Eur Radiol DOI 10.1007/s00330-014-3423-3
EXPERIMENTAL
Assessment of diabetic peripheral neuropathy in streptozotocin-induced diabetic rats with magnetic resonance imaging Dongye Wang & Xiang Zhang & Liejing Lu & Haojiang Li & Fang Zhang & Yueyao Chen & Jun Shen
Received: 17 May 2014 / Revised: 12 August 2014 / Accepted: 28 August 2014 # European Society of Radiology 2014
Abstract Objective To determine the role of magnetic resonance (MR) imaging and quantitative T2 value measurements in the assessment of diabetic peripheral neuropathy (DPN). Methods Sequential MR imaging, T2 measurement, and quantitative sensory testing of sciatic nerves were performed in streptozotocin-induced diabetic rats (n=6) and normal control rats (n=6) over a 7-week follow-up period. Histological assessment was obtained from 48 diabetic rats and 48 control rats once weekly for 7 weeks (n=6 for each group at each time point). Nerve signal abnormalities were observed, and the T2 values, mechanical withdrawal threshold (MWT), and histological changes were measured and compared between diabetic and control animals. Results Sciatic nerves in the diabetic rats showed a gradual increase in T2 values beginning at 2 weeks after the induction (P=0.014), while a decrease in MWT started at 3 weeks after the induction (P=0.001). Nerve T2 values had a similar time course to sensory functional deficit in diabetic rats. Histologically, sciatic nerves of diabetic rats demonstrated obvious endoneural oedema from 2 to 3 weeks after the induction, followed by progressive axonal degeneration, Schwann cell proliferation, and coexistent disarranged nerve regeneration. Conclusion Nerve T2 measurement is potentially useful in detecting and monitoring diabetic neuropathy. Key points • Sciatic nerves in diabetic rats showed a gradual increase in T2 values D. Wang : X. Zhang : L. Lu : H. Li : F. Zhang : Y. Chen : J. Shen (*) Department of Radiology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, No. 107 Yanjiang Road West, Guangzhou, Guangdong 51012, China e-mail: [email protected]
• Nerve T2 values were negatively correlated with sensory function impairment • Longitudinal T2 values can be used to monitor the disease progress • Nerve degeneration contributed mainly to progressive prolongation of nerve T2 values Keywords Peripheral nervous system diseases . Diabetes mellitus . Magnetic resonance imaging . Rats
Introduction Diabetic peripheral neuropathy (DPN) is one of the most common complications of diabetes mellitus (DM), with a high incidence, and greatly affecting patients’ quality of life [1]. Nonetheless, up to 50 % of neuropathic patients can be asymptomatic. At present, the clinical diagnosis of DPN is mainly based on assessments of neurological deficits, electrophysiology, quantitative sensory testing (QST), and intraepidermal nerve fibre density (IENFD). These diagnostic methods are useful tools to help diagnose and assess the progression of diabetic neuropathy [2]. However, assessments of neurological deficits have recently been shown to have poor diagnostic reproducibility [3]. QST is possibly useful in identifying small or large fibre sensory abnormalities in diabetic neuropathy and may be more sensitive but lacks specificity [4]. IENFD in skin-punch biopsies can accurately quantify nerve fibre damage and repair, but is an invasive procedure [5]. It is anticipated that newer, noninvasive techniques to directly assess nerve fibre damage will be developed and that these will replace nerve or skin biopsies [6]. Magnetic resonance (MR) imaging can directly display peripheral nerves, and has become an invaluable, widely used, noninvasive tool to diagnose and monitor the injury and
Eur Radiol
recovery in the peripheral nervous system [7–11]. To clarify the sequence of imaging changes following nerve injury and how they correlate with histological changes and functional deficit, various animal models of peripheral nerve injuries have been established to correlate MR imaging findings with histological and functional recovery [7–11]. The results of these studies demonstrated that peripheral nerves with traction injury or crush injury were featured as increased T2 signal intensities and prolonged T2 values, followed by gradual recovery of T2 signal intensity or T2 normalization over time. Quantitative measurements of nerve T2 values can predict the severity of injuries and are indicative of functional improvement [9, 11]. Recently, a set of consensus criteria for the phenotyping of rodent models of diabetic neuropathy has been developed relating to the status of commonly used rodent models of diabetes, nerve structure, electrophysiological assessments of nerve function, behavioural assessments of nerve function, and the role of biomarkers in disease phenotyping [12]. To date, there is a paucity of data in terms of MR imaging of DPN. Initially, MR spectroscopy of sural nerves of diabetic patients demonstrated a marginal increase in the coefficient of nerve hydration [13]. Further study of a rat diabetes model induced by intravenous injection of streptozotocin (STZ) indicated that peripheral tissue perfusion was clearly reduced at 2 weeks after induction, as assessed by dynamic contrastenhanced MR imaging [14]. However, the role of nerve MR signal change pattern, in particular quantitative T2 values in DPN, remains unknown. In this study, longitudinal MR imaging was performed in the sciatic nerves of streptozotocininduced diabetic rats and was correlated to functional assessment and histology. The purpose of our study was to determine the role of MR signal abnormalities and quantitative T2 value measurements in the assessment of DPN.
