JNENS

Introduction

Compression of a peripheral nerve can cause functional disorders ranging from mild paresthesia to complete sensory loss, and from slight motor weakness to total paralysis [1]. The severity of the nerve injury depends on several factors, with the level of trauma caused by the compressive injury being the most significant one. Neuropathy of the sciatic nerve is a crucial focus for exploring pain mechanisms, addressing the challenges of neuropathic pain (NP), and advancing axonal regeneration and remyelination [24]. Experimental models designed to simulate NP typically arise from various forms of peripheral trauma. These meticulously crafted models effectively replicate sensory disorders, leveraging peripheral neuropathy to elicit pain responses.

[5-7]. However, researchers must rely on indirect measures to gauge pain intensity since NP cannot be directly quantified in animal subjects. First-degree nerve injury is defined as a nerve conduction block with segmental demyelination8. With recovery, this class of nerve injury can recover function through remyelination. Second-degree nerve injury is defined as damage to the axon [8]. Electrophysiological studies show a decrease in amplitude due to this nerve damage [8,9]. However, the nerve can recover and regain function if the axon is damaged and limited scar tissue forms in the endometrium [8,9]. It is considered a third-degree injury. Unfortunately, full recovery is not possible in such cases. Nerve injuries are classified into five degrees based on the severity of damage. First and second-degree injuries are usually caused by acute nerve compression, while third -, fourth -, and fifth-degree injuries are associated with chronic nerve compression [8]. A third-degree injury is characterized by damage to the myelin sheath, while a fourth-degree injury results in extensive scar tissue formation with no possibility of recovery. Lastly, a fifth-degree injury is defined as a complete severance of the nerve [8]. Past research has indicated that electrophysiological analyses of sensory nerve action potentials and compound motor action potentials (CMAPs) are the most effective techniques for evaluating peripheral nerve dysfunction [5-12]. However, these methods and their outcome measures in assessing sciatic nerve dysfunction have been inconsistent and varied significantly in their reporting [3-13]. Therefore, it is difficult to accurately determine sciatic nerve function electrophysiologically across all NP experimental models. In the work by Hideroni et al. [14]. Their proposed electrophysiological analysis of CMAPs, SNAPs, and SCV in the sciatic nerve follows the clinical electrophysiological methods. This approach addresses a significant limitation in prior studies [10-13].

Gasser & Erlanger established that compression pressure produces a size-dependent conduction block order that may eventually lead to nerve injury. This relationship depends on three primary parameters involved in nerve conduction block and injury: the magnitude, duration, and location of the pressure. Denny-Brown and Schlapp conducted experiments using metal clips to compress rabbits’ sciatic nerves [15]. Their study found that applying slight pressure for two hours did not cause a notable decline in nerve conduction. In contrast, more considerable pressures applied for two hours resulted in paralysis that lasted up to 3 weeks [16]. Other parameters that determine susceptibility to injury include nerve diameter, conduction speed, and myelination. Specifically, it was observed that fast and larger-diameter myelinated axons are more vulnerable to conduction block than slower and smaller-diameter myelinated axons [16,17]. In previous studies, two significant consequences of compression nerve injuries have been identified. The first consequence is mechanical deformation, a well-studied manifestation of this type of injury. According to Fern and Harrison, nerve compression creates a pressure gradient between compressed and uncompressed nerve segments. This gradient causes improper shifting of axonal materials. Changes in fiber morphology also indicate mechanical deformation. The epineurium and perineurium surrounding the peripheral nerve and fascicles are thickened. Within the nerve structure, there is a movement of the nuclei to the periphery and an inappropriate scattering of Nissl substance [18]. Additionally, the nerve conduction block has been hypothesized to result from the displacement of the Node of Ranvier [19]. Causey and Palmer stated that early tissue deformation due to pressure applied to a nerve results in a decrease in the nerve’s cross-sectional area. This association is defined by the pressure magnitude being inversely proportional to the change in cross-sectional area. The change in cross-sectional area is primarily due to a decrease in the interstitial space. The mean fiber size was also decreased to a lesser extent [17]. Another significant consequence of nerve injury is the inappropriate regulation of intraneural blood flow. Mainly, the venous blood flow in the epineurium was found to be impaired. This impedes intracellular axonal transport. Impaired blood flow has been attributed to changes in the permeability of intraneural blood vessels. Consequently, this increases the intraneural interstitial fluid pressure, further impeding venous blood flow [20]. Studies examining the impact of compression pressure on nerve injuries have primarily used invasive techniques that directly compress the nerves. These techniques involve exposing an animal’s muscles and soft tissue, isolating the nerve, and applying pressure over the epineurium or myelin sheath. However, such nerve lesions do not provide clinical insight into how nerves respond to real-world injuries involving muscle, soft tissue, and skin. In the present study, we investigate whether variations in the magnitude of compressive trauma, produced by altering external compressive pressure in an in vivo rat model, result in clinically detectable differences in functional disruption. We directly assess functional loss using electrophysiological measures, including the compound motor action potential (CMAP) and nerve conduction velocity (NCV), before, during, and after the injury. Functional loss is compared across four tested compressive pressures as applied to the triceps surae region of both male and female rats.

