A novel marker for identifying and studying the membranes, barriers, and compartments surrounding peripheral nerves microscopically

Abstract

Recent anatomical discoveries indicate the importance of identifying membranes and compartments surrounding peripheral nerves into which local anesthetic agents can be injected and continuous nerve block catheters placed during regional anesthetic procedures. However, current markers used in anatomical studies have multiple drawbacks, specifically extravasation into noninjected locations, which can result in inadequate treatment. We studied a readily-available new marker, heparinized blood solution (HBS), which is easy to identify by microscopy and can remain in the nerve compartment into which it is deposited without distorting the tissue. We collected blood from 22 patients and prepared it as HBS. This was then injected into four fresh cadavers as in routine clinical practice for ultrasound-guided nerve blocks to form a so-called "doughnut" by "hydro-dissecting" at 32 sites. All samples, including nerves and neighboring tissues, were then prepared and examined by light microscopy. Although no deliberate intraneural injection was attempted, the marker was identified inside all the nerve compartments except the fascicles. Apart from leaking through the needle entry site in some instances, there was no extravasation of the HBS into neighboring nerve compartments in either direction. The tissues were not distorted and the erythrocytes did not form a thrombus. Nerve membranes and compartments could be clearly identified with routine staining. This technique enabled us to study the longitudinal and circumferential spread in all nerve compartments and to collect data for better interpretation of factors influencing an anesthetic nerve block and situations in which complications could possibly arise. HBS seemed superior to other markers because it did not leave the compartments into which it had been injected, did not distort the tissue, and was easily visible under the light microscope. Clin. Anat., 31:1050-1057, 2018. © 2018 Wiley Periodicals, Inc.

India ink: a time-tested histological marker

“…While we applaud and share their interest in better understanding the impact of neural microanatomy on clinical care, they produce no evidence for their remarks regarding India ink, referencing only one study using lidocaine radiolabeled with carbon-14 to determine its intraneural concentration. In their recent paper,2 the work by Orebaugh and colleagues is the only reference cited,3 omitting a broad review of the use of India ink in pathology, as well as its use as a marker of needle tip position in studies of ultrasound guided peripheral nerve blocks 4…”

“…In their study,2 50 mL samples of blood, with an initial hematocrit of 32%–40%, were diluted with heparin to 75 mL resulting in an injectate hematocrit of 21%–26%. The authors acknowledge that the heparinized solution does not resemble local anesthetic molecular size so that its diffusion may differ.…”

“…The authors clearly state in the abstract that ‘this technique enabled us to study the longitudinal and circumferential spread in all nerve compartments and to collect data for better interpretation of factors influencing an anesthetic.’ They injected relatively large volumes, 5–10 mL of blood, and showed still sonograms of nerves surrounded by fluid filled ‘doughnuts.’ After admitting in the discussion of limitations, that the injectate is significantly different from local anesthetic, they now change the aim of their research and state the purpose was ‘not to examine local anesthetic spread and final diffusion, but to evaluate a marker that could better reveal the complexities of the nerve membranes and help us better study the microscopic architecture of nerve membranes.’2 If that is the case, there was no need to inject a volumeof 5-10 mL, which may distort nerve architecture.…”

“…When blood is the marker in the presence of H & E stain, dye color is no longer helpful, and there is a requirement for high magnification to assess the morphology of all cells to determine where the injectate is located. Hence, all histology figures provided in both the letter to the editor1 and the study2 required labeling of the dye to differentiate for the reader the difference between red colored connective tissue and the red injectate. This is largely unnecessary when India ink is used.…”

India ink: a time-tested histological marker

“…We continue to explore different options to perfect the technique and improve the performance of the new marker in an attempt to optimize the results. In the first experiment, we diluted the heparinized blood to a hematocrit of 25% 3. In the samples published in our Letter to the Editor,2 the hematocrit was between 10% and 15%, and in our current experiments, we are working with hematocrits less than 10%.…”