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Streptozotocin-induced diabetic hyperalgesia in rats is associated with upregulation of toll-like receptor 4 expression
Neuroscience Letters 526 (2012) 54–58
Contents lists available at SciVerse ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Streptozotocin-induced diabetic hyperalgesia in rats is associated with upregulation of toll-like receptor 4 expression Jian-e Yan a , Wei Yuan b , Xiaoli Lou c , Tao Zhu d,∗ a
Department of Anesthesia, Guiyang Medical College, Guiyang, China Department of gynecology and obstetrics, Songjiang Branch, Shanghai First People’s Hospital Affiliated to Shanghai Jiaotong University, Shanghai, China Central Laboratory, Songjiang Branch, Shanghai First People’s Hospital Affiliated to Shanghai Jiaotong University, Shanghai, China d Department of Anesthesia, Songjiang Branch, Shanghai First People’s Hospital Affiliated to Shanghai Jiaotong University, Shanghai, China b c
h i g h l i g h t s
Role of TLR4 in diabetes-induced hyperalgesia in rats was evaluated. Streptozotocin-induced diabetic rats show robust hyperalgesia. Association between upregulated TLR4 mRNA expression and neuropathic pain. Protein levels of TNF-␣ and IL-1 are also increased along with hypersensitivity. In summary, TLR4 and its signaling pathway are associated with STZ-induced diabetic hyperalgesia in rats.
a r t i c l e
i n f o
Article history: Received 11 June 2012 Received in revised form 24 July 2012 Accepted 8 August 2012 Keywords: Toll-like receptor Tumor necrosis factor-alpha Interleukin-1 beta Diabetes Neuropathic pain
a b s t r a c t Neuropathic pain is one of the common complications of diabetes mellitus, and current treatments often do not meet medical needs. Toll-like receptor 4 (TLR4) has been implicated as a potential therapeutic target in neuropathic and other pain models. In this study, we investigated whether TLR4 expression in spinal cord would be altered in streptozotocin-induced diabetic rat model, which had persistent mechanical and thermal hypersensitivity. The results showed that the mRNA expression of TLR4 was upregulated in streptozotocin-treated animals. Furthermore, TLR4 expression was associated with both paw-pressure withdrawal threshold toward mechanical stimulus and paw withdrawal latency toward thermal stimulus. The protein levels of TNF-␣ and IL-1, two downstream proinflammatory cytokines of TLR4 signaling pathway, were also significantly raised and correlated with mechanical/thermal hypersensitivity in diabetic rats. Together, these data have demonstrated that TLR4 and its signaling pathway are associated with neuropathic pain in a diabetic model. It may imply that TLR4 could be a novel target for treating diabetic neuropathy. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Peripheral diabetic neuropathy (PDN) is one of the most common complications of diabetes mellitus (DM), affects more than 50% of diabetic patients in the United States, and is the leading cause of foot amputation [3,12]. In clinic, symptoms encountered in PDN include abnormal sensations such as paresthesias, hyperalgesia, allodynia and spontaneous pain. Although it is thought to be associated with peripheral demyelination, decrease inperipheral nerve
Abbreviations: DM, diabetes mellitus; IL-1, interleukin-1 beta; PDN, peripheral diabetic neuropathy; PPWT, paw-pressure withdrawal threshold; PWL, paw withdrawal latency; STZ, streptozotocin; TLR, toll-like receptor; TNF-␣, tumor necrosis factor-alpha. ∗ Corresponding author. Tel.: +86 18918289046; fax: +86 02167722251. E-mail address: [email protected] (T. Zhu). 0304-3940/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2012.08.012
