Neuroscience
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Use of In Vivo Single-fiber Recording and Intact Dorsal Root Ganglion with Attached Sciatic Nerve to Examine the Mechanism of Conduction Failure
Chapters
Summary August 27th, 2019
Single-fiber recording is an effective electrophysiological technique that is applicable to the central and peripheral nervous systems. Along with the preparation of intact DRG with the attached sciatic nerve, the mechanism of conduction failure is examined. Both protocols improve the understanding of the peripheral nervous system's relationship with pain.
Transcript
Single-fiber Recording plays an important role in recording the activities of nerve fibers. Especially activities transmitting peripheral sensation from receptive fields to neurons in dorsal root ganglion. In wavel of fiber recording, provides length of duration time with capacity to record responses to nature stimuli, and was later disturbance of the intercellular environment.
In a single fiber recording requires neutron automatic good for the situation. Therapist calls the requirement for the preparation of integrated DRG tests the sciatic nerve is integrated. The result of demo situation shows almost all the details of nerves in good, physical decal stage.
And third, entire DRG preparation as are integrated. To begin this procedure, prepare and disinfect all surgical instruments prior to surgery. Then prepare one to two liters of normal Ringer's extracellular solution and store at four degrees Celsius until use.
To expose the sciatic nerve trunk for recording, first, cut open the anesthetized rat's skin and muscles on the dorsal part of the thigh. Then, perform a blunt dissection along the femoral biceps. Carefully isolate the sciatic nerve trunk using ophthalmic scissors and a glass separation needle, and keep the tissue moist using Ringer's solution.
Next, fix the animal on a home-made metal hoop by sewing the skin into the slot around it. Pull the skin up slightly to establish a fluid bath. Expose one centimeter of sciatic nerve trunk at the proximal side.
Place a small, brown platform under the nerve trunk to enhance the contrast in order to observe the fine nerve trunk clearly. Subsequently, apply some warm liquid paraffin on the top of the nerve trunk to prevent drying of the fiber surface. Remove the fluid if there is an exudation around the nerve trunk.
To perform recording, select the platinum filament as the recording electrode. Eat and create a small hook at the very end. Afterward, attach the electrode to a micromanipulator.
In the bath, place a reference electrode next to the subcutaneous tissue. Split the spinal dura and the Pia mater and obtain the sciatic nerve. Under a stereo-microscope at 25%magnification, pick up a fine fascicle and suspend the approximal end of the axon on the hook of the recording electrode.
Identify the receptive field of a single, nonconceptive c fiber using a mechanical simulis and thermal stimulus. If the firing of the nerve fiber responds to the mechanical stimuli and hot water, then consider it as a polymoto, nonconceptive c fiber. Next, insert two needle stimulus electrodes with a two millimeter interval into the skin of the identify field for the delivery of the electrical stimuli.
Display the wave form of an action potential on the oscilloscope, and employ computer a d-board with a signal sampling rate of 20 kilohertz. Then, record the spikes using data acquisition software and analyze it later with professional software. To measure conduction failure, deliver the repetitive electrode stimuli in different frequencies to a c fiber for 60 seconds.
Allow a 10 minute interval for the fiber to relax between stimuli. Then, calculate the ratio of the number of failures to the number of delivered repetitive stimulus pulses and multiply by 100%to obtain the degree of conduction failure. To expose the dorsal root ganglion, first cut open the skin from the midline of the back at the l4 to l5 segment.
Next, remove the muscles spine process vertebral bor and transverse process using a bone rongeur, and expose the spinal cord NDRG body. Cover the exposed spinal cord, NDRG, with cotton soaked with normal Ringer's extracellular solution to maintain neural activity. Stop the bleeding and remove the blood as necessary.
Subsequently, using ophthalmic scissors, remove l4 to s1 bone structure above the vertebral foramen in order to expose the DRG and connected spinal nerve. Make a cut on the skin to expose the sciatic nerve at the middle thigh. Separate and disconnect the sciatic nerve from the distal end of the nerve where it goes inside the muscle.
And ligate the nerve trunk with surgical line at the end of the nerve prior to cutting. Then, separate the sciatic nerve from the underlying connective tissue by lifting the nerve ligation point. Remove for dura from the spinal cord, and separate the DRG from the underlying connective tissue until it reaches the adjacent part of the sciatic nerve.
Thus, isolate the whole preparation of DRG with an attached sciatic nerve. To clear the surface of the DRG, at 4x magnification, carefully remove the spinal dura on the surface of l4 to l6 DRG using tweezers. Place the DRG with attached sciatic nerve in a glass tube containing one milliliter of mixed enzymes.
Digest in a 37 degree Celsius water bath for 15 minutes. After 15 minutes, lift the end of the surgical line and move the preparation to a dish filled with normal Ringer's extracellular solution to wash out the enzyme. Then, transfer the digested DRG to a container filled with oxygenated Ringer's extracellular solution for recording.
To perform recording, prepare intracellular solution, and store it at zero degree Celsius until use. Using a slice anchor, stabilize the ganglia and connect the nerve end to a suction stimulating electrode. At 40x magnification, visualize and select a DRG neuron with a water emersion objective.
Fold an electrode and fill it with intracellular solution. Attach the electrode on the holder, and apply positive pressure in the pipet with a final resistance of four to seven megaohms. Next, bring the electrode to the cell surface.
Then, apply negative pressure to the pipet to form a seal. Once a gigaohm seal is reached, set the membrane potential at about minus 60 millivolts and establish all cell recording mode. Subsequently, deliver repetitive stimuli of 5 to 50 hertz to the sciatic nerve the through the suction electrode to screen for conduction failure.
Measure the amplitude of AHP from baseline to peak and the 80%AHP duration. This figure shows the original consecutive recordings of single c5 re-firings from rats in response to 10 hertz electrical stimulation. Every twentieth sweep is shown and displayed top to bottom.
The insert shows a representative action potential. Here are the recordings of single c fibers from CFA injected rats in response to the same stimulation as the previous panel. This figure shows the continuous recordings of series firing responses to five hertz stimulation under control conditions, or administration of different concentration of ZD7288 in a small diameter DRG neuron from CFA rated rats.
The insets show expanded traces for the specified recording periods. Dark spots represent spike failures. The AHP showed a bigger rising slope in the control.
While a smaller rising slope was observed after 125 micromolars at ZD7288 application. When a time single fiver recording, I think it's important to cut the fiber will maintaining the animals is good any safety condition. And microenvironment around the neutron.
The combination of single fiber recording and application of intact DRG attached with the sciatic nerve, improved our understanding of the peripheral nervous system pertaining to pain.
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