Hindpaw Withdrawal from a Painful Thermal Stimulus after Sciatic Nerve Compression and Decompression in the Diabetic Rat

Abstract

Introduction Although the effect of chronic compression and surgical decompression of the diabetic rat sciatic nerve has been evaluated by walking track analysis, the measurement of sensory function by response to thermal nociceptive stimulation has not been investigated.

Methods Fifteen male Wistar rats with streptozotocin-induced diabetes underwent sciatic nerve compression through a 10-mm silicone band. Five rats had histology done 60 days after confirming chronic nerve compression. Pain threshold was measured using hindlimb withdrawal times (HLWT) from a heat stimulus. After 60 days of compression, the silicone tube was removed. Five nondiabetic, nonbanded rats were used as controls.

Results Control mean HLWT was 9.7 ± 1.5 sec. In the diabetes group (60 days of compression), mean HLWT was 23.6 ± 2.4 sec. (p < 0.001). Thirty days after removal of the silicone, mean HLWT to painful stimuli was 14.9 ± 1.5 sec. (p < 0.001).

Conclusion Chronic compression of the diabetic rat sciatic nerve increases (worsens) the threshold to heat (pain) perception. Decompression reverses this effect (improves nociception).

Keywords

diabetes - pain - nerve compression

It has been shown that the diabetic rat is susceptible to nerve compression and that elimination of compression permits recovery[1] of sensory and motor function. Several tests used in experimental studies confirm the loss of motor function after nerve compression and diabetes mellitus (DM) by analyzing the movement of rats,[2] [3] [4] but in the same pathological conditions no studies have evaluated the perception of thermal pain stimulation. Our experimental model evaluates the lesions after nerve compression in diabetic rats and the response of subsequent neurolysis upon the perception of pain as determined by the withdrawal of the limb from a painful thermal stimulus.

Methods

Animals

The experimental study was performed at the Pius Branzeu Center for Laparoscopic Surgery and Microsurgery with the approval of the ethics committee for animal experimentation of the Victor Babes University of Medicine and Pharmacy, Timisoara, Romania. Twenty male Wistar rats, weighing between 250 and 300 g, were followed for 90 days. They were housed in metal cages and given food and water ad libitum.

Plantar Test Assay

Plantar Test (Ugo Basile, Comerio VA, Italy) is an instrument that measures sensitivity to pain in laboratory rats.[5] It consists of a controller that can be set and calibrated for different parameters, an infrared (IR) light source that induces pain in the pelvic limb paw, and a transparent support on which the rat is placed in testing position. Briefly, prior to testing, each rat was placed on this support, on which it adopted a reflex sitting position after a few minutes. An IR light source was placed under the pelvic limb paw,[6] and the IR source intensity was set. IR intensity is expressed in milliwatts (mW) per square centimeter (cm²) of body surface (BS) area acting in a unit time (sec.).[6] [7] [8] The intensity was set at 50 mW/cm² BS throughout the study. Thermal sensibility threshold, or pain (nociceptor) sensitivity evaluation, was measured (sec.) from the beginning of stimulation to the moment of hindfoot withdrawal. Because of reduced perception of painful stimuli through peripheral neuropathy, prolonged exposure of plantar skin to IR can induce burns. To avoid this, an automatic cut-off time was set to 30 sec. In the absence of reaction (hindfoot withdrawal) in the examined rat within the 30-sec. timeframe, stimulation was turned off automatically with a latency of 1 sec.[9] [10] [11] [12] [13] [14]

Plantar test assay was applied in group A on day 0, day 60, and day 90 and compared with controls (group C). Each rat was tested 10 times/examination, and mean response time to the painful stimulus on day 0 was considered normal value.

Experimental Groups

The rats were divided into three groups: A (n = 10), B (n = 5) and C (n = 5 nondiabetic, control group). On day 0, group C was tested with Plantar Test and the normal value of the reaction to the painful stimuli was determined and used as control. In group A and B, DM was induced chemically by a single intraperitoneal injection of 60 mg/kg/body weight streptozotocin (STZ). The STZ solution was prepared from sterile lyophilized substance (Streptozocin, S0130, Sigma Aldrich, St. Louis, MO) dissolved in 0.1 M citrate, pH = 4.6.[15]

Starting with day 3 after injection, the glucose level of the rats in both groups was monitored. Blood samples were collected through tail vein access and the automatic analysis of the glucose concentration was performed with a standard glucometer. Diabetes was diagnosed individually, after recording two consecutive glucose values higher than 200 mg/dL (normal level: 70 to 110 mg/dL). To prevent ketosis and death, rats with glucose levels above 400 mg/dL received 1 to 2 UI/day INSULATARD (Novo Nordisk A/S, Bagsvaerd, Denmark) administered subcutaneously.

For nerve compression, animals were anesthetized using mask-driven inhalatory 2% isoflurane + 4 l/min O2 through a tabletop vaporizer (Harvard Apparatus, Holliston, MA). Clean but not sterile conditions were used. The rat is placed in ventral position and its right pelvic limb immobilized with elastic strings. The anatomical reference points for the sciatic nerve were the sciatic notch, the femur, and the knee joint. The anatomical projection of the sciatic nerve is marked from its origin down to the level of the knee joint. A longitudinal incision of ∼20 millimeters (mm) was made in the sciatic nerve parallel and posterior to the cutaneous projection of the femoral diaphysis by separating the flexor femoral muscle mass of the thigh.[16] Once the sciatic nerve was identified (approximately 1.2 mm diameter) and dissected, a 10-mm silicone tube (internal diameter 1.25mm, MasterFlex, Cole-Parmer, Vernon Hills, IL) was wrapped circumferentially around the middle third of the nerve and fastened with 8–0 nylon sutures ([Fig. 1]).[17] [18] After 60 days, in group A the silicone tube was removed (decompression of sciatic nerve).

