The potencies and neurotoxicity of intrathecal levobupivacaine in a rat spinal model: Effects of concentration

Abstract

This study was aimed at examining the anesthetic effects and spinal cord injuries in the rats by intrathecal injection of levobupivacaine at different concentrations. Rats with successful intrathecal cannulation were selected and randomly divided into six groups (n = 72), and administered 0.1 mL of 0.125%, 0.25%, 0.5%, or 0.75% levobupivacaine, saline or 5% lidocaine via intrathecal catheters. The potency of levobupivacaine was evaluated by walking behavior. To identify the motor and sensory function, walking behavior and paw withdrawal thresholds (PWTs) were measured once a day. After 7 days, the L4–5 spinal cord segments were removed for histological examination. The onset time of 0.125% levobupivacaine intrathecal injection was 70.0 ± 8.9 s, and the maintenance time was 9.5 ± 1.8 min. The onset time of 0.75% levobupivacaine intrathecal injection was significantly shortened to 31.0 ± 5.5 s, and the maintenance time was significantly extended to 31.3 ± 5.4 min. The severe injury was observed in the 5% lidocaine group, while milder injury was observed in the 0.75% levobupivacaine group. The damage in the 0.5% levobupivacaine group was mild, and there were no histological abnormalities in the 0.125%, 0.25% levobupivacaine and saline groups. The neurotoxicity of intrathecally administered levobupivacaine was concentration dependent. In addition, higher concentrations of levobupivacaine were associated with shorter onset and longer maintenance times. The clinical concentration of levobupivacaine should not exceed 0.5% to avoid potential damage.

The potencies of different concentrations of levobupivacaine.

Abbreviation

PWT

paw withdrawal threshold

1. INTRODUCTION

Levobupivacaine is a local anesthetic that is the levo‐S‐enantiomer of racemic bupivacaine and has low‐potential toxicity to the central nervous system and the heart. ¹ Levobupivacaine has a similar sensory block time to bupivacaine and does not cause significant changes in haemodynamics. ² Compared with ropivacaine, levobupivacaine has an earlier onset of sensory and motor blockade, a longer duration of sensory and motor blockade and more stable haemodynamic parameters. ³ In addition, the concentration of local anesthetic drugs is closely related to the potential for the motor blockade. ⁴ A previous study found that high concentrations of intrathecal levobupivacaine cause axonal degeneration of the posterior root and extension to the posterior column. ⁵

A study of intrathecal levobupivacaine for spinal cord toxicity in neonatal rats employed a levobupivacaine concentration of 0.5%, which is also the concentration that produces reliable motor and sensory blockade in neonatal rats. ⁶ To meet the requirements of anesthesia and analgesia, intrathecal levobupivacaine is commonly used at a concentration of 0.5% in the clinic, ⁷ while 0.125% levobupivacaine intrathecal injection combined with the epidural block is also commonly used for labour analgesia. ⁸ A study on the neurotoxicity of intrathecal ropivacaine in rabbits started at the concentration of ED50, which caused the motor block in the hind limbs of rabbits. ⁹ The ED50 of intrathecal injection of levobupivacaine for caesarean section in clinical practice is 6.2 mg (95% CI: 2.6–7.6 mg), ¹⁰ and the corresponding experimental concentration of levobupivacaine in rats was calculated to be 0.1267% by normalizing the body surface area. ¹¹

This trial was dedicated to the early detection of levobupivacaine toxicity and is helpful for estimating the safe concentration range of levobupivacaine use in humans.

2. METHODS

The study was conducted in accordance with the Basic & Clinical Pharmacology & Toxicology policy for experimental and clinical studies. ¹²

2.1. Animals

All the research protocols and procedures were approved by the Ethics Committee for Animal Research of North Sichuan Medical College. All the experiments were performed in adult Wistar rats (provided by the Laboratory Animal Center, North Sichuan Medical College, n = 72, weighting 210–220 g). The animals were housed three per cage for 1 week before the experiment and were allowed free access to food and water. They were maintained individually after intrathecal catheterization.

2.2. Surgical procedure for intrathecal catheterization

Rats were anesthetized with an intraperitoneal injection of 1% pentobarbital sodium (40 mg/kg). Referring to the method of Yaksh and Rudy ¹³ an incision of 1.5–2 cm was made longitudinally above the foramen magnum of the occipital bone, the subcutaneous tissues and neck muscles were bluntly separated, and the occipital atlas was exposed. Then the subarachnoid space was cannulated with a polyurethane microspinal catheter (ID 0.12 mm, OD 0.35 mm, USA) through a puncture of the atlanto‐occipital membrane. The tip of the catheter was advanced 6–6.5 cm caudally. The polyurethane tubing was connected to the catheter hub and was firmly mounted to the skin of the hind neck. The rats were observed for 7 days after surgery, with free access to water and food. Rats that had sensory and motor dysfunction were excluded.