Materials and methods Animal model All interventions and animal care procedures were performed in accordance with the Guidelines and Policies for Animal Surgery provided by our university and were approved by the Institutional Animal Use and Care Committee. Adult healthy Sprague-Dawley female rats weighing a mean of 250±20 (standard deviation) g were obtained from the Animal Experiment Center of our university and were housed in a standard animal facility with 12-h on-off light conditions and were allowed free access to standard food and water. After overnight fasting, animals were induced by a single intraperitoneal injection of a freshly prepared solution of STZ (Sigma Chemical Co., USA) in 0.1 M cold citrate buffer (pH 4.5) at a dosage of 50 mg/kg body weight to establish
diabetic mellitus, as previously described [15]. Animals injected with the same volume of citrate buffer alone were used as controls. Body weight and blood glucose levels were measured after overnight fasting before the injection (week 0) and at 1, 2, 3, 4, 5, 6, and 7 weeks after the injection. Glucose levels in the blood sampled from the lateral vein of the tail were measured by using a glucometer (Onetouch Ultra, JNJ, USA). Rats whose blood glucose levels were above 250 mg/ dL 1 week after STZ administration were accepted as diabetic, as previously described [15]. There were 54 diabetic rats and 54 control rats. The diabetic rats were randomly assigned into groups A (n=6) and C (n=48), and the control rats were randomly divided into groups B (n=6) and D (n=48). Animals in groups A and B underwent sequential MR imaging and sciatic nerve function tests before the injection (0 week) and at 1, 2, 3, 4, 5, 6, and 7 weeks after the injection to yield longitudinal data. At each time point, six rats each in groups C and D were randomly selected and sacrificed for histological examination of the sciatic nerves. The anaesthetic used in all cases consisted of an intraperitoneal injection of sodium pentobarbital (30 mg/kg body weight; Sigma Chemical Co., USA) MR imaging MR imaging was performed with a 1.5-T unit (Intera; Philips Medical Systems, Best, Netherlands) and a 5-cm linearly polarized birdcage radio-frequency mouse coil (Chenguang Medical Technologies, Shanghai, China). After induction of anaesthesia, animals were placed in the prone position, and the two hind limbs of each animal were positioned symmetrically. Longitudinal T2-weighted images and T2 relaxation data of the sciatic nerves were acquired. T2-weighted imaging was performed by using three-dimensional turbo spin-echo sequence with fat-suppression using spectral presaturation inversion recovery (repetition time/echo time, 1200/60 ms; number of signal averages, 1; slice thickness, 1 mm; no intersection gap), and T2 relaxation data were obtained by using a single-section multi-spin-echo sequence (repetition time/echo time, 1600/20–160 ms; echo spacing, 20 ms; number of signal averages, 2; slice thickness, 2 mm). Both sequences were obtained by using the following parameters: field of view, 60×60 mm; acquisition matrix, 256×256. Image evaluation MR images obtained at each time point were randomly presented. The nerve signal abnormalities were observed and T2 relaxation time measurements were performed in a blinded manner and in isolation at each time by two authors (J.S., with more than 10 years of experience with musculoskeletal MR imaging, and F.Z., with 5 years of experience with MR imaging technology). T2 relaxation times were calculated by using
Eur Radiol
the available software tools provided by the MR imaging unit manufacturer. For each measurement, a region of interest with a minimal size of 85 pixels covering whole nerve structure was placed at the midportion of the nerve trunk (Fig. 1), as previously described [10].
