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. 2023 Aug 2;11(8):2170.
doi: 10.3390/biomedicines11082170.

The Effect of Local Anesthetics on Neutrophils in the Context of Different Isolation Techniques

Sara Sixt  1 , Michael Gruber  1 , Gesche Kolle  1 , Thies Galla  1 , Diane Bitzinger  1
Affiliations

The Effect of Local Anesthetics on Neutrophils in the Context of Different Isolation Techniques

Sara Sixt et al. Biomedicines. .

Abstract

Various functions of polymorphonuclear neutrophils (PMNs) are related to diseases and postoperative plasma changes. The influence of some local anesthetics (LAs) on PMNs obtained by conventional isolation methods and their functions has already been demonstrated. This study investigates the effect of selected LAs on PMNs, comparing a new isolation method with conventional ones. To obtain the PMNs, we performed either gelafundin sedimentation, hypotonic lysis or density gradient centrifugation. Subsequently, PMNs were mixed with different concentrations of bupivacaine, levobupivacaine, lidocaine or ropivacaine. Live cell imaging and flow cytometry were performed to quantify the migration, ROS production, NETosis and antigen expression of PMNs. We found the inhibition of chemotaxis and ROS production by LAs. PMNs showed a strong reduction in time to half maximal NETosis in response to bupivacaine and lidocaine, but not to levobupivacaine and ropivacaine. We also found distinct differences in survival time and migration duration between the isolation methods. This suggests that the careful selection of LAs has a short-term impact on in vitro PMNs.

Keywords: PMN functions; PMNs; bupivacaine; isolation; levobupivacaine; lidocaine; local anesthetics; neutrophils; ropivacaine.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Tested combinations of PMN isolation techniques and LAs used for incubation.
Figure 2
Figure 2
Schematic representation of (a) 3D-µ-slide and (b) µ-Slides VI 0.1. The 3D-µ-slide consists of three equal units, each with a central channel containing 6.5 µL and a reservoir to the left and right of it. Each of the reservoirs contains 65 µL. The µ-Slides VI 0.1 consists of six equal units (numbers 1–6), each with a central channel containing 1.7 µL and a reservoir above and below it. Each reservoir contains 60 µL. In both types of slides, there is no barrier between the reservoirs and the associated channel of the facility.
Figure 3
Figure 3
TLs of PMNs treated with (a) bupivacaine, (b) levobupivacaine and (c) lidocaine in all three periods arranged by concentration group. The time periods are divided into 0–10 h, 10–15 h and 15–21 h. Data are presented as boxplots. Not all outliers (circles) and extreme values (asterisks) are displayed.
Figure 4
Figure 4
Impact of bupivacaine, levobupivacaine and lidocaine on ET50NETosis after gelafundin sedimentation presented as boxplots. Significant differences are indicated by asterisks (★) and the corresponding p-values. Outliers (circles) and extreme values (asterisks (*)) are displayed.
Figure 5
Figure 5
Effects of LAs on the ET50NETosis separated by isolation method. The different LAs are displayed via colors. The isolation methods are displayed via line type.
See this image and copyright information in PMC

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