2.3. Intrathecal drug administration

Each solution was prepared freshly before injection. Levobupivacaine at different concentrations was made from 0.75% levobupivacaine hydrochloride injection (Henrui) in normal saline. 5% lidocaine was made from lidocaine hydrochloric acid powder in normal saline.

At Day 7, 72 rats with successful intrathecal cannulation were randomly divided into six groups (n = 12 in each group): Group N was a negative control group with intrathecal saline injection; Group L was a positive control group with intrathecal 5% lidocaine; and Groups A, B, C, and D received intrathecal injections of 0.125%, 0.25%, 0.5%, and 0.75% levobupivacaine, respectively. Each rat received intrathecal medication or saline at a dose of 0.1 mL in addition to 10 μL of saline for the dead space of the catheter.

2.4. Neurological function evaluation

An investigator who was blinded to the grouping evaluated the motor function of each rat by analyzing its ability to walk. The walking function was scored as follows: score 0 = no blockage, normal ambulation, score 1 = asymmetric walking of hind limbs, score 2 = inability to walk but the perceptible movement of hind limbs, and score 3 = complete paralysis of both hind limbs. After intrathecal administration, the walking function score was recorded every 15 s until the maximal block was achieved, and then every 5 min until the motor function was completely recovered. The drug onset time was defined as the time from the end of intrathecal administration to the attainment of the maximal motor block of the hind limbs. The time from reaching the maximal motor block to complete recovery of locomotion was regarded as the duration time.

The paw withdrawal threshold (PWT) was measured 10 min before and every 5 min after injection until sensation returned, and every morning after injection by mechanical stimulation (Dynamic Plantar Aesthesiometer).

The systolic pressure of the rats was monitored using an RBP‐1 sphygmomanometer at 10 min before injection, and 2, 5, 15, 25, 35, 45, 55, and 65 min after intrathecal injection. Considering the damage of hypotension to the spinal cord. If the blood pressure dropped more than 20%, the rat was withdrawn from the experiment. PWT and walking ability were observed once a day from the day of administration to assess and record motor and sensory recovery in the rats.

2.5. Tissue preparation

At the end of the behavioral assessment, rats were euthanized with pentobarbital sodium. and the lumbar spinal cords were quickly removed en bloc. Then the lumber spinal cords (L3–L4) were divided into two samples on ice: (1) a transverse section of the L4 spinal cord was fixed in 10% neutral formalin, embedded in paraffin, and sliced into 4–7 μm thick sections for hematoxylin and eosin staining; (2) 1 mm ³ of the posterior spinal cord was immersed in 3% glutaraldehyde phosphate buffer and stored at 4°C for electron microscope observation. All the tissue samples were processed and observed by professionals in the electron microscopy department of the Sichuan University.

2.6. Spinal cord injury evaluation

Spinal cord injury was assessed by light and electron microscopy examination. The damage scores were quantified as follows: the grade of damage observed visually by light microscopy was classified as L0 = no lesion, slight dilation and hyperaemia of meningeal vessels without inflammatory cell infiltration, L1 = slight dilation and hyperaemia of meningeal vessels with infiltration of several inflammatory cells, L2 = dilation and hyperaemia of meningeal vessels with some inflammatory cell infiltration, and L3 = severe dilation and hyperaemia of meningeal vessels with infiltration of numerous inflammatory cells. The grade of damage observed visually by electron microscopy was classified as E0 = no lesion, E1 = slight demyelination of several nerve fibers, E2 = moderate demyelination of some nerve fibers with some cytoplasmic vacuoles, and E3 = severe demyelination of nerve fibers with large cytoplasmic vacuoles and mitochondrial swelling.

2.7. Statistics

The results are presented as the number or mean (± SD), and SAS statistics software version 8.0 was used for the statistical analysis of the data. The scores of motor function and spinal cord injury were analyzed using one‐way ANOVA, followed by Dunn's test. A value of p ≤ .05 was regarded as statistically significant.

3. RESULTS

3.1. Drug onset and duration time

There were partial decreases in blood pressure in some of the rats in this experiment, but these decreases did not exceed 20%, which may be due to our slower rate of administration, so the data from all rats in this experiment were involved in the statistical analysis. Rats in the saline group (Group N) showed no motor blockage after intrathecal injection. Rats injected with levobupivacaine showed a significant concentration‐dependent duration time. The onset time was shortened with increasing concentration. All of the rats recovered in 2 h and showed no motor or sensory dysfunction after recovery (Figure 1).

FIGURE 1.

Effect of concentration on onset time (A) and duration time (B) from rats. Onset time (A) and duration time (B) evaluated by motor block duration. When the concentration of levobupivacaine increases, the onset of action of the drug is shortened and the maintenance time increases. The time between the end of intrathecal injection and maximum motor block of the hind limb is the time of drug onset. The time between maximum hind limb motor block and full recovery of hind limb movement is the drug maintenance time. Results are mean ± SD, 12 rats per group, 72 rats in total. *p < .05 compared with 0.125% levobupivacaine group.

3.2. Structural and ultrastructural injury

Most of the 12 rats in the low‐concentration group had a spinal cord tissue injury score of 0 or 1. A few rats in the high‐concentration levobupivacaine group had a tissue injury score of 3, and more than half of the rats in the 5% lidocaine group had an injury score of 3 (Table 1). There was no significant histological injury in the lumbar spinal cord of rats in the saline group (Group N) and 0.125% levobupivacaine group (Group A). Spinal injuries were observed in several rats in 0.25% levobupivacaine (Group B) under light microscopy (Figure 2). The spinal cords of rats receiving >0.50% levobupivacaine showed structural and ultrastructural injuries. The structural changes included meningeal vascular dilation and hyperaemia and lymphocytic infiltration (Figure 3). The ultrastructural abnormalities involved demyelinoclasis, local valuoles, and mitochondrial swelling.

TABLE 1.

Number of rats with different levels of spinal cord injury.

Groups	Light microscopy rating				Electron microscopy rating
	L0	L1	L2	L3	E0	E1	E2	E3
NS	12	‐	‐	‐	12	‐	‐	‐
0.125% levobupivacaine	12	‐	‐	‐	12	‐	‐	‐
0.25% levobupivacaine	7	5	‐	‐	12	‐	‐	‐
0.5% levobupivacaine	3	9	‐	‐	8	4	‐	‐
0.75% levobupivacaine	‐	3	6	3	‐	4	5	3
5% lidocaine	‐	‐	3	9	‐	‐	6	6

Note: The neural tissue damage demonstrated by the sections was rated by a pathologist who was unaware of the experimental subgroup. “‐” indicates that no rats are at that rating.

FIGURE 2.

Light micrograph of spinal cord showing neurotoxicity. Light microscopic images of spinal cord. Some mild histological changes was shown in Group N and Group A. Meningeal vascular dilation and hyperemia and lymphocytic infiltration are indicated by arrows in Group N and levobupivcaine groups. Massive lymphocytic infiltrations are highlighted by arrows in Group L. N group normal saline, A group 0.125% levobupivacaine, B group 0.25% levobupivacaine, C group 0.5% levobupivacaine, D group 0.75% levobupivacaine, L group 5% lodocaine.

FIGURE 3.

Electron micrograph of spinal cord showing neurotoxicity. Electron microscopic images of spinal cord. No obvious lesion was shown in Group N and Group A and Group B. Black arrow indicate mild demyelinoclasis in Group C. Demyelinoclasis and edematous degeneration of mitochondria and local vacuole are indicated by arrows in Group D and Group L. N group normal saline, A group 0.125% levobupivacaine, B group 0.25% levobupivacaine, C group 0.5% levobupivacaine, D group 0.75% levobupivacaine, L group 5% lodocaine.

4. DISCUSSION

Local anesthetics have been used for spinal anesthetics, which mainly act on nerve fibers and primary neurons of the spinal cord. Hence, the clinical symptoms of local anesthetic‐induced neurotoxicity manifest as injuries to nerve fibers and primary neurons of the spinal cord, involving reversible injuries such as aching and pain in the buttocks and lower extremities and irreversible injuries such as urination disorders and intestinal and sexual dysfunction. ¹⁴ , ¹⁵ , ¹⁶

0.126% is the concentration corresponding to the ED50 of levobupivacaine. Therefore, 0.125% is the minimum dose for this experiment, while the concentrations of levobupivacaine in the other experimental groups are 0.25%, 0.5%, and 0.75%. The drug volume was constant in this experiment, and elevated drug concentrations corresponded to increasing drug doses. The efficacy of intrathecal anesthesia increases with the concentration of local anesthetic drugs. Some experiments have shown that the ED50 of intrathecal bupivacaine is higher at high concentrations than at low concentrations, but the difference in the volume administered affects the level of spinal anesthesia. ¹⁷ , ¹⁸ Moreover, for clinical reasons the experimenter evaluated the motor block earlier. Pathak et al. ¹⁹ found that the duration of motor blockade and analgesia was shorter with low concentrations of ropivacaine than with high concentrations of ropivacaine. This experiment also found that an increase in intrathecal levobupivacaine concentration resulted in a shorter onset and longer duration of motor block in rats.