conduction, impaired cutaneous endothelium-related vasodilation, and degeneration of myelinated sensory fibers, the exact cause and mechanism of PDN still remain unclear [23]. Therefore, the treatment of diabetic neuropathic pain often has only limited efficacy and/or are associated with intolerable side effects [30]. In rodents, symptoms of PDN such as mechanical allodynia and thermal hyperalgesia can be studied after induction of diabetes with streptozotocin (STZ) [21]. Assessment of behavioral responses to external stimuli in this diabetic model has led to identification of a number of mechanisms of abnormal sensation and pain in diabetes [23]. Evidence from both animal models and humans indicates that systemic inflammation is involved in the pathophysiological processes of DM and is facilitated by innate immune responses [7]. Toll-like receptors (TLRs) are a class of proteins that play a key role in the innate immune response. Interactions between TLRs and their ligands expressed by microbial pathogens can induce a
J.-e. Yan et al. / Neuroscience Letters 526 (2012) 54–58
cascade of intracellular signaling events, culminating in the upregulation of proinflammatory pathways [20]. TLRs are expressed in various cell types in the nervous system and contribute to infectious as well as non-infectious disorders [10,11,13]. After tissue insult and nerve injury, TLRs can induce the activation of microglia and the production of the proinflammatory cytokines in the spinal cord, leading to the development of inflammatory pain and neuropathic pain [14,22,29]. Although TLR4 expression is increased in many inflammatory disorders [10,11,19,28], the effects of metabolic aberrations on TLR4 and its role in diabetes and its complications are still poorly understood. In current study, by establishing the timeline of TLR4 and two pro-inflammatory cytokines (TNF-␣ and IL-1) levels in the rat spinal cord following streptozotocin injection, we aimed to characterize the role of TLR4 in peripheral diabetic neuropathy. 2. Materials and methods 2.1. Animal Two-month old male Sprague-Dawley rats weighing 180–200 g were obtained from the Animal Care Facility of Shanghai First people’s hospital and housed in light-controlled (12-h light–dark cycle) and temperature-controlled (20 ± 2 ◦ C) room with food and water available ad libitum. Diabetes was induced by a single intraperitoneal injection of 1% STZ (Sigma–Aldrich, USA) at 65 mg/kg body weight after 14 h of food deprivation. STZ was dissolved in 10 mM citrate buffer, pH 4.38. Rats injected with citrate buffer alone without STZ served as the normal control. Establishment of diabetes was confirmed at one week after STZ induction and at weekly intervals by measuring fasting (>8 h) blood glucose levels (>250 mg/dl) with a glucometer (Roche, Swiss). All procedures were approved by the Ethical Committee of Animal Research of the hospital and performed in accordance with the NIH’s Guiding Principles in the Care and Use of Laboratory Animals. 2.2. Mechanical hyperalgesia and thermal hyperalgesia Pain responses were evaluated in pre- and post-diabetic rats. Tests were conducted in the morning between 9:00 and 12:00 AM in a noise-free environment. For mechanical testing, animals were placed in a hanging cage with metal mesh floor and allowed to acclimate for at least 30 min. Mechanical hyperalgesia was assessed by measuring the paw-pressure withdrawal threshold (PPWT) when exposed to increasing mechanical stimulation with an analgesiometer (Sunny Instruments, China). The computer converted the applied stimulus intensity into grams, and the pressure at which a paw withdrawal occurred was recorded. On a given test, the same procedure was repeated three times at 5 min intervals, the mean withdrawal pressure was calculated by averaging the three measurements. To quantify thermal sensitivity, rats were placed on a clear Plexiglas chamber and given 5–10 min to acclimate. The radiant heat source in II T336 paw-flick instrument (life science, USA) delivered a heat stimulus to the plantar surface of the hind paw. The reflective photocell sensor detected when the rat moved or lifted its paw. The time between onset of the stimulus and paw flick was defined as the paw withdrawal latency (PWL). On a given test and for each hind paw, the same procedure was repeated three times at 5 min intervals, the mean withdrawal pressure was computed by averaging the six measurements. 2.3. Spinal cord transection Rats were deeply anesthetized with 10% chloral hydrate (300 mg/kg, intraperitoneal injection) and sacrificed rapidly.
