Guidance of Block Needle Insertion by Electrical Nerve Stimulation: A Pilot Study of the Resulting Distribution of Injected Solution in Dogs

The study was conducted in compliance with animal research regulations of the Medical College of Wisconsin and with the approval of the Institutional Animal Care and Use Committee (Milwaukee, Wisconsin). To limit the use of animal subjects while avoiding sequential survival surgeries in any one subject, mongrel dogs (n = 11, both sexes, 25–35 kg) were used from another study in a manner unlikely to affect the findings of either study. Specifically, injections were performed during the terminal, organ harvesting phase of open-chest cardiovascular research protocols during general anesthesia (200 mg/kg barbital sodium, 15 mg/kg pentobarbital) and mechanical ventilation. Coronary occlusion was followed by 3 h of reperfusion, during which animals showed no cardiogenic shock or failure, after which the nerve injection experiment was initiated. The cardiovascular study protocols variously included administration of intravenous sildenafil or simvastatin more than 3 h before the nerve injections. No local anesthetics were injected during the cardiovascular or nerve injection protocols. Four additional dogs were studied in which hyperglycemia had been induced by treatment with intravenous alloxan and streptozotocin.12These animals developed sustained hyperglycemia (blood glucose 250–350 mg/dl) without ketosis or renal insufficiency during the 3-week period between initiation of hyperglycemia and the terminal experiment.

Hemodynamic conditions were stable throughout the experimental period. With the dog in a lateral position, a stimulating needle (22 gauge, 2-inch length, Stimuplex A needle; B. Braun, Bethlehem, PA) was inserted from the medial or lateral aspect at the mid-thigh level 1–2 cm posterior to femur. Stimulation through the needle was provided from a source (Digi-stim Plus 3 nerve stimulator; CCR Medical, Inc., St. Petersburg, FL) with the cathode lead on the needle and the anode lead clipped to muscles at the thoracic incision. This device produces a square wave monophasic pulse with a duration of 0.2 ms, provides continuing readout of the stimulating current, and was tested on an oscilloscope for accuracy.

Animals were randomly assigned to one of two groups. In both, initial advancement of the needle was performed with 1-mA stimulation. When a distal motor response was achieved, typically extension at the ankle and/or plantar flexion of the toes, needle advancement was immediately halted, and its position was stabilized. The current was diminished until the motor response vanished and was then increased again to identify the minimal current sufficient to produce a motor response, which was tabulated as the “final threshold.” In the high-current group, injection was performed at this point. In the low-current group, the current was set at 0.5 mA, further advancement or needle redirection was performed until motor stimulation was achieved, and the final threshold was determined as described above and injection was performed. In all animals, 0.5 ml of particulate black or orange ink was injected over 5 s at the final needle location, with 0.1 ml of this total being injected during needle withdrawal to mark the needle track. This small volume was used because our goal was to accurately record the needle tip location and to identify whether solution injected through the needle at this location would enter the nerve. Therefore, the ink left behind served as a proxy for the tissue location of the needle tip at the moment of injection. A larger volume, such as a dose proportionate to a clinical injection, would tend to obscure this detail and substantially distort the anatomy. In most animals, both sciatic nerves were injected.

Immediately after injection, the animals were killed by removal of the heart. The posterior thigh tissues were removed en masse  with care not to distort the tissues. The resulting specimen was marked with sutures for orientation and stored in a −80°C freezer. The material was cryosectioned without fixation in 15-μm increments, and photographs were taken of the block every 300 μm using a digital camera (d70, micro 105 lens, Nikon, Melville, NY; and a 1:2 teleconverter, Tamron Inc., Commack, NY) mounted on a tripod, with a scale included in the image. Sections were performed in a plane perpendicular to the axis of the sciatic nerve.

Photographic series were examined without knowledge of the stimulation setting or presence of hyperglycemia. A standard process was used in which the injection site was first identified. Features identifying the section representing the needle position included accumulation of ink as a pool rather that occupying interstices in the tissue, a tapering pattern of ink in adjacent images, bleeding, and the residue of a track through the adjacent muscle marked by ink. Because of displacement of adjacent structures during palpation for injection and shift of layers after harvest, the needle track did not reliably indicate the exact path to the nerve, but was considered a reliable indicator that the injection site was within the sample. We thereafter examined images both proximally and distally to identify the deepest penetration of ink contiguous with that at the injection site. By this approach, even when the tissue layers at the point of injection were hard to discern exactly, we could determine the closest proximity of the needle to the sciatic nerve or its distal divisions at the moment of injection, assuming that the ink was unable to cross tissue barriers because of its particulate nature. Because our imaging does not allow reliable recognition of the perineurial layer, we categorized ink location as within the epineurium of the nerve, in contact with the epineurium, or distant from the nerve. In this final case, we measured the distance between the nerve and the closest ink.