glutaraldehyde, washed, postfixed, dehydrated, and embedded in araldite. Ultrathin 50-nm cross sections were then made and stained with Mg-uranyl acetate and lead citrate for transmission electron microscopic examination (FEI Tecnai G2 Spirit; Hillsboro, OR, USA), as described [18]. The nerve ultrastructure changes were visually assessed.
Quantitative sensory testing To quantify sensory function, Von Frey testing was performed at each time point before MR imaging in groups A and B to assess nerve tactile allodynia by two authors (X.Z. and L.J.L., both with 3 years of experience with sciatic nerve functional assessment) in a blinded manner, and the mechanical withdrawal threshold (MWT) [16] was obtained as an indicator of nerve sensory function. Histological examination At each time point, six rats each in groups C and D were randomly sacrificed by means of anaesthetic overdose. The midportion of sciatic nerves was harvested and each tissue sample was divided into two parts. One part was fixed in 10 % paraformaldehyde. Semithin 0.5-μm cross sections were obtained and stained with toluidine blue for light microscopic examination (Nikon ECLIPSE Ti; Japan), and the myelinated nerve fibre density and the average cross-sectional area of myelin sheath per nerve segment on the toluidine bluestained cross sections were measured with the use of NISElements BR 3.00 Imaging Software (Nikon, Japan), as previously described [17]. The other part was fixed in 2.5 %
Statistical analysis All data are presented as means±standard deviations. The interobserver variability of MWT or T2 values between the two observers was assessed by calculating the intraclass correlation coefficient (ICC), considering any two-way random effects (observer effect and measurement effect). The averaged values from two observers were used for statistical analysis. Body weights, blood glucose levels, MWT, and T2 values obtained at specific acquisition points between group A and group B were separately compared by using a repeatedmeasures one-way analysis of variance. The myelinated nerve fibre density and the average cross-sectional area of myelin sheath obtained at specific acquisition points in groups C or D were compared by using one-way ANOVA followed by a LSD post hoc test, and Student’s t-test was used for between-groups comparisons. Pearson's correlation analysis was used to investigate the relationship between the individual data of T2 values and MWT. A two-sided P value of less than 0.05 was considered to indicate a significant difference. All statistical tests were performed using SPSS 17.0 software (SPSS Inc., Chicago, IL, USA).