Methods

This study used Sprague Dawley female (n = 8) and male rats (n = 8). These rats were acquired at 200-250 grams (Charles River Laboratories, Chicago, IL, United States) and acclimated to approximately 300-450 grams before use in experiments. All experimental procedures were approved by the University of Illinois at Chicago Institutional Care and Use Committee. A custom pneumatic compression cuff was devised by modifying a small-animal blood pressure cuff to a size of 2 cm × 12 cm. It was connected to a manual inflation bulb and an analog pressure gauge via a two-way valve system, allowing inflation of the cuff and maintenance of the experimental pressure. Four compression pressures-0, 120, 180, and 240 mmHg-were chosen for experimental exploration. The lower test pressure limit was used as the control, and the remaining pressures were assigned to the experimental groups. Anesthesia was maintained throughout the experiment using 3% isoflurane in oxygen. Induction was achieved in a chamber box with 0.8-1.5 L/min of concomitant oxygen. Maintenance anesthesia was administered via a mask connected to a Bain circuit, with a concomitant oxygen flow rate of 0.4 to 0.8 L/ min. To prevent heat loss, animals were kept draped over a waterrecirculating heating pad for the duration of the experiment until they recovered from anesthesia. The animal’s experimental limb was secured in full extension before wrapping the pneumatic cuff around the proximal aspect of the gastrocnemius muscle. CMAP recordings were obtained before cuff inflation and the start of the experiment (Figure 1).

Figure 1: Rat hindlimb with pneumatic tourniquet and CMAP electrodes.

Continuous single-channel CMAP recordings were obtained by placing 32-gauge reading needle electrodes at four different locations: one recording electrode each at the muscle belly of the (electrode 1) lateral and (electrode 2) medial heads of the gastrocnemius, above the limb compression site, and (electrodes 3-4) two within the Achilles tendon, distal to the compression site. To stimulate the Sciatic nerve with an intermittent current pulse, two bipolar stimulating needle electrodes were inserted proximal to the compression site. To appropriately target the Sciatic nerve proximal to its trifurcation, palpation of the rat’s lateral femoral condyle was used as a horizontal and vertical landmark - stimulating electrodes were placed on either side of the hind leg with the distance between cathode and anode approximately ≥ 1 cm. CMAP recordings were captured using a PowerLab modular data-acquisition device, LabChart recording software, and a BioAmp (ADInstruments). Once recording started, the data were processed with a low-pass filter at 1 kHz, a high-pass filter at 1 Hz, and an active primary filter with a 3-second delay to eliminate 60 Hz interference, before being displayed in real time on the screen (Figure 2).

Figure 2: Schematic demonstrating hardware requirements for recording CMAP.

After complete anesthesia was reached, the experimental animal was positioned correctly and secured, and the pneumatic cuff was placed around the rat’s limb at the anticipated site of compression. Initially, the cuff was secured to the animal’s limb but was not yet pressurized. All electrodes were then placed in their proper positions. Only after real-time CMAP recordings were established and running continuously did the compression cuff pressure match the pressure being studied in the respective experiment. Changes in CMAP amplitude (in mV) before and after pressure application were noted. A baseline CMAP amplitude, measured within 5 minutes of cuff pressurization, was calculated as the average of 11 consecutive CMAP peaks. CMAP recordings were maintained continuously for a subsequent 180 minutes. During this time, limb compression pressure was maintained within +/- 5 mmHg. CMAP amplitudes were measured at 10-minute intervals throughout the experiment. Thus, with the baseline value, 18 additional experimentally captured amplitude values allowed us to trend changes in CMAP throughout the experiment.