Cross-tabulation and the Pearson chi-square were used for nonparametric analysis of ink distribution patterns (Statistica 7.0; StatSoft, Tulsa OK). Significance levels were set at P < 0.05. Stimulation thresholds are presented as mean ± SD, and the significance of differences between groups was determined by the Student t  test.

Distal motor stimulation was achieved in all animals. The injection site could be identified in 24 of 30 specimens, thus assuring that we did not miss the critical portion of the nerve in the harvest. Data were not used from the remaining 6 specimens. The pattern of ink distribution was readily categorized from examination of the photographed sections (figs. 1–3).

In the normoglycemic animals, the actual measured stimulation thresholds were higher in the high-threshold group compared with the low-threshold group (P < 0.001; table 1). The distribution patterns of ink (table 1) were not different between low and high stimulation threshold groups (P = 0.462), with both groups showing a mix of injections distant from the nerve and in contact with the nerve. (We acknowledge that this study's sample size cannot determine statistical similarity regarding this parameter but can only confirm differences.) In addition, one intraneural injection occurred in the high-threshold group. For injections that were distant from the nerve, the average distances in the low- and high-threshold groups were not different.

The six needle insertions in the four hyperglycemic dogs were all performed using a low-threshold technique. No insertions in the hyperglycemic dogs used a high-threshold technique. The average stimulation threshold in the hyperglycemic group was the same as in the low-threshold normoglycemic group, but the ink distribution patterns were significantly different (P < 0.001), with only intraneural patterns occurring in the hyperglycemic animals.

Although monitoring a motor response during passage of current through the nerve block needle is a standard technique for guiding placement of the needle, there is minimal understanding of the final relation between needle and nerve when using this method. Even whether the solution is deposited within or outside the nerve has not been established when using various stimulation parameters. In our study, a high threshold of 1 mA resulted in a mix of distribution patterns comparable to that which occurs using a much lower threshold level of 0.33 mA. While many cases in each category showed contact of the small volume of injected solution (0.5 ml) with at least part of the nerve, others showed no solution actually reaching the epineurium. Because in all cases the solution reached within 0.5 mm of the nerve, successful block would likely have been achieved in all injections regardless of final stimulation threshold, because the larger volumes of clinical injections would provide a source from which diffusion of local anesthetic would ultimately block conduction. These anatomical results suggest that threshold differences in this range are not clinically relevant, as has been reported in studies of efficacy in patients.9,13,14 

Although our study is not powered adequately to prove the similarity of the ink tracer location after insertion with high and low stimulation threshold, the lack of any trend showing a difference makes it unlikely that a large effect would be found with a larger sample size. The counterintuitive result that a lower final threshold does not deliver solution closer to the nerve may be due to the inherent inhomogeneity of the electrical conditions immediately surrounding the nerve. Consistent with this hypothesis, we have noted that minute movements of the stimulating needle that are too small to penetrate into a new tissue layer nonetheless may have substantial effects on the ability to produce a distal motor event, which probably represents contact of the needle tip with tissues of different resistance such as fat, fibrous tissue, or vascular elements.

Although a larger number of subjects in this study may have produced a difference between groups, we note that there was substantial variability in both the high- and low-threshold distribution patterns. Our results may also have been different if a noninsulated needle had been used. However, conduction through the tip alone is thought to produce a more precise placement than is achieved with a noninsulated shaft.15Because the epineurium provides a relatively large resistive barrier to stimulation,16variations in the thickness of this layer between subjects or on different fascicles that might have been approached by the stimulating needle may also account for variability in results that overwhelms any influence of stimulation current levels in the two groups. Our technique did not identify the location of the needle tip per se  but rather the location of a small amount of solution injected through it. We used this approach because this leaves a secure trace of the needle's location at the moment of stimulation, and the toxic effect of injected anesthetic is considered the main contributor to nerve damage rather than mechanical damage from needle penetration per se .1,2Finally, use of a stimulus duration other than the 0.2 ms used here may have resulted in different depths of needle placement. However, block success rates are indistinguishable even when 0.1- and 0.3-ms durations are compared.17 

Two previous studies have examined the relation of stimulation current level to nerve/needle distance in animal models.15,18The needle location was determined in both these studies by dissection, making accurate determination of injection depth relative to the nerve uncertain. The reported findings indicate that the needle was within 3 mm for thresholds of 0.37–0.47 mA in rabbits18and within 2 mm for thresholds of 0.9–1.2 mA in cats.15The small size of the animals and decreased thickness of their epineurium may account for the greater distances at these stimulation levels. Also, our model duplicated a clinical technique, whereas a micromanipulator was used in these other studies.

