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Anesthesia & Analgesia:
doi: 10.1213/ANE.0000000000000142
Regional Anesthesia: Brief Report

Intraneural and Perineural Inflammatory Changes in Piglets After Injection of Ultrasound Gel, Endotoxin, 0.9% NaCl, or Needle Insertion without Injection

Pintaric, Tatjana Stopar MD, PhD, DEAA*; Cvetko, Erika DMD, PhD; Strbenc, Malan DVM, PhD; Mis, Katarina PhD§; Podpecan, Ozbalt DVM, PhD; Mars, Tomaz MD, PhD§; Hadzic, Admir MD, PhD

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From the *Clinical Department of Anesthesiology and Intensive Therapy, University Medical Center Ljubljana, Ljubljana, Slovenia; Institute of Anatomy, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia; Bia Ltd. Ljubljana, Slovenia; §Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia; Savinian Veterinary Polyclinic, Zalec, Slovenia; and College of Physicians and Surgeons, Columbia University, New York, New York.

Accepted for publication January 8, 2014.

Funding: This work was funded by the Slovenian Research Agency (P3-0043-0381) and the tertiary funding of the Clinical Department of Anesthesia and Intensive Therapy, University Medical Center Ljubljana.

Conflict of Interest: See Disclosures at the end of the article.

Reprints will not be available from the authors.

Address correspondence to Tatjana Stopar Pintaric, MD, PhD, DEAA, Clinical Department of Anesthesiology and Intensive Therapy, University Medical Center Ljubljana, Zaloska 7, 1000 Ljubljana, Slovenia. Address e-mail to tatjanas38@gmail.com.

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BACKGROUND: Ultrasound gel nerve inflammation has been reported. We evaluated the extent and nature of inflammation after gel injection with endotoxin (positive), saline, or dry needle puncture (negative) controls after peripheral blocks in piglets.

METHODS: Selected nerves of 12 piglets were localized by landmarks and nerve stimulator. Forty-eight hours after injection, specimens were examined for immunohistochemical cell differentiation/quantification and cytokine expression by using quantitative polymerase chain reaction.

RESULTS: Both gel and endotoxin injections resulted in a significantly higher density of inflammatory cells (lymphocytes/granulocytes) as compared with needle insertions and/or saline injections (both P < 0.001). Cytokines were not detected in any of the specimens.

CONCLUSIONS: Perineural gel injections cause significant inflammation. The lack of cytokines suggests injectate-related changes rather than mechanical trauma.

To optimize image quality during ultrasound (U.S.)-guided nerve blocks, clinicians apply an aqueous gel as a coupling medium between the U.S. transducer and the skin. However, gel in the needle lumen may be injected peri- or intraneurally.1–3 We reported inflammatory changes after injection of gel into the neuraxial space.4 Recently, inflammation was reported after direct application of gel on canine nerves; however, surgical trauma of the applied open-tissue model might have contributed to the inflammation.5 Therefore, we performed a study in piglets, a model immunologically close to humans6 by using a closed-tissue model and percutaneous injections, to evaluate the extent of inflammatory response induced by U.S. gel injection as compared with needle trauma by using histoimmunological quantification of inflammation and cytokine detection.

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After approval by Review Board for Animal Research (N° 34401–25/2011/4, 34401–30/2012/6), 12 piglets (Sus scrofus domesticus) (15–25 kg, 4 months) were studied in this double-blind, prospective, randomized trial. The animals were free of swine fever, Aujeszky disease, porcine respiratory and reproductive syndrome, and salmonellosis, and they were vaccinated against mycoplasmosis 4 to 6 weeks before the experiment. Only clinically healthy animals with no visible skin lesions were included in the study. On the day of the experiment, the piglets were sedated with azaperon (Stresnil, Janssen Pharmaceutica, Bfeerse, Belgium) 0.5 mg/kg IM and anesthetized via facemask under spontaneous ventilation with 2% to 4% isoflurane in 50% air–oxygen mixture. The anesthetized animals were placed in a lateral decubitus position with a forelimb slightly abducted. The areas of intervention were washed with clear water and soap, shaved, and disinfected with chlorhexidine. Two nerves from the front limb were selected because of their accessibility and size: a deep branch of radial nerve and ulnar nerve. The nerves were located by using a 22-gauge, 5 cm long, short-beveled, insulated nerve block needle (Stimuplex A, B. Braun Melsungen AG, Melsungen, Germany) with a nerve stimulator set to 1 mA (0.1-millisecond pulse duration, f = 2 Hz). For the deep radial branch, the needle was inserted a third of the distance from the inner elbow angle to the olecranon and advanced through the radial carpal extensor muscle until the evoked motor response (extension of all 4 digits) of 0.3 mA was elicited. For ulnar nerve identification, the needle was inserted through the antebrachial fascia until the flexion of digits was elicited.