Results Body weight and blood glucose level
Fig. 1 Nerve T2 measurement. Fat-suppressed T2-weighted image (a) shows the sciatic nerve (arrowheads) of a diabetic rat. The corresponding T2 map (b) shows that the rectangular region of interest (rectangle) positioned on the midportion of the sciatic nerve trunk to derive the T2 value
Body weights in groups A and C (diabetic rats) decreased at 1 week (218.1±11.7 g), then remained stable from 2 weeks (202.4±13.5 g) to 7 weeks (190.6±17.0 g) after the induction compared with 0 week (271.3±6.8 g), whereas they increased gradually from 262.1±7.2 g at 0 week to 397.5±20.3 g at 7 weeks in groups B and D (control rats) during the entire study period. After the injection of STZ average blood glucose levels rapidly increased above 400 mg/dL and remained elevated up to 7 weeks in diabetic rats (22.3± 2.5 mmol/L to 24.5±4.2 mmol/L), while the blood glucose levels in control rats were not significantly changed throughout the study period (5.4±0.6 mmol/L to 5.7± 0.3 mmol/L). The body weights and blood glucose levels were significantly different between diabetic and control rats after STZ induction (P=0.011–0.026; P
EXPERIMENTAL
Assessment of diabetic peripheral neuropathy in streptozotocin-induced diabetic rats with magnetic resonance imaging Dongye Wang & Xiang Zhang & Liejing Lu & Haojiang Li & Fang Zhang & Yueyao Chen & Jun Shen
Received: 17 May 2014 / Revised: 12 August 2014 / Accepted: 28 August 2014 # European Society of Radiology 2014
Abstract Objective To determine the role of magnetic resonance (MR) imaging and quantitative T2 value measurements in the assessment of diabetic peripheral neuropathy (DPN). Methods Sequential MR imaging, T2 measurement, and quantitative sensory testing of sciatic nerves were performed in streptozotocin-induced diabetic rats (n=6) and normal control rats (n=6) over a 7-week follow-up period. Histological assessment was obtained from 48 diabetic rats and 48 control rats once weekly for 7 weeks (n=6 for each group at each time point). Nerve signal abnormalities were observed, and the T2 values, mechanical withdrawal threshold (MWT), and histological changes were measured and compared between diabetic and control animals. Results Sciatic nerves in the diabetic rats showed a gradual increase in T2 values beginning at 2 weeks after the induction (P=0.014), while a decrease in MWT started at 3 weeks after the induction (P=0.001). Nerve T2 values had a similar time course to sensory functional deficit in diabetic rats. Histologically, sciatic nerves of diabetic rats demonstrated obvious endoneural oedema from 2 to 3 weeks after the induction, followed by progressive axonal degeneration, Schwann cell proliferation, and coexistent disarranged nerve regeneration. Conclusion Nerve T2 measurement is potentially useful in detecting and monitoring diabetic neuropathy. Key points • Sciatic nerves in diabetic rats showed a gradual increase in T2 values D. Wang : X. Zhang : L. Lu : H. Li : F. Zhang : Y. Chen : J. Shen (*) Department of Radiology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, No. 107 Yanjiang Road West, Guangzhou, Guangdong 51012, China e-mail: [email protected]
• Nerve T2 values were negatively correlated with sensory function impairment • Longitudinal T2 values can be used to monitor the disease progress • Nerve degeneration contributed mainly to progressive prolongation of nerve T2 values Keywords Peripheral nervous system diseases . Diabetes mellitus . Magnetic resonance imaging . Rats
Introduction Diabetic peripheral neuropathy (DPN) is one of the most common complications of diabetes mellitus (DM), with a high incidence, and greatly affecting patients’ quality of life [1]. Nonetheless, up to 50 % of neuropathic patients can be asymptomatic. At present, the clinical diagnosis of DPN is mainly based on assessments of neurological deficits, electrophysiology, quantitative sensory testing (QST), and intraepidermal nerve fibre density (IENFD). These diagnostic methods are useful tools to help diagnose and assess the progression of diabetic neuropathy [2]. However, assessments of neurological deficits have recently been shown to have poor diagnostic reproducibility [3]. QST is possibly useful in identifying small or large fibre sensory abnormalities in diabetic neuropathy and may be more sensitive but lacks specificity [4]. IENFD in skin-punch biopsies can accurately quantify nerve fibre damage and repair, but is an invasive procedure [5]. It is anticipated that newer, noninvasive techniques to directly assess nerve fibre damage will be developed and that these will replace nerve or skin biopsies [6]. Magnetic resonance (MR) imaging can directly display peripheral nerves, and has become an invaluable, widely used, noninvasive tool to diagnose and monitor the injury and
Eur Radiol
recovery in the peripheral nervous system [7–11]. To clarify the sequence of imaging changes following nerve injury and how they correlate with histological changes and functional deficit, various animal models of peripheral nerve injuries have been established to correlate MR imaging findings with histological and functional recovery [7–11]. The results of these studies demonstrated that peripheral nerves with traction injury or crush injury were featured as increased T2 signal intensities and prolonged T2 values, followed by gradual recovery of T2 signal intensity or T2 normalization over time. Quantitative measurements of nerve T2 values can predict the severity of injuries and are indicative of functional improvement [9, 11]. Recently, a set of consensus criteria for the phenotyping of rodent models of diabetic neuropathy has been developed relating to the status of commonly used rodent models of diabetes, nerve structure, electrophysiological assessments of nerve function, behavioural assessments of nerve function, and the role of biomarkers in disease phenotyping [12]. To date, there is a paucity of data in terms of MR imaging of DPN. Initially, MR spectroscopy of sural nerves of diabetic patients demonstrated a marginal increase in the coefficient of nerve hydration [13]. Further study of a rat diabetes model induced by intravenous injection of streptozotocin (STZ) indicated that peripheral tissue perfusion was clearly reduced at 2 weeks after induction, as assessed by dynamic contrastenhanced MR imaging [14]. However, the role of nerve MR signal change pattern, in particular quantitative T2 values in DPN, remains unknown. In this study, longitudinal MR imaging was performed in the sciatic nerves of streptozotocininduced diabetic rats and was correlated to functional assessment and histology. The purpose of our study was to determine the role of MR signal abnormalities and quantitative T2 value measurements in the assessment of DPN.