The following mathematical expression ascertained NCV:

NCV (mm/msec) = ∆D stimulating-recording electrodes (mm) / ∆t (msec), where ∆D stimulating-recording electrodes represent the length measured in millimeters from a point equidistant to the stimulating cathode and anode to a point midway between the two most distal recording electrodes (within the Achilles tendon). ∆t represents the time in milliseconds when an action potential travels the distance ∆D, stimulating-recording electrodes, as determined on the x-axis of a CMAP recording using the Lab Chart software. After proper electrode placement, the distance between the stimulating and recording electrodes was measured with a tape measure, and baseline CMAP measurements were obtained (Table 1).

Table 1: Rat Leg Circumference and Probe Distance

All captured electrophysiological data, representing the duration before, during, and after limb compression in each experimental trial, were extracted from LabChart recording software (macOS, Version 8.1.17; AD-Instruments Inc.). Average CMAP amplitude values at each point of interest were determined as the average of 11 consecutively recorded CMAP peaks, with the point of interest set to the median, and 5 preceding and 5 subsequent peaks. The duration of compression was defined as the period from the baseline CMAP to the point at which pressure was maintained within ±5 mmHg of the target. Data analysis was performed using SPSS (IBM SPSS Statistics for macOS, Version 24.0; IBM Corp.), the Python programming language (version 3.10 for Windows, Python Software Foundation: https://www.python. org/), MATLAB (version R2022a for Windows; MathWorks, Inc.), and Excel (Microsoft Excel for macOS, Version 16.47; Microsoft Corp.).

Results

The baseline CMAP amplitude before initiating limb compression (>0 mmHg) was 71.2 ± 14.4 mV, 72.8 ± 4.5 mV, and 69.8 ±19.9 mV for all trials involving male and female rats, respectively. For all trials, the average CMAP amplitude decreased to 60.9 ± 14.5 mV after approximately 3 hours of limb compression, corresponding to a 14.5 ± 0.7% decrease from baseline. Stratified by gender, trials involving male rats decreased to an average amplitude of 58.6 ± 11.7 mV, and those involving female rats decreased to 62.9 ± 17.2 mV. These correspond to decreases of 19.5 ± 160.0% and 9.9 ± 13.6% from baseline, respectively.

Table 2: Summary of Data Points and Statistical Analysis Results for 0 mmHg (Control, N=2).

Two trials, 1 with a male and 1 with a female animal, were conducted at a limb compression pressure of 0 ± 2 mmHg. This was considered the control cohort, as the pneumatic cuff was applied gently to the animal’s limb, flush with the skin, but not inflated. The average baseline CMAP amplitude of the control group was 53.1 ± 33.7 mV. This decreased to 51.2 ± 30.5 mV after 1 hour of continuous cuff application, corresponding to a 3.6 ± 9.5% decrease (t-statistic = -0.087; p-value = 0.945). At the second hour of continuous cuff application, CMAP amplitude was 49.8 ± 31.6 mV, corresponding to a 6.2 ± 6.2% decrease from baseline (t-statistic = -0.149; p-value = 0.906). After 3 hours of compression, CMAP amplitude was 52.3 ± 32.9 mV, corresponding to a 1.5 ± 2.4% decrease from baseline (t-statistic = -0.036; p-value = 0.977) (Table 2).

Table 3: Summary of Data Points and Statistical Analysis Results for 120 mmHg (N=3).

Three trials were performed with the pneumatic cuff applied to the animal’s limb and inflated to a compression pressure of 120 mmHg (Table 3): 2 trials with male rats and 1 with a female rat. The average baseline CMAP amplitude recorded immediately after inflation of the cuff (within <5 minutes from cuff application to the limb) was 77.6 ± 3.4 mV. After 1 hour of continuous limb compression, amplitude decreased to 75.2 ± 4.0 mV, corresponding to a 3.1 ± 17.7% decrease from baseline (t-statistic = -1.015; p-value = 0.417). At 2 and 3 hours of continuous compression, CMAP amplitude was found to be 75.3 ± 5.1 mV and 72.6 ± 5.0 mV, corresponding to decreases from baseline of 3.0 ± 50.0% (t-statistic = -0.777; p-value = 0.519) and 6.4 ± 47.1% (t-statistic = -1.765; p-value = 0.220), respectively.

Table 4: Summary of Data Points and Statistical Analysis Results for 180 mmHg (N=4).

Four trials were performed at a compression pressure of 180 mmHg, with two trials per sex. The average baseline CMAP amplitude, recorded immediately after cuff inflation, was 76.1 ± 11.8 mV. At 1, 2, and 3 hours of continuous compression, CMAP amplitudes were 68.8 ± 10.9 mV, 66.9 ± 9.2 mV, and 62.7 ± 11.1 mV, respectively. This corresponded to decreases from baseline of 9.6 ± 7.6% (t-statistic = -1.347; p-value = 0.271), 12.1 ± 22.0% (t-statistic = -1.987; p-value = 0.141), and 17.6 ± 5.9% (t-statistic = -2.412; p-value = 0.095), respectively (Table 4).