This study included 4 groups (U.S. gel, saline, endotoxin, and needle insertion without injection). According to the random assignment to either side, 1 mL either U.S. gel (demineralized water (96%) and propylene glycol (3%); Eko gel, Ultrasound transmission gel, Eurocamina Srl., Salerno, Italy) (gel samples), 0.9% NaCl (negative), or endotoxin (Lipopolysaccharides from E. coli 026:B6 [Sigma Aldrich; L2654], 1 μg/mL) (positive) controls was injected (Fig. 1). An injection was immediately aborted in case of abnormal resistance (opening injection pressure ≥15 psi).7 “Dry needle insertions” were performed to control for needle-induced inflammatory changes.

Figure 1
Figure 1
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Before and after the experiment, behavior and motor function were evaluated in all piglets daily and recorded by using a 4-grade scale as follows:8

* Grade 0: The animal is able to rise, walk, and run without difficulties and feeds normally.

* Grade 1: The animal is able to rise and walk, feeds normally, but shuffles one or both rear limbs, walks with a slight limp.

* Grade 2: The animal is able to rise with difficulty, refuses to walk, limps, and feeds poorly.

* Grade 3: The animal is unable to rise, stand, walk, and eat and lies in a sternal or lateral recumbent position.

Piglets were euthanized 48 hours after the experiment. Nerve specimens were excised and divided into 3 portions; one was placed in liquid nitrogen (at −80°C), a second in fixative (4% neutral-buffered formalin) for immunohistochemical analysis, and a third in RNA Stabilization Reagent (RNAlater; Qiagen, Hilden, Germany) for quantitative polymerase chain reaction (PCR). The specimens were numbered, and key numbers were kept in sealed envelopes to ensure blinding.

Five-micrometer thick transverse sections were stained with hematoxylin/eosin and processed for immunohistochemistry. Paraffin-embedded sections were used for labeling T-lymphocytes with an anti-CD3 antibody (NCL-L-CD3-565, Leica Biosystems Newcastle Ltd, Newcastle upon Tyne, United Kingdom) and frozen specimens to demonstrate macrophages and monocytes with CD14 antibody (MCA1218, AbDSerotec, Oxford, United Kingdom), and monocytes and granulocytes (PMNL) with anti-monocyte + granulocyte antibody (ab24991, Abcam, Cambridge, United Kingdom). All cross-sectional nerve-areas from differently stained serial sections were captured by a digital camera (DXM1200F, Nikon, Tokyo, Japan) connected to the microscope (Eclipse E800, Nikon, Tokyo, Japan). For each specimen, 5 sections separated by 1 mm were examined. With the use of the quantification software (Ellipse programme, ViDiTo, Kosice, Slovakia), the outer border, for example, epineurium of the nerve, and the border of individual nerve fascicles, for example, perineurium were delineated, and the areas for both calculated. The surface (area) of nerve fascicles was subtracted from the whole cross-section of the nerve and inflammatory infiltration expressed as the number of immunopositive cells per square millimeter between nerve fascicles. The overall count of immunopositive cells was the summation of immunopositive cells from all 3 stainings.