Levobupivacaine is less neurotoxic and has a higher safety profile for the intrathecal application. ¹⁰ , ²⁰ Three rats in the lidocaine group failed to fully recover motor function and showed various degrees of dyskinesia in the hindlimbs. Rats administered lidocaine developed lesions in both the posterior roots and posterior columns, which are involved in the transduction of sensory information. Therefore, the sensory impairment caused by lidocaine may lead to behavioral limitations. In contrast, all the rats recovered full motor and pain‐sensory function after subarachnoid space administration of levobupivacaine. Previous studies on the effects and neurotoxicity of levobupivacaine have tended to focus on visceral anti‐injury perception, ²¹ and since intrathecal injections of local anesthetics are commonly used in lower limb surgery, the present study is particularly concerned with lower limb motor and sensory blockade. Nerve damage can be caused by a variety of clinical factors, including an inadequate blood supply that may initiate apoptosis. ²² This study measured blood pressure in rats and excluded the effect of hypotension leading to inadequate vascular perfusion in the soft spine.

Animal models in this study were established using the method of Yaksh and Rudy ¹³ in this study. ²³ The lumber spinal cords and nerve roots close to the tip of the catheter were harvested for histopathological observation, which avoided any interference or damage caused by the catheter. Varying degrees of histological alterations, including focal thickening of the meninges and meningeal vascular dilation and hyperaemia with infiltration of several inflammatory cells, were observed in all groups. Previous studies demonstrated that inflammatory changes could be a reaction to tissue degeneration following catheter insertion. ⁹ The current study shows that massive lymphocytic infiltration of the spinal cord appeared in the lidocaine group, suggesting that lidocaine could cause a spinal inflammatory response. No lesions were found in the 0.125% group and 0.25% group under electron microscopy. However, demyelination, local vacuoles and even swollen mitochondria were found in the high‐concentration levobupivacaine groups, which suggests that the degree of spinal cord injury was highly correlated with concentration. Many studies have shown that high concentrations of local anesthetics are neurotoxic, ⁹ , ²⁴ manifesting as axonal degeneration and motor dysfunction, sensory function damage and even irreversible neurodysfunction. ²⁵ , ²⁶ , ²⁷ Local anesthetics may cause nerve injury through decreased blood flow to perineural vessels. Bouaziz et al. ²⁸ reported that significant histopathological changes were not observed despite 0.25% and 0.5% levobupivacaine inducing a significant reduction in peripheral nerve blood flow in regional anesthesia. Nerve blood flow was not observed in this study; thus, it remains unclear whether the damage caused by 0.75% levobupivacaine was connected to its reduction in nerve blood flow.

5. CONCLUSION

In summary, the article confirms that the efficacy and neurotoxicity of intrathecal Levobupivacaine in rats are concentration‐dependent and that intrathecal injection of levobupivacaine at concentrations below 0.5% is noninvasive to the spinal cord, which means that the concentration of levobupivacaine may not exceed 0.5% to avoid potential damage to the spinal cord.

AUTHOR CONTRIBUTIONS

All the persons who meet authorship criteria are listed as authors, and all authors certify that they have participated sufficiently in the work to take public responsibility for the content, including participation in the concept, design, analysis, writing, or revision of the manuscript. Luyue Gao was responsible for the design and implementation of the experiment, the statistics and sorting of data, and the drafting of articles. Zhen Yang was responsible for part of the implementation of intrathecal catheters in rats. Sisi Zeng was responsible for collecting behavioral data from animal experiments. Jiabei Li performed the data analyses. Na Wang was responsible for raising animals. Funjun Wang was responsible for the review and improvement of the experimental scheme and revision of the article.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest [or state‐specific conflicts].

ETHICS STATEMENT

All research protocols and procedures were approved by the Ethics Committee for Animal Research of North Sichuan Medical College [No. 2021093].

Gao L, Yang Z, Zeng S, Li J, Wang N, Wang F. The potencies and neurotoxicity of intrathecal levobupivacaine in a rat spinal model: Effects of concentration. Pharmacol Res Perspect. 2023;11:e01116. doi: 10.1002/prp2.1116

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.