Materials and methods Animal model All interventions and animal care procedures were performed in accordance with the Guidelines and Policies for Animal Surgery provided by our university and were approved by the Institutional Animal Use and Care Committee. Adult healthy Sprague-Dawley female rats weighing a mean of 250±20 (standard deviation) g were obtained from the Animal Experiment Center of our university and were housed in a standard animal facility with 12-h on-off light conditions and were allowed free access to standard food and water. After overnight fasting, animals were induced by a single intraperitoneal injection of a freshly prepared solution of STZ (Sigma Chemical Co., USA) in 0.1 M cold citrate buffer (pH 4.5) at a dosage of 50 mg/kg body weight to establish
diabetic mellitus, as previously described [15]. Animals injected with the same volume of citrate buffer alone were used as controls. Body weight and blood glucose levels were measured after overnight fasting before the injection (week 0) and at 1, 2, 3, 4, 5, 6, and 7 weeks after the injection. Glucose levels in the blood sampled from the lateral vein of the tail were measured by using a glucometer (Onetouch Ultra, JNJ, USA). Rats whose blood glucose levels were above 250 mg/ dL 1 week after STZ administration were accepted as diabetic, as previously described [15]. There were 54 diabetic rats and 54 control rats. The diabetic rats were randomly assigned into groups A (n=6) and C (n=48), and the control rats were randomly divided into groups B (n=6) and D (n=48). Animals in groups A and B underwent sequential MR imaging and sciatic nerve function tests before the injection (0 week) and at 1, 2, 3, 4, 5, 6, and 7 weeks after the injection to yield longitudinal data. At each time point, six rats each in groups C and D were randomly selected and sacrificed for histological examination of the sciatic nerves. The anaesthetic used in all cases consisted of an intraperitoneal injection of sodium pentobarbital (30 mg/kg body weight; Sigma Chemical Co., USA) MR imaging MR imaging was performed with a 1.5-T unit (Intera; Philips Medical Systems, Best, Netherlands) and a 5-cm linearly polarized birdcage radio-frequency mouse coil (Chenguang Medical Technologies, Shanghai, China). After induction of anaesthesia, animals were placed in the prone position, and the two hind limbs of each animal were positioned symmetrically. Longitudinal T2-weighted images and T2 relaxation data of the sciatic nerves were acquired. T2-weighted imaging was performed by using three-dimensional turbo spin-echo sequence with fat-suppression using spectral presaturation inversion recovery (repetition time/echo time, 1200/60 ms; number of signal averages, 1; slice thickness, 1 mm; no intersection gap), and T2 relaxation data were obtained by using a single-section multi-spin-echo sequence (repetition time/echo time, 1600/20–160 ms; echo spacing, 20 ms; number of signal averages, 2; slice thickness, 2 mm). Both sequences were obtained by using the following parameters: field of view, 60×60 mm; acquisition matrix, 256×256. Image evaluation MR images obtained at each time point were randomly presented. The nerve signal abnormalities were observed and T2 relaxation time measurements were performed in a blinded manner and in isolation at each time by two authors (J.S., with more than 10 years of experience with musculoskeletal MR imaging, and F.Z., with 5 years of experience with MR imaging technology). T2 relaxation times were calculated by using
Eur Radiol
the available software tools provided by the MR imaging unit manufacturer. For each measurement, a region of interest with a minimal size of 85 pixels covering whole nerve structure was placed at the midportion of the nerve trunk (Fig. 1), as previously described [10].