In a small group of injections in hyperglycemic animals, we found that use of a low stimulation threshold uniformly resulted in intraneural injection. Although constraints on the availability of animals did not permit examination of a high-threshold group in hyperglycemic dogs, it is not likely that improved safety would be guaranteed, considering the lack of difference we found in normal animals using high and low thresholds. Clinical reports have observed unexpectedly high stimulation thresholds in diabetic patients,10even when ultrasound imaging shows contact of the needle with the nerve.11These observations imply that low or normal thresholds could require intraneural needle placement to achieve motor stimulation, as we observed in hyperglycemic dogs. Conduction velocity in diabetics is slowed because of numerous factors.19Because longitudinal conduction of action potentials represents sequential excitation of segments of neuronal membrane, factors that diminish conduction velocity would also depress excitation by an external source of depolarization such as a needle electrode. Reduced excitability of peripheral nerves by depolarizing current has been confirmed in diabetics by clinical electrophysiologic examination.20 

Clinical diabetes mellitus consists of a variety of pathogenic processes that accrue over time. Although the duration of hyperglycemia in the model we used was relatively brief, it nonetheless resulted in decreased conduction velocity in the timeframe we tested, attributable to disrupted neuronal metabolism21and reduced endoneurial blood flow.22,23Particularly important aspects of diabetic neuropathy are directly attributable to the effect of hyperglycemia via  increased activity of the sorbitol pathway of glucose metabolism, which is activated in sensory neurons within hours of exposure to hyperglycemia and produces excitability deficits and sensory neuron dysfunction within 1–4 weeks.24–26 

Our findings in addition indicate that intraneural injection may ensue during blockade guided by needle stimulation even in normoglycemic animals using a high stimulation threshold. Furthermore, compared with our protocol using a pulse duration of 0.2 ms, it is possible that a commonly used shorter pulse duration of 0.1 ms13would increase the incidence of intraneural injection, because a shortened pulse duration results in a decreased potency for motor stimulation.27The threshold for motor stimulation with needles placed intraneurally in surgically exposed pig nerves averages 0.43 mA and may range up to 1.8 mA.28Although it is accepted that awake human subjects may experience pain during intraneural injection, there are no data to indicate what fraction of intraneural injections produce such a response, and it is known that intraneural injections may occur without a sensory event.5,6,29,30Together, our data and these observations suggest that partial intraneural injection may be a common feature of nerve blocks using needle stimulation. In diabetics, a decreased sensitivity to electrical stimulation combined with diminished sensory function and increased sensitivity to local anesthetic toxicity31may particularly elevate the risk and consequence of unrecognized intraneural injections during nerve blocks guided by needle stimulation.

Certain limitations of our study must be emphasized. Although there is no direct manner in which previous coronary occlusion and sildenafil or simvastatin might alter the responsiveness of nerves to stimulation, secure conclusions will require confirmation without these potentially confounding influences. More importantly, the small number of subjects, particularly in the hyperglycemic group, means that these results must be considered preliminary and serve primarily to generate hypotheses. A much larger study with animal subjects subjected solely to block experiments, and confirmation in a study of patients, would be necessary before a change in clinical practice could be recommended on these matters. Finally, application of our findings to a clinical setting may be limited by an inability of some stimulation systems in clinical use to accurately duplicate the stimulation intensities described here.

We conclude that under study conditions that resemble clinical peripheral nerve blockade, both high- and low-current stimulation levels may result in needle placement close to the target nerve. The PNS technique does not preclude intraneural injection, especially in subjects with hyperglycemia. It is possible that additional monitoring and/or imaging techniques may be necessary to avoid intraneural injection.

The authors thank John Tessmer, B.S., and John Krolikowski, B.S. (Laboratory Technicians, Medical College of Wisconsin, Milwaukee, Wisconsin), for expert assistance.