Associations between Sensorimotor, Autonomic and Central Neuropathies in Diabetes Mellitus

Background: Patients with long-standing diabetes mellitus (DM) are exposed to hyperglycemia and metabolic disorders associated with extensive neuronal damage. Using a bedside applicable setup, the aim was to explore whether diabetes patients suffering from sensorimotor neuropathy had co-existing autonomic and central neuropathy. Methods: Twenty DM patients (10 women, average age 58.3 ± 12.0 years) with sensorimotor polyneuropathy and 16 healthy volunteers (HV) (9 women, age 62.6 ± 10.5 years) were recruited. Heart rate variability was recorded. Peripheral tactile detection threshold and sensation to pinprick was assessed by Von Frey-filaments. Sensitivity to pressure of the forearm extensor digitorum muscle was measured before and after conditioning pain modulation (CPM) induction by immersing the contra lateral hand into ice water in 180 seconds. Results: In comparison to HV, DM patients had lower variance (7.1 ± 5.6 vs. 13.3 ± 8.1 beats pr. min.; P=0.02) and mean standard deviation (3.3 ± 2.0 vs. 5.6 ± 2.8; P=0.01) of heart rate. They also had peripheral hypoesthesia to tactile stimulation: Median (3.0 g (0.8-12.5) vs. 1.0 g (0.4-1.4), P=0.03) and less efficacious CPM (13.9 ± 14.8% vs. 37.4% ± 28.9%; P=0.005). In patients, peripheral hypoesthesia was associated to mean heart rate (P=0.01), standard deviation of heart rate (P=0.004), and to CPM (P=0.046). Conclusion: DM patients with sensorimotor neuropathy showed generalized polyneuropathy evident as peripheral hypoesthesia, autonomic neuropathy and impaired CPM. Von Frey tactile detection threshold was associated with heart rate variability and CPM. The clinical approach and combined testing procedures, may serve as a prognostic platform to quantitatively evaluate the severity, extension and progression of diabetic neuropathy.


Introduction
Diabetes Mellitus (DM) is a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both. Chronic hyperglycemia is associated with long-term damage, dysfunction and failure of various organs, especially targeting the eyes, kidneys, heart, blood vessels and the nerves [1]. Hence, severe peripheral sensorimotor, autonomic and central neuropathies are common complications to longstanding DM. Advanced peripheral diabetic neuropathy classically manifests as chronic progressive thick (Aβ) and thin (Aδ) fiber neuropathy affecting myelinated axons and later affecting also the unmyelinated C-fibers [2,3]. The natural history of the disease varies from intermittent mild symptoms to manifest neuropathic pain, the latter being present in up to one fourth of the patients diminishing their quality of life [4][5][6]. The typical appearance is length dependent sensorimotor neuropathy caused by chronic progressive symmetric deafferentation and Schwann cell degeneration affecting axons of the distal lower extremities [2,7]. Sensorimotor neuropathy can be reliably assessed by use of vibration assessment, quantitative sensory testing measuring decreased sensitivity to pinprick and von Frey tactile detection threshold and electrophysiological techniques determining alterations of sensory nerve conduction velocity [8].
Accurate detection and quantification of small fiber neuropathy in the skin is challenging, even though different methods are available. Quantitative sensory testing are limited by involvement of larger fibres, however the more objective assessments including skin biopsies quantifying nerve fiber density or corneal confocal microscopy, are limited to specialized centers [9][10][11]. Diabetic autonomic neuropathy shares same etiology as peripheral neuropathy. Clinically autonomic neuropathy can be present with or without presence of large fiber neuropathy. Symptoms are often mild; nevertheless it has been shown that approximately 50% of diabetes type 1 patients and 75% of diabetes type 2 patients had subtle objective autonomic impairments [12]. Generalized autonomic disease, resulting in a variety of troublesome symptoms including orthostatic hypotension, nausea, abnormal gastrointestinal motility and erectile dysfunction was reported in 10%. A common approach to assess the generalized severity of autonomic neuropathy is based on cardiac Heart Rate Variability (HRV), which is diminished in autonomic neuropathy.
The pain systems exert specialized descending Conditioning Pain Modulation (CPM) from the brainstem and supra-spinal structures, which modulates the upstream afferent transmission. The CPM efficacy is likely related to the generalized status of the central nervous system, and hence diabetes induced central impairment may likely interfere with this complex network [13][14][15]. Changes in pain modulation processes, as reflected by dynamic psychophysical tests, are now increasingly recognized as clinically relevant. Hence, CPM has been shown to predict the efficacy of duloxetine in treatment of diabetic neuropathy [16]. However, a comprehensive neurophysiological profile including the different levels and the interaction between them has not been studied in DM patients with peripheral neuropathy. Thus, in order to determine the degree of generalized neuropathy in DM patients, we established a clinical applicable platform providing the possibility of investigating coexisting sensorimotor, autonomic and central neuropathies.
We hypothesized that DM patients, due to generalized neuropathy, would present with coexisting sensorimotor, autonomic and central neuropathy. Hence, the specific aims were to compare DM patients with Healthy Volunteers (HV) in terms of 1) tactile detection and pain to pinprick using von Frey monofilaments, 2) assessment of heart rate variability, 3) CPM, and 4) the interaction between the different parameters.