glutaraldehyde, washed, postfixed, dehydrated, and embedded in araldite. Ultrathin 50-nm cross sections were then made and stained with Mg-uranyl acetate and lead citrate for transmission electron microscopic examination (FEI Tecnai G2 Spirit; Hillsboro, OR, USA), as described [18]. The nerve ultrastructure changes were visually assessed.
Quantitative sensory testing To quantify sensory function, Von Frey testing was performed at each time point before MR imaging in groups A and B to assess nerve tactile allodynia by two authors (X.Z. and L.J.L., both with 3 years of experience with sciatic nerve functional assessment) in a blinded manner, and the mechanical withdrawal threshold (MWT) [16] was obtained as an indicator of nerve sensory function. Histological examination At each time point, six rats each in groups C and D were randomly sacrificed by means of anaesthetic overdose. The midportion of sciatic nerves was harvested and each tissue sample was divided into two parts. One part was fixed in 10 % paraformaldehyde. Semithin 0.5-μm cross sections were obtained and stained with toluidine blue for light microscopic examination (Nikon ECLIPSE Ti; Japan), and the myelinated nerve fibre density and the average cross-sectional area of myelin sheath per nerve segment on the toluidine bluestained cross sections were measured with the use of NISElements BR 3.00 Imaging Software (Nikon, Japan), as previously described [17]. The other part was fixed in 2.5 %
Statistical analysis All data are presented as means±standard deviations. The interobserver variability of MWT or T2 values between the two observers was assessed by calculating the intraclass correlation coefficient (ICC), considering any two-way random effects (observer effect and measurement effect). The averaged values from two observers were used for statistical analysis. Body weights, blood glucose levels, MWT, and T2 values obtained at specific acquisition points between group A and group B were separately compared by using a repeatedmeasures one-way analysis of variance. The myelinated nerve fibre density and the average cross-sectional area of myelin sheath obtained at specific acquisition points in groups C or D were compared by using one-way ANOVA followed by a LSD post hoc test, and Student’s t-test was used for between-groups comparisons. Pearson's correlation analysis was used to investigate the relationship between the individual data of T2 values and MWT. A two-sided P value of less than 0.05 was considered to indicate a significant difference. All statistical tests were performed using SPSS 17.0 software (SPSS Inc., Chicago, IL, USA).
Results Body weight and blood glucose level
Fig. 1 Nerve T2 measurement. Fat-suppressed T2-weighted image (a) shows the sciatic nerve (arrowheads) of a diabetic rat. The corresponding T2 map (b) shows that the rectangular region of interest (rectangle) positioned on the midportion of the sciatic nerve trunk to derive the T2 value
Body weights in groups A and C (diabetic rats) decreased at 1 week (218.1±11.7 g), then remained stable from 2 weeks (202.4±13.5 g) to 7 weeks (190.6±17.0 g) after the induction compared with 0 week (271.3±6.8 g), whereas they increased gradually from 262.1±7.2 g at 0 week to 397.5±20.3 g at 7 weeks in groups B and D (control rats) during the entire study period. After the injection of STZ average blood glucose levels rapidly increased above 400 mg/dL and remained elevated up to 7 weeks in diabetic rats (22.3± 2.5 mmol/L to 24.5±4.2 mmol/L), while the blood glucose levels in control rats were not significantly changed throughout the study period (5.4±0.6 mmol/L to 5.7± 0.3 mmol/L). The body weights and blood glucose levels were significantly different between diabetic and control rats after STZ induction (P=0.011–0.026; P