Total RNA, extracted with the RNeasy Mini Plus Kit (Qiagen, Hilden, Germany), was reversely transcribed with a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Quantitative PCR was performed on an ABI PRISM SDS 7500 (Applied Biosystems) by using TaqMan chemistry: TaqMan Universal PCR Master Mix (Applied Biosystems) and the following Gene Expression Assays (Applied Biosystems): tumor necrosis factor-α (Ss03391318_g1), interferon-γ (Ss03391054_m1), interleukin-6 (Ss03384604_u1), interleukin-1β (Ss03393804_m1) and for the internal control: β-actin (Ss03376081_u1) and GAPDH (Ss03375435_u1). Relative (ΔΔCt) quantification was performed to assess expression levels of target genes.

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Statistical Analysis

The primary objective of this study was to quantify the inflammatory response to injection of U.S. gel in a closed-tissue animal model that is immunologically similar to humans. Our previous data showed that the density of immunopositive cells (number of cells/mm2) increased to 8.2 ± 2.4 (gray matter) and 3.7 ± 1.4 (white matter) when aqueous gel was injected into the subarachnoidal space in piglets. No immunopositive cells were present in the subarachnoidal space under the control condition.4 For the current study, no previous data on which to estimate the sample size were available for the radial and ulnar nerves in piglets. Thus, a post hoc power analysis estimated from our empirical findings was conducted. For the gel condition, immunopositive cell density was approximately 30 cells/mm2 higher compared with the saline condition. Moreover, the largest variability among the 4 study conditions was approximately 15 cells/mm2. Therefore, for a 2-tailed test at α 0.01, δ 30, and standard deviation (SD) 15, the study had 97.5% power to test a difference by using 12 piglets and was not likely to be reduced substantially when testing differences among the 4 conditions (power of 92% when estimated with α 0.0025). Variables are presented as mean ± SD. Differences in the number of immunopositive cells, peri- or intraneurally amount the 4 groups, were tested by analysis of variance. Differences were considered statistically significant if P < 0.05. The statistical package for the social sciences (SPSS, version 19, 2011; SPSS Inc, Chicago, IL) was used for statistical analysis.

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All injections were performed as planned; no injection had to be aborted because of high resistance on injection. On emergence from anesthesia, limited grade 1 dysesthesias were apparent in all groups. Nevertheless, in spite of the presence of inflammatory changes, there was no actual evidence of any long-lasting dysesthesias in any of the piglets. Forty-eight hours after injections and/or needle insertions, 48 specimens were obtained, of which 22 exhibited hematoma infiltrating surrounding muscle, fibrous, and fat tissue regardless of the solution injected.

Nerve fascicles comprised 41.8% to 52.2% of nerve cross-sectional areas. We found no architecture damage or hematoma. The inflammatory cells were observed only interfascicularly with no signs of perineural infiltration.

Specimens receiving gel injection or endotoxin exhibited scattered or clustered inflammatory cells (Fig. 2, A–C, E–F), while after saline injections or needle punctures, only solitary inflammatory cells were observed (Fig. 2, G–I, J–L). The most prevalent cell types were lymphocytes T (57%) and granulocytes (39%); macrophages were rare (4%).

Figure 2
Figure 2
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In the gel group, the density of immunopositive cells between the nerve fascicles was significantly higher (50.86 ± 15.22) compared with the saline (mean 22.11 ± 11.22) or needle puncture group (18.17 ± 10.78) (P = 0.001), respectively, and significantly lower compared with endotoxin-exposed samples (100.7 ± 7.37) (P = 0.001) (Fig. 3).

Figure 3
Figure 3
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The tested levels of cytokines’ mRNAs were below detection limit.

Equation (Uncited)
Equation (Uncited)
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Under the conditions of this study, the injection of U.S. gel near the peripheral nerves in piglets resulted in significant intraneural inflammation. The observed inflammation consisted of lymphocyte T/granulocyte accumulation between the nerve fascicles. The inflammation appeared gel-related rather than mechanical because “dry needle insertions” did not lead to intraneural hematoma formation or/and monocytic infiltration.9 Moreover, the absence of cytokine detection suggests that needle trauma did not significantly contribute to the inflammation.10