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A dorsal midline incision was made from L2–S2. Once exposed, L4–L6 spinal cord were harvested and placed in the liquid nitrogen immediately. 2.4. RT-PCR Total RNA was extracted from spinal tissue with Trizol reagent (Invitrogen, USA) according to the manufacturer’s recommendations. Complementary DNA sequences of -actin and TLR4 were obtained from the database of NCBI. The following PCR primers (Sangon Biotech, China) were used: TLR4 (forward: 5 -GCCGGAAAGTTATTGTGGTGGT-3 ; reverse: 5 ATGGGTTTTAGGCGCAGAGTTT-3 ), the anticipated length was 356 bp; -actin (forward: 5 -CACGATGGAGGGGCCGGACTCATC-3 ; reverse: 5 -GTAGCCCACGTCGTAGCAAA-3 ), and the anticipated length was 240 bp. The amplification protocol was: 10 min at 95 ◦ C; followed by 30 cycles 30 s at 95 ◦ C, 50 s at 62 ◦ C and 50 s at 72 ◦ C; then 10 min at 72 ◦ C for extension. Amplification products were separated by agarose gel electrophoresis and photographed. 2.5. ELISA The spinal cord was collected and homogenized followed by centrifugation. TNF-␣ and IL-1 concentrations were quantified by ELISA (Neobioscience, China). According to the manufacturer’s instructions, the absorbance (A) was detected at 450 nm (A450 ). The content of each sample was obtained according to the standard curve. 2.6. Statistics All the data are presented as mean ± standard deviation (SD). To assess the significance of difference between groups, summed effects of STZ over the course of an experiment were used to compare treatment by analysis of variance (ANOVA) followed by Fisher’s least significant difference. Two-way ANOVA was used to evaluate the interaction between different groups. p < 0.05 was considered statistically significant. The regression and correlation analysis was based on least squares method. Once a regression model has been constructed, the goodness of the fit was checked by R-squared, analyses of the pattern of residuals and hypothesis testing. Statistical significance was checked by an F-test of the overall fit, followed by t-test of individual parameters.
3. Results 3.1. Induction of diabetes with STZ Since the primary indicator for the successful induction of diabetes using animal model was the development of hyperglycemia, it was necessary to measure the levels of glucose in blood circulation. As shown in Fig. 1A, one week after STZ injections, treated animals developed clear diabetes. Glucose concentrations in all STZ-treated diabetic rats (DM group) were greater than 250 mg/dl, a standard level defined as diabetic hyperglycemia, at week 1, week 2, week 3 and week 4 (n = 6 animals per time point). Meanwhile the glycemia levels in citrate-treated non-diabetic rats (control group) remained relatively stable and typically ranged from 100 to 110 mg/dl. The body weights of the DM animals did not show any differences from the controls before treatment (Fig. 1B). After STZ injections, the rats in DM group lost their weights
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Fig. 1. Time course of blood glucose concentration (A) and body weight (B) after streptozotocin (DM) or citrate buffer (control) treatment. Data are presented as mean ± SD (n = 6 animals per time point), ***p < 0.001 by two-way ANOVA analysis.
Fig. 2. Time course of paw-pressure withdrawal threshold to mechanical stimuli (A) and paw withdrawal latency to thermal stimuli (B) in DM (open symbols) and control (closed symbols) animals. Data are presented as mean ± SD (n = 6 animals per time point), *p < 0.05, **p < 0.01, ***p < 0.001 by two-way ANOVA analysis.
gradually while the body weights of the control animals elevated progressively.
3.2. Assessment of mechanical hyperalgesia and thermal hyperalgesia Pain reactions were characterized in both DM rats and their respective controls for diabetes-induced polyneuropathic pain symptoms. The PPWT toward mechanical stimulus and the PWL toward thermal stimulus in control animals were relatively stable and typically ranged around 11–13 g (Fig. 2A) and 10–12 s (Fig. 2B), respectively. On the contrary, in the DM rats PPWT and PWL decreased gradually after STZ injection and showed significant difference since the second week after treatment. Both of them reached their minima at week 4 (Fig. 2).
3.3. TLR4 expression The expressions of TLR4 in L4–L6 spinal cord from DM rats were upregulated gradually but significantly in week 1–4 and the peak was observed four weeks after STZ injection while the TLR4 levels in control group remained relatively stable (Fig. 3A). Further analysis showed that TLR4 expressions in these DM animals (n = 30) were negatively linear correlated with PPWT (r = −0.55, p < 0.01; Fig. 3B) and PