Persistent neurological dysfunction is a rare but feared complication after spinal anesthesia (1). Functional impairment and morphological changes of the spinal cord and nerve roots after intrathecal administration of local anesthetics have been investigated in various animal models (2–6). Although local anesthetics administered intrathecally have been reported to have neurotoxic potential, the precise mechanism of neuronal injury is still obscure (2–6).
Adams et al. (2) administered etidocaine or tetracaine intrathecally in rabbits and showed degenerative changes in the posterior column and nerve roots in the animals treated with 2% tetracaine. Ready et al. (3) administered local anesthetics intrathecally in rabbits and observed dose-related neuronal injury, including damage to the cauda equina with axonal degeneration, areas of central necrosis within the spinal cord, and subpial vacuolation. However, they did not find a correlation between lesions in the cauda equina and the extent or type of functional loss (3). Drasner et al. (4) continuously administered local anesthetics through an implanted catheter with the tip located at the level of cauda equina in rats. They demonstrated that morphological damage to the nerve roots was consistent with the observed functional impairment and that there was very little damage in the spinal cord or dorsal root ganglia (7).
In our laboratory, we have investigated the neurotoxicity of large concentrations of local anesthetics administered intrathecally through an implanted catheter with the tip located at the level of cauda equina in rabbits (6,8,9). The characteristic histopathological findings in the animals with neurological dysfunction were vacuolation that was restricted in the dorsal funiculus in the spinal cord and central chromatolysis of the motor neurons. The vacuolation was thought to be a result of Wallerian degeneration because the dorsal funiculus consists of primary afferent fibers without synapsing in the dorsal horn. Sensory dysfunction correlated well with the degree of vacuolation in the dorsal funiculus. Central chromatolysis of the motor neurons occurs after axonal injury of motor neurons. Thus, we hypothesized that the injury of nerve fibers of both sensory and motor neurons is the main feature of neuronal injury after intrathecal administration of local anesthetics in our model. Takenami et al. (5) have suggested that the nerve root entry zone into the spinal cord, known as the Obersteiner-Redlich zone, may be highly vulnerable to large concentrations of tetracaine given intrathecally in rats. However, morphological changes of the nerve root entry zone have not been evaluated.
In the present study, we sought to obtain the morphological evidence of the injury of nerve fibers at the nerve root entry zone with various concentrations of tetracaine administered intrathecally in rabbits. In addition, the degree of injury was evaluated at the dorsal funiculus, distal part of nerve roots, dorsal root ganglia, and cauda equina.
The protocol of this experiment was reviewed and approved by the Committee of the Ethics on Animal Experiments in Yamaguchi University School of Medicine.
We used 25 New Zealand white rabbits weighing 2.5–3.0 kg. The methods for implantation of intrathecal catheters for drug administration in this experiment were similar to those reported in our previous studies (6,8,9). Briefly, under general anesthesia with isoflurane, the animal was positioned prone and the sixth lumbar spinous process, ligamentum flavum, and epidural fat were sequentially removed. The dura mater was exposed, and then the dura mater and arachnoid were pulled up with 7–0 Prolene. A small slit was made in the dura mater and arachnoid at the L5-6 interlaminar space, and PE-10 catheter for drug administration was implanted intrathecally through the slit after confirmation of the outflow of cerebrospinal fluid. The tip of this catheter was located at the level of cauda equina.
Three days after implantation, animals that showed no sign of neurological dysfunction were randomly assigned to one of the following groups: a control group (n = 6), a 1% tetracaine group (1%T group, n = 6), a 2% tetracaine group (2%T group, n = 6), or a 4% tetracaine group (4%T group, n = 6).
The control group received 0.3 mL of saline (0.9% NaCl solution) and tetracaine groups received 0.3 mL of 1%, 2%, or 4% tetracaine solution (crystalline tetracaine hydrochloride dissolved in saline; Kyorin Pharmaceutical, Tokyo, Japan), respectively. The drug was administered through the catheter over 90 s under general anesthesia maintained by 5% inhaled sevoflurane with a nonsealing face mask device. After administration of drugs, anesthesia was discontinued. An antibiotic (cephazolin 30 mg/kg IM) was administered once daily for 6 days after the implantation of a catheter.
The rabbits were neurologically assessed daily until 1 wk after the administration of local anesthetics by an observer unaware of the treatment group. Sensory function was evaluated by seeking an aversive response to pinprick stimulation with a 23-gauge needle progressing from sacral to thoracic dermatomes. The score of sensory function was assessed by a three-point grading scale: 2 = normal; 1 = the region with diminished response is present; 0 = the region with no response is present (8,9). The hindlimb motor function was assessed with a five-point grading scale proposed by Drummond and Moore (10): 4 = normal motor function; 3 = ability to draw legs under body and hop but not normally; 2 = some lower-extremity function with good antigravity strength but inability to draw legs under body; 1 = poor lower-extremity motor function, weak antigravity movement only; 0 = paraplegic with no lower-extremity motor function.
After completion of the neurological function scoring at 1 wk, the animals were reanesthetized and transcardiac perfusion and fixation with 10% phosphate-buffered formalin were performed. After adequate fixation, the spinal cord was removed and divided into the following samples: 1) the lumbar enlargement of the spinal cord, 2) the nerve root entry zone of ventral and dorsal roots, 3) the distal part of ventral and dorsal roots, 4) dorsal root ganglia, and 5) cauda equina. The nerve root entry zone, distal part of the roots, and dorsal root ganglia were obtained at the level of the lumbar enlargement of the spinal cord. The lumbar enlargement of spinal cord and dorsal root ganglia were embedded in paraffin, cut transversely at a thickness of 6 μm, and stained with hematoxylin-eosin. Other samples were postfixed with 1% osmium tetroxide and dehydrated in a series of graded alcohol solutions and embedded in Epok 812 (Oken Shoji, Tokyo, Japan). These samples were cut longitudinally at a thickness of 1 μm by ultramicrotome and stained with toluidine blue.
Histopathological changes were assessed at a magnification of 40× to 200× by an observer unaware of the treatment groups. The nerve fiber degeneration at the nerve root entry zone of ventral and dorsal roots, distal part of the ventral and dorsal roots, and cauda equina was assessed with the longitudinal sections by calculating the percentage of the degenerated nerve fibers in one fascicle. The degenerated nerves were defined as those that showed remarkable edema, demyelination, a disintegration of the myelin, or axonal degeneration. We evaluated the vacuolation of the dorsal funiculus and chromatolytic change of the motor neurons with the transverse section of the spinal cord. The degree of the vacuolation in the dorsal funiculus was assessed by calculating the percentage of the area of the vacuolation in the dorsal funiculus using Adobe Photoshop version 5.5 (Adobe, San Jose, CA) and National Institutes of Health image software. The neurons with chromatolytic change were identified by the round-shaped cytoplasm with loss of Nissl substance from the central part of the cell and eccentric nuclei. The dorsal root ganglia were also evaluated. The histopathological assessments were done in two sections for each part and averaged.
Parametric data are presented as mean ± sd. We used the Kruskal-Wallis test followed by Mann-Whitney U-test to determine differences among the groups in the degree of neurological function, the percentage of the degenerated nerve fibers, and the percentage of the vacuolation area of the dorsal funiculus. P < 0.05 was considered significant.
One rabbit was excluded from the study because it showed neurological dysfunction after implantation of an intrathecal catheter. In the other rabbits, an intrathecal catheter did not cause any irritating behavior or loss of appetite.
The animals receiving saline did not show any behavioral disturbance. All animals treated with tetracaine showed hindlimb motor paralysis when the animals emerged from general anesthesia. The cephalad analgesia level was at Th 9-12 in the 1% T group, Th 3-11 in the 2% T group, and Th 3-10 in the 4% T group. In the 1% T group, anesthetic effects disappeared by 5 h after the tetracaine administration. However, in 3 animals in the 2% T and all animals in the 4% T groups, sensory disturbance persisted until 1 wk after the tetracaine administration.
All animals in the control and 1%T groups showed normal sensory and motor function 1 wk after administration (Fig. 1). The sensory function score was significantly worse in the 4%T group than in the control, 1%T, and 2%T groups. The area of sensory dysfunction included buttocks and hindlimbs in most animals, and extended to lower thoracic levels in 2 animals in the 4%T group. The motor function was significantly worse in the 2%T and 4%T groups than in the control and 1%T groups.
Typical light microphotographs of the nerve root entry zone are shown in Figure 2, and the percentage of the degenerated nerve fibers at the nerve root entry zone are shown in Figure 3. In the control group, there was no apparent change at the nerve root entry zone in both ventral and dorsal roots (Fig. 2). In the 1%T group, the myelin sheaths made by oligodendrocytes were damaged at the both ventral and dorsal nerve root entry zones (Figs. 2 and Fig. 3), whereas the axons were almost intact and the myelin sheaths made by Schwann cells showed minimal changes. When the concentrations of tetracaine increased to 2% and 4%, the axons myelinated by oligodendrocytes also became damaged at the nerve root entry zone of both ventral and dorsal roots (Fig. 2). In the 2%T and 4%T groups, the nerve fibers myelinated by Schwann cells at the nerve root entry zone of dorsal roots became moderately to severely damaged (Fig. 2) compared with those in the control and 1%T groups (Fig. 3), whereas those at the nerve root entry zone of ventral roots appeared to be mildly damaged (Fig. 2) and did not show significant difference among the four groups (Fig. 3).
Typical light microphotographs of the distal part of the ventral and dorsal roots and cauda equina are shown in Figure 4, and the percentage of the degenerated nerve fibers of those are shown in Figure 3. The damage in the distal part of the ventral and dorsal roots, and cauda equina was only minimally detected in the 1%T group (Fig. 4). The damage was detected in the 2%T and 4%T groups with a dose-related tendency of its grade, but the difference did not reach statistical significance among the four groups. The vacuolation of the dorsal funiculus was not detected in the control group but increased significantly in the three tetracaine groups in a dose-dependent manner (Figs. 5, 6). The central chromatolysis of the motor neurons was not detected in the control and 1%T groups (Fig. 5). In 2 animals in the 2%T group and 2 animals in the 4%T group, there were 2, 8, 3, and 6 neurons with chromatolytic change in the ventral horn, respectively. In all animals there was no apparent change in dorsal root ganglia.