Table 5: Summary of Data Points and Statistical Analysis Results for 240 mmHg (N=4).

Four trials were performed at a compression pressure of 240 mmHg (Table 5), with two trials per sex. The average baseline CMAP amplitude, recorded immediately after cuff inflation, was 70.4 ± 4.0 mV. At 1, 2, and 3 hours of continuous compression, CMAP amplitude was 61.9 ± 6.7 mV, 57.5 ± 9.4 mV, and 54.8 ± 10.1 mV, respectively. This corresponded to decreases from baseline of 12.1 ± 67.5% (t-statistic = -2.535; p-value = 0.069), 18.3 ± 135.0% (t-statistic = -2.747; p-value = 0.051), and 22.2 ± 152.5% (t-statistic = -3.086; p-value = 0.044), respectively.

Table 6: Summary of Data Points and Statistical Analysis Results for NCV.

SNAP conduction velocity is measured by calculating the distance and time required for the action potential to propagate. SNAP conduction velocity is expressed in millimeters per second (mm/s). No final change was observed for the control and 120 mmHg cohorts, but alterations occurred at various intervals before returning to the baseline. In contrast, subjects within the 180 mmHg and 240 mmHg cohorts revealed alterations. One rat in the 180-mmHg cohort declined in conduction velocity from 1380 mm/ms to 690 mm/ms after three hours (t-statistic = -1.385; p-value = 0.591). Two rats in the 240-mmHg cohort showed a decline from 1180 mm/ms to 590 mm/ms (t-statistic = -1.092; p-value = 0.532) and from 1440 mm/ms to 720 mm/ms (t-statistic = -1.265; p-value = 0.492). Overall, any changes observed in either cohort were deemed insignificant (Table 6).

Discussion

In the present study, we employed a standardized approach to induce peripheral nerve injuries by applying circumferential compression pressure to the lower extremities of Sprague-Dawley rats. We defined trauma severity based on the magnitude of the force and its duration. The experiments consisted of four groups: 0 mmHg (control), 120 mmHg, 180 mmHg, and 240 mmHg. The durations for the control and experimental groups were 1, 2, and 3 hours of continuous compression [17]. The effects of graded compression were analyzed in situ and in real-time. The nerve conduction studies used CMAP and NCV to explore motor and sensory function. We demonstrate incremental declines in motor-neuron competency of the rat sciatic nerve with increasing magnitudes of compressive trauma. Although motor nerve function steadily decreases with increasing injury severity, no statistically significant declines are observed at compressive pressures of 0, 120, or 180 mmHg. In contrast, conduction of injury at a pressure of 240 mmHg is demonstrated to be the critical threshold value above which loss of motor function becomes statistically and clinically meaningful. Moreover, clinically relevant nerve injury was shown to occur only after 2 hours of continuous trauma (Figure 3). In previous studies, a discernible trend emerges, with the data showing considerable inconsistency. These studies predominantly focus on direct pressure injury, neglecting careful consideration of the diverse tissues and anatomical structures that exist between nerves and the external environment of the limbs.

Figure 3: Percent Change on EMG v. Time.

Our study supports and extends previous research by establishing quantitative measures of nerve function decline due to compressive injury. Stecker et al. constructed a model in which they applied compressive pressures up to 2000 mmHg at 200-second intervals for up to 20 minutes. The CMAP amplitude reduction was approximately 50% from baseline at the highest pressures tested [21]. Therefore, a positive correlation is evident between higher pressure magnitudes and greater reductions in CMAP amplitudes. Our findings support this conclusion by demonstrating a reduction in CMAP amplitude with increasing pressure and longer continuous pressure durations (Table 7 and Figure 4). Unlike Stecker et al., our experiment did not lead to significant changes in NCV. The NCV fluctuation was minimal, with any decreases in velocity plateauing over the three-hour interval. This finding suggests that the nerve’s microenvironment is adaptable, enabling it to dynamically recalibrate and establish a new baseline in response to external compression and subsequent mechanical damage.

Table 7: Amplitude Percent Change from Baseline for Each Group after 1, 2, or 3 Hours of Compression.

 Figure 4: Amplitude Change v. Time for 240 mmHg.