Fig. 1 Sciatic nerve compression with a silicone tube was applied in group A and B at day 0. After induction of diabetes mellitus and 60 days of compression, the silicone tube was removed.

In group B, DM and chronic compression were induced exclusively for the histopathological examination. The sciatic nerve in this group was sampled on day 60. The rats were euthanized by short inhalatory anesthesia as described above, followed by cardiac injection of T61 (MSD Animal Health, Unterschleisheim, Germany). The sciatic nerve was sampled using the same approach as described above. The samples that were collected at a distal point from the compression level were introduced in 1% formalin and then fixed in paraffin blocks. The samples were stained with three methods: Masson's trichrome, hematoxylin-eosin, and toluidine blue, and the examination was performed with a microscope (Multizoom AZ100, Nikon Instruments, Inc., Melville, NY). The histopathological examination was performed in the laboratory of the Morphopathology Department of the Victor Babes University of Medicine and Pharmacy Timisoara, Romania. The examination searched for pathological changes in the nerve fiber components.

After 60 days, in group A, sensitivity to pain was determined a second time with Plantar Test and the decompression of the sciatic nerve was performed by removal of the silicone tube and epiperineural neurolysis. After 90 days, at the end of the study, group A, was re-evaluated with Plantar Test and the recovery of pain sensitivity in the pelvic limb was monitored.

Statistical Analysis

The data were recorded and analyzed statistically by help of the OpenEpi (software version 2.3.1), which calculated the mean average and standard error mean (SEM) for quantitative variables, the statistical comparison of means average for each group by paired Student t-test, and finally estimated the statistical significance using p < 0.05.

Results

The results of the histopathological examination revealed endoneural and subperineural edema consistent with diabetes. The nerve fibers after 60 days of compression demonstrated perineural fibrosis, thinning of the myelin, and some axonal loss consistent with chronic compression from the silicone tube.

The hindpaw withdrawal times demonstrated that in the control period, day 0, the mean reaction time was 9.67sec. ± 1.49 SEM. Sixty days after inducing DM and the chronic compression of the sciatic nerve, in the rats of group A (n = 10) pain sensitivity decreased (withdrawal times increased) in comparison with day 0, reaching a mean of 23.63 sec. ± 2.39 SEM. The statistical analysis revealed a significant difference in the pain sensitivity values before and after peripheral nerve compression (p < 0.001). These 10 rats in Group A then had a removal of the silicone tube to decompress the sciatic nerve, followed by a 30-day recovery period. The hindpaw withdrawal times showed (compared with day 60, 23.63 sec. ± 2.39 SEM) improvement in sensitivity toward normal, with the mean pain sensitivity reaching 14.99 sec. ± 1.50 SEM ([Fig. 2]), p < 0.001.

Fig. 2 The average response to painful stimulus in the control group without diabetes (Non DM [diabetes mellitus] rats) showed normal value and is compared with the average response of group A after 60 days of diabetes mellitus and chronic compression (DM rats). After 90 days of diabetes and decompression at day 30, there is a significant decrease of the pain sensitivity threshold (*p< 0.001) in the same group. Values presented as mean ± standard error mean.

Discussion

The most plausible mechanism for the susceptibility of the peripheral nerve in DM to chronic nerve compression is the double-crush syndrome.[1] This concept was applied in the experimental study of the present paper, with a view to monitor the histological changes of the compressed sciatic nerve in Wistar rats and the evolution of pain sensitivity in the plantar surface of the hindlimb. Results show that at 60 days of sciatic nerve chronic compression, the reaction to the painful stimulus decreased (9.69 sec. in day 0 versus 23.63 sec. in day 60; p < 0.001).

Lundborg[19] has proved that applying low-intensity compression on a peripheral nerve generates increased intraneural pressure by the formation of edemas, which decreases the blood and the axoplasmic flow. The histopathological study of the present paper reveals significant changes in the nerve structure, such as vacuolizations in the cytoplasm and neural edema, atrophy and advanced Wallerian degeneration, chronic inflammatory infiltrate with intra- and perifascicular disposition, reduced myelin layer and perivascular fibrosis, marked hyperemia, and dystrophic calcifications in vessel walls at the level of the vasa nervorum. The experimental model continued with decompression and assessment of postoperative pain reactivity to stimulation, day 90. The studies in humans have shown that neurolysis of chronic nerve compression in the lower extremity can relieve pain in ∼80% of patients with diabetic neuropathy.[20] [21] [22] [23] [24] [25] The decompression in the present study demonstrated the reversal of the pain sensitivity perception, the average reaction time being 14.98 sec. in day 90, against 23.63 sec. ± 2.39 SEM in day 60, p < 0.001.

Conclusion

This study demonstrated that chronic compression of the diabetic Wistar rat sciatic nerve for 60 days with a silicone tube causes an increase in the hindpaw withdrawal time to a heat stimulus (decrease in the nociceptive threshold) and, further, that decompression of the sciatic nerve (removal of the silicone band) will result in recovery toward the normal threshold by 1 month after initial compression.

Acknowledgments

This study was supported, in part, by the Romanian Research Grant ID92/2006.

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