In our previous studies, we demonstrated that tetracaine administered intrathecally caused neuronal in-jury in the spinal cord in a dose-dependent manner (6,8,9). Morphological features, including vacuolation of the dorsal funiculus and central chromatolysis of the motor neurons, suggested that the nerve fibers of both sensory and motor neurons are vulnerable to large concentrations of local anesthetics (6,8,9). However, it was not determined what part of nerve fibers was most vulnerable. In the present study, we demonstrated for the first time that the myelin sheaths made by oligodendrocytes at the nerve root entry zone were highly vulnerable to large concentrations of tetracaine given intrathecally.
Takenami et al. (5) investigated morphological changes after intrathecal administration of various concentrations of tetracaine (0.5%–20%) through an implanted catheter with the tip placed at the level of Th-13 in rats. They demonstrated that rats injected with 3% or more tetracaine developed lesions, which began in the dorsal roots close to the spinal cord and extended to the posterior white matter, and that the distal portion of both roots and cauda equina did not show any significant abnormality (5). Because the ventral roots close to the spinal cord showed little change, they have suggested that the initial target of intrathecal tetracaine neurotoxicity may be the dorsal roots at their entry into the spinal cord (5). However, they did not show morphological changes at the nerve root entry zone.
The nerve root entry zone where Schwann cell myelin changes to oligodendrocyte myelin has been considered to be vulnerable to mechanical stretch, including operative maneuvers and trauma (11,12). However, the morphological evidence of the neuronal injury at the nerve root entry zone after intrathecal administration of local anesthetics has not been reported. This is thought to be partly attributable to the difficulty in the assessment of the nerve root entry zone. In the present study, the most difficult part of the experimental technique was to obtain the section that included the nerve root entry zone. However, once the proper section was obtained, it was easy to differentiate myelin sheaths made by oligodendrocytes from those made by Schwann cells with toluidine blue staining as suggested in the literature (13).
Neurotoxicity was detected histopathologically with 1% tetracaine at the nerve root entry zone where the degeneration was selectively found in the myelin sheaths made by oligodendrocytes, although neurological dysfunction was not detected in our scoring system. When the concentrations of tetracaine increased to 2% and 4%, neurological dysfunction became apparent and histopathological changes extended to the dorsal funiculus, distal part of the roots, and cauda equina. Therefore, we recommend including the assessment of the nerve root entry zone when investigating neurotoxicity of local anesthetics administered intrathecally.
As stated above, it has been suggested that the initial target of intrathecal tetracaine neurotoxicity may be the posterior roots at the nerve root entry zone in rats (5). In the present study, the nerve fibers myelinated by Schwann cells at the nerve root entry zone of the dorsal roots appeared to be more severely injured compared with those of the ventral roots. These changes were observed with 3% and 5% tetracaine in Takenami et al.’s study and with 2% and 4% tetracaine in the present study. However, we found that the myelin sheaths made by oligodendrocytes at the nerve root entry zone of both ventral and dorsal roots were equally damaged with 1% tetracaine. Therefore, it seems likely that the initial target of intrathecal tetracaine neurotoxicity is the myelin sheaths made by oligodendrocytes at the nerve root entry zone of both ventral and dorsal roots.
There is no clear explanation why the myelin sheaths made by oligodendrocytes at the nerve root entry zone are highly vulnerable. We have demonstrated that tetracaine administered intrathecally increases glutamate concentrations in the cerebrospinal fluid in a dose-dependent manner although the origin of glutamate has not been determined (6,8,9). Large concentrations of glutamate are neurotoxic. Oligodendrocytes from the forebrain have been reported to be highly vulnerable to AMPA/kainate receptor-mediated neurotoxicity in mice (14). Therefore, glutamate neurotoxicity might be involved in the degeneration of the myelin sheaths made by oligodendrocytes.
The concentrations of tetracaine that showed neurotoxicity in the present study were larger than those used clinically. Nevertheless, the current results may have clinical relevance. Transient neurologic symptoms characterized by pain, dysesthesia, or both in the buttocks, thighs, or lower limbs have been known to occur after recovery from spinal anesthesia (15). Although the relation between persistent neurological injury and transient neurologic symptoms remains to be clarified, it is possible that transient neurologic symptoms represent the lower end of a spectrum of toxicity (16). One of the risk factors postulated to increase the incidence of transient neurologic symptoms has been reported to be a lithotomy position (17). In a lithotomy position, nerve roots may be stretched (18). Mechanical stretch might increase the vulnerability of the nerve root entry zone to local anesthetics.
In summary, we demonstrated that the myelin sheaths made by oligodendrocytes at the nerve root entry zone were highly vulnerable to large concentrations of tetracaine given intrathecally. Our results may provide some insight into the mechanism of neurotoxicity of local anesthetics when administered intrathecally.
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