In a previous study in a dog model, the U.S. gel, applied on the nerve surface after surgical exposure, induced inflammatory changes that increased from mild to moderate within a day.5 Nevertheless, the occurrence of inflammatory changes in perineural tissues as a result of surgical trauma that is used to accomplish nerve exposure may have confound the results, which we tried to overcome by a closed-tissue percutaneous model. We chose piglets because their anatomy, physiology, and immunologic response may be closer to those of humans.6 In addition, we included positive and negative controls for gel-induced versus tissue trauma to control for inflammation that may be caused by needle insertion alone. Finally, we used immunohistochemistry and PCR to better differentiate and quantify inflammatory cells in an attempt to discern the etiology of inflammatory changes.9,10

Our study has several important limitations. For instance, the relevance of our findings to clinical practice remains speculative. First, longer observation studies are necessary to determine the duration, magnitude, and long-term neurological consequences of inflammation. Since the injections in our study were made percutaneously, one cannot be entirely certain of the exact location intra versus perineural. However, similar to common clinical practice, we used nerve stimulation and avoided injections when an evoked motor response was present at <0.3 mA or when an excessive resistance to injection was encountered to help reduce the chance of intraneural/intrafascicular injection. In addition, the lack of architectural nerve damage and intrafascicular hematoma also suggests that the injections did not occur intraneurally. Finally, this study did not examine the dose–response relationship of U.S. gel amount and the degree of nerve inflammation or nerve reaction to different gel substances.

In conclusion, this study adds additional evidence that application of U.S. gel near peripheral nerves may cause peri/intraneural inflammation. The relevance of our findings to clinical practice, however, remains unknown.

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Name: Tatjana Stopar Pintaric, MD, PhD, DEAA.

Contribution: This author helped design and conduct the study, collected the data, and prepared the manuscript.

Attestation: Tatjana Stopar Pintaric approved the final manuscript and attested to the integrity of the original data and the analysis reported in this manuscript and is the archival author.

Conflicts of Interest: The author has no conflicts of interest to declare.

Name: Erika Cvetko, DMD, PhD.

Contribution: This author helped conduct this study, analyzed the data, and prepared the manuscript.

Attestation: Erika Cvetko attested to the integrity of the original data and the analysis reported and approved the final manuscript.

Conflicts of Interest: The author has no conflicts of interest to declare.

Name: Malan Strbenc, DVM, PhD.

Contribution: This author helped design the study, collected the data, and prepared a manuscript.

Attestation: Malan Strbenc attested to the integrity of the original data and the analysis reported.

Conflicts of Interest: The author has no conflicts of interest to declare.

Name: Katarina Mis, PhD.

Contribution: This author analyzed the data.

Attestation: Katarina Mis attested to the integrity of the original data and the analysis reported.

Conflicts of Interest: The author has no conflicts of interest to declare.

Name: Ozbalt Podpecan, DVM, PhD.

Contribution: This author helped conduct the study and collected the data.

Attestation: Ozbalt Podpecan attested to the integrity of the original data and the analysis reported.

Conflicts of Interest: The author has no conflicts of interest to declare.

Name: Tomaz Mars, MD, PhD.

Contribution: This author analyzed the data.

Attestation: Tomaz Mars attested to the integrity of the original data and the analysis reported.

Conflicts of Interest: The author has no conflicts of interest to declare.

Name: Admir Hadzic, MD, PhD.

Contribution: This author helped design the study, analyzed the data, and prepared the manuscript.

Attestation: Admir Hadzic reviewed the original data and the analysis reported and approved the final manuscript.

Conflicts of Interest: Dr. Hadzic has consulted and advised for Skypharma, GE, Sonosite, Codman & Shrutleff, Inc (Johnson and Johnson), Cadence, Pacira, Baxter and BBraun Medical. His recent industry-sponsored research includes Glaxo Smith-Kline Industries, Pacira, Baxter. Dr Hadzic is an equity holder at Macosta Medical USA.

This manuscript was handled by: Terese T. Horlocker, MD.

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Skilful technical assistance of Ivan Blažinovič, Majda Črnak-Maasarani, Andreja Omahen, Nataša Pollak, Marko Slak, Friderik Štendler, Milan Števanec and Dane Velkavrh is acknowledged.

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