Subjects
Data were collected from June 2010 until October 2011, and data regarding visceral, cardiac and sensorimotor neuropathies have been reported previously by Søfteland et al. [17]. Patient characteristics are presented in Table 1 For comparison sixteen HV, matched for age and gender (9 women, age 62.6 ± 10.5 years) were recruited in Bergen through newspaper advertisement. Subjects were examined by a doctor to rule out any disease. Oral and written informed consent was obtained from all participants, and the study was approved by the local ethical committees at Haukeland and St. George Hospitals. Experiments were carried out according to the Helsinki declaration.

Experimental protocol
All patients and HV fasted for 6 hours. To standardize influence of glucose and insulin levels on sensory assessments, a hyperinsulinemiceuglycemic clamp technique ensured continuous adjustment of the blood glucose level to 5-6 mmol/L throughout the entire study procedure.
Most sensory afferents likely encode both non-painful and painful sensations. Hence, we instructed subjects in use of the modified 0-10 electronic Visual Analogue Scale (VAS), which has been widely used in sensory experiments [19]. The use of VAS was facilitated through anchor words, where 0=no perception; 1=first perception, 3=vague perception of moderate sensation; 5=pain detection threshold; 7=moderate pain and 10=worst perceivable pain. VAS was recorded continuously during testing.

Von frey tactile detection threshold
Von Frey filaments (Marstock Nervtest, Schriesheim, Germany) made of optic glass fibers, are highly elastic and bends to a certain pressure. Endings are epoxy coated to ensure constant contact surface for fibers with different diameters. Subjects were tested at the base of the dorsum of the first toe on the dominant side and the weight (i.e. thickness of the filament) corresponding to tactile detection threshold was noted.

Sensory response to pinprick
Von Frey filaments with the size of 26 gram were also used to assess the sensory score to a pinprick.

Autonomic nervous system tests
The autonomic nervous system was investigated by examining heart rate variability using the Heart Rhythm Scanner PE (Biocom Technologies, Poulsbo, WA, USA). The system investigates both timeand frequency domain measures of the HRV and its use has been described and validated elsewhere [20]. Data are presented in Table 1. Mean variance of heart rate at rest (beats pr. minute (bpm) pr. ms) and standard deviation of heart rate (consecutive RR intervals) was used for further analysis.

