.
The retaining current varies with the size of the micropipet tip and agonist concentration, but is highly reproducible for defined parameters. Next, using the micro manipulator controls while viewing through the microscope carefully position the tip of the micropipet next to an arteriolar wall. With practice, this can be accomplished without breaking the tip or damaging the tissue.
To eject a subtle choline trigger the micro iontophoresis programmer with a TTL pulse of at least five volts. Using commercial software, an electronic trigger can be sent to the computer that simultaneously sends the TTL pulse and starts the recording of digital images from the camera. The duration of the stimulus pulse is controlled by the software.
The micro phoresis programmer controls the ejection current. When it receives the TTL pulse, the retaining current is withdrawn and the ejection current is delivered. This mouse arterial has endothelial cells expressing calcium sensitive GFP, which fluoresces proportionally to rises in intracellular calcium above a threshold level, a subtle choline triggered a fluorescence response as the duration of the acetylcholine stimulus increased.
Endothelial cell calcium fluorescence in the arterial also increased. The intensity of fluorescence increased at the site of stimulation and fluorescence spread over progressively greater distances along the arterial. When performing this procedure, it is important to remember that the effectiveness of micro phoresis is determined by the internal diameter of the tip, the concentration of agonist within the micro pipette, and the intensity and the duration of the ejection current.
After watching this video, you should have a good understanding of how to use micro pheresis to deliver a charged molecule from a micro pipette at a designated site. In the experimental preparation.
Overview
Microiontophoresis is a technique used to deliver charged substances, such as acetylcholine, to specific sites in biological preparations. This method allows researchers to study cellular signaling events related to blood flow control and synaptic transmission.
Key Study Components
Area of Science
- Neuroscience
- Physiology
- Cellular Biology
Background
- Microiontophoresis involves the movement of ions from a micropipette due to electrical potential differences.
- It is essential for studying endothelium-dependent vasodilation in microcirculation.
- The technique requires precise preparation and manipulation of micropipettes.
- Acetylcholine is commonly used as a neurotransmitter in these studies.
Purpose of Study
- To deliver acetylcholine to specific sites to observe its effects on blood flow.
- To understand the mechanisms of endothelium-dependent vasodilation.
- To explore applications in neurophysiology and synaptic transmission.
Methods Used
- Preparation of micropipettes from glass capillary tubes.
- Backfilling micropipettes with acetylcholine chloride solution.
- Using a micro manipulator to position the micropipette tip accurately.
- Applying electrical currents to control the delivery of acetylcholine.
Main Results
- Calcium fluorescence responses were recorded in endothelial cells upon acetylcholine delivery.
- Fluorescence intensity increased with the duration of acetylcholine stimulation.
- The technique demonstrated reproducibility in controlling drug delivery parameters.
- Insights into cellular signaling events related to blood flow control were gained.
Conclusions
- Microiontophoresis is a valuable tool for studying neurotransmitter effects in biological systems.
- Effective micropipette preparation and current adjustment are crucial for successful experiments.
- The method can be applied to various fields, including neurophysiology and vascular biology.
Frequently Asked Questions
Executive Industry Relevance
Microiontophoresis enables precise, localized delivery of charged compounds to study vasomotor responses in the microcirculation without systemic hemodynamic perturbations. This capability supports mechanistic de-risking of vascular targets by isolating endothelial signaling pathways in vivo. The technique enhances predictive confidence in target validation by linking agonist stimulation to calcium-dependent endothelial responses in arterioles.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Interrogates therapeutic hypotheses by delivering agonists like acetylcholine to defined microvascular sites to assess receptor-mediated vasodilation.
- Operational Value: Enables reproducible stimulus-response relationships through controlled ejection current intensity and duration.
- Predictive Value: Supports target confidence by measuring intracellular calcium fluxes as a functional readout of endothelial activation.
Screening & Assay Development
- Assay Readiness: Prepares validated biological systems for downstream compound evaluation by establishing baseline vasomotor reactivity.
- Quantitative Outputs: Generates fluorescence and diameter measurements that enable dose-response characterization under defined electrical parameters.
- Reproducibility: Standardizes delivery via retaining and ejection currents, minimizing variability in agonist exposure across experiments.
Translational & Preclinical Research
- Disease Relevance: Studies intrinsic microvascular properties in models like the mouse cremaster muscle, relevant to hypertension and endothelial dysfunction.
- Translational Continuity: Connects discovery-stage mechanism probing to preclinical validation by preserving physiological context in vivo.
- Risk-Adjusted Advancement: Informs go/no-go decisions by revealing whether observed effects are endothelium-dependent and localized.
Pipeline & Workflow Integration
The method fits within the discovery continuum from target hypothesis testing to lead optimization by providing localized, quantifiable readouts of vascular reactivity.
- Discovery Biology: Supports pathway clarification by enabling site-specific agonist delivery to dissect signaling cascades in endothelial cells.
- Screening: Delivers assay readiness through standardized micropipette preparation and current-controlled substance ejection.
- Analytics: Provides quantitative calcium fluorescence and diameter responses that allow comparison of compound potency and efficacy.
- Translational Research: Maintains physiological relevance by operating in intact microcirculation, avoiding artifacts from isolated tissue preparations.
- Enterprise Reuse: Establishes a reusable platform for probing multiple agonists or antagonists across vascular beds with minimal systemic interference.
Operational & Enterprise Impact
- Scientific Value: Delivers predictive confidence by linking agonist stimulation to endothelial calcium signaling, reducing mechanistic ambiguity in vascular target validation.
- Operational Value: Ensures standardization and reproducibility through precise control of ejection and retaining currents, minimizing delivery variability.
- Strategic Value: Improves capital efficiency by enabling early de-risking of targets whose effects are confounded by systemic hemodynamic changes.
- Portfolio Impact: Supports risk-adjusted prioritization by distinguishing direct endothelial effects from indirect or reflexive vascular responses.
Implementation Considerations
- Requires expertise in micropipette fabrication, micro-manipulation, and electrophysiological current control.
- Depends on instrumentation including micro pipette pullers, micro manipulators, and iontophoresis programmers with TTL triggering capability.
- Necessitates standardization of current parameters across users and labs to ensure reproducible agonist delivery.
- Involves adaptation considerations for different vessel sizes, agonist solubilities, and ionization states affecting retention and ejection efficiency.
- Limited by the difficulty in quantifying exact solute concentration delivered, though stimulus-response relationships remain reproducible under fixed conditions.