Efficacy of descending pain modulation
Noxious heterotopic conditioning painful stimulus was applied by immersing the right hand until the wrist in cooled circulated water (2 ± 0.3°C), the so-called cold pressor test. Study subjects were encouraged to withstand the 180 seconds immersion, however instructed to remove the hand, if pain was considered intolerable. A test stimulus was applied before and immediately after the cold pressor test consisting of: Pressure tolerance threshold (7 on the visual analogue scale) assessed in the mid part of the extensor digitorum muscle 10 cm distal from the left elbow joint, by use of a handheld electronic pressure algometer with a standard probe of 1 cm2 (Somedic AB, Stockholm, Sweden). The pressure was increased with a rate of 30 kPa/s. The efficacy of the CPM was presented as absolute (kPa) and relative (%) change in muscle sensitivity.

Statistical Analysis
Descriptive data are presented as mean with Standard Deviation (SD) or median with Inter-Quartile Range (IQR) where appropriate. To test differences between patients and HV, comparison of demographics, HRV and glucose levels were done by Student's t-tests. Von Frey filament size between patients and HV were compared with a Kruskal-Wallis One-Way Analysis Of Variance (ANOVA) as data were not normally distributed. To compare efficacy of descending pain modulation between patients and HV a one way ANOVA was used. Correlation analyses were done using Spearman´s test to investigate associations between peripheral hypoesthesia, HRV and CPM efficacy. The software package Sigma Stat v.3.0 (SPSS Inc., Chicago; IL, USA) was used in the analysis and P-values ≤ 0.05 were considered significant.

Results
All subjects underwent the hyperinsulinemic clamp without any adverse events. No difference between patients and HV in clamped mean glucose levels: 6.1 ± 1.1 vs. 5.5 ± 0.6 mmol/L (P=0.2) was found

Sensory response to pinprick with 26 gram Von Frey filament
There was no difference between patients and HV in the sensory response to pinprick with a 26 gr monofilament: Mean VAS of 1.9 ± 0.8 vs. 2.6 ± 1.3 (P=0.1).

Assessments of autonomic nervous system
Patients had lower mean variance of heart rate (7.1 ± 5.6 vs. 13.3 ± 8.1 beats pr. minute/ms, P=0.02) and mean standard deviation of heart rate (3.3 ± 2.0 vs. 5.6 ± 2.8 P=0.01) in comparison to HV, indicating autonomic neuropathy in DM patients.

Cold pressor test
All subjects tolerated immersion of the hand into cooled water for 180 seconds. However, one patient was withdrawn due to a severe vaso-vagal response immediately after the cold pressor test. Thus, data analyses were obtained in 19 patients. No difference between patients and HV was seen in mean pain perception of the cold pressor test: VAS score of 5.3 ± 2.0 vs. 6.1 ± 2.0 (P=0.2).

Clinical correlations
Associations between von Frey tactile detection threshold and cardiac autonomic parameters in patients are shown in Figure 3. Significant negative correlations were found to both mean variance in heart rate (r=-0.63, P=0.01) and mean standard deviation of heart rate (r=-0.69, P=0.004).
In the combined cohort of patients and healthy volunteers, an association between sensory score to pinprick and efficacy of CPM was found (r=0.52, P=0.001), which also was present in the patient population (r=0.36, P=0.048).

Discussion
Patients with longstanding DM showed evidence of generalized neuronal damage manifested as sensorimotor, autonomic and central neuropathies. For the first time reported, to our best knowledge, it was shown that the degree of peripheral hypoesthesia was associated with heart rate variability and impaired conditioned pain modulation.

Diabetes induced neuropathies
Diabetes induced neuronal damage leads directly and indirectly to neurodegeneration, which present as Schwann cell dysfunction, decreased neurotransmission and diminished neurotrophic axonal support [21]. However, the generalized co-existing glia cell activation and neuroinflammation is often under-diagnosed.
CPM influences primarily dorsal horn convergent neurons through a delicate balance involving descending pathways from brainstem nuclei. In comparison to HV, DM patients in the present study showed non-dynamic and less efficacious CPM, which could indicate dysfunctional brainstem regulation. These findings could be supported by the fact that a similar group of DM patients have previously shown altered brain processing -likely centrally induced neuropathy-evident as a delay in the activation of a specific brain network involving brainstem/operculum/frontal cortex and finally, microstructural neurodegenerative changes in pain specific brain areas of DM patients have been identified by use of diffusion tension imaging [23]. Impairment of CPM are previously reported in chronic painful conditions such as fibromyalgia, painful osteoarthritis, temporomandibular joint disease, chronic low back pain, inflammatory bowel disease and chronic pancreatitis, where it is considered to predispose development and persistence of chronic pain [24][25][26][27][28][29][30]. DM patients with clinical suspicion of sensorimotor neuropathy may have concomitant peripheral Aδ-and C-neuropathy and consequently decreased afferent transmission. Therefore, it must be considered if the observed changes in CPM are caused by deafferentation. If so, the decreased involvement of convergent dorsal horn neurons would lead to lead to CPM patterns which could be misinterpreted as central impairment. However, in this study there was no difference in sensory response between patients and HV to von Frey pinprick and muscle pressure at baseline (indicating comparable afferent input), it is plausible that concerns regarding alterations inafferent transmission may be ruled out.
Taken together, DM patients with clinical suspicion of peripheral neuropathy in this study showed co-existence of autonomic and altered central processing, which gives rise to speculate whether diabetes patients more often than expected, suffer from generalized polyneuropathy. In that case, future treatment possibilities could include agents which directly increase anti-oxidative capacity, support neurotrophic properties or enhance reparative processes after injury [21,31].

Methodological considerations
Patient recruitment was based on clinical suspicion of diabetes evident as peripheral sensorimotor neuropathy, however not all patients fulfilled the Toronto criteria, which was put forward after the study onset, and hence only a portion of the patients had had nerve conduction velocities assessed. HRV was assessed and interpreted without controlling for hypertension [2]. As the patient group has higher prevalence of hypertension than healthy, the study can be criticized for having interpreted HRV in the groups without controlling for possible confounding by hypertension. With only 19 subjects, data showed larger variety than expected, which gave rise to speculate whether the study was underpowered. Indeed the findings must be interpreted cautiously. However, even with this small study group, consistent differences between patients and HV were found, and thus we believe that the obtained results are fairly applicable in diabetes patients with sensorimotor neuropathy.
Quantitative Sensory Testing (QST) is psychophysical in nature with an objective physical stimulus (von Frey monofilaments or pressure algometry) and a subjective report as the response [8]. Hence, QST may be too dependent on compliant cooperation from the subject, otherwise possibly biases the outcome. Furthermore, even though the Von Frey filament endings are rounded to avoid Aβnociceptor activation, first tactile detection activates non-specific nociceptors as well as low threshold mechanoreceptors, possibly blurring the sensation [32]. Nevertheless, hypoesthesia was convincingly shown at the dorsum of the first toe on the dominant foot. In DM patients CPM induction has, to the best of our knowledge, not been induced by the cold pressor test, even though it has been widely used to explore central pain mechanisms in other diseases and in healthy controls [29,30]. The handheld pressure algometer has previously been validated, and to familiarize the subjects with the scoring procedure, participants were trained by applying muscle pressure in the femoral quadriceps muscle [33,34]. All patients and HV withstood immersion of their hand in 180 seconds. This likely reflect that these DM patients did not experience neuropathic pain as it is in contrast to chronic pain patients who cannot withstand the complete immersion in the cold pressor test, likely because they have developed central sensitization [29]. On the other hand, the finding raised speculations whether DM patients with small fiber neuropathy perceived less conditioning pain, resulting in less efficacious CPM. However, no differences between patients and HV were found in perceived pain during the cold pressor, and hence we believe CPM was sufficiently induced.

Conclusion
DM patients with suspicion of sensorimotor neuropathy showed generalized neuronal damage evident as peripheral hypoesthesia, autonomic neuropathy and impaired conditioned pain modulation. Increased Von Frey tactile detection threshold were associated with reduced heart rate variability and reduced efficacy of CPM. The clinically applicable platform presented in the present study may be used as a prognostic tool to quantitatively evaluate the extension, severity and progression of diabetes induced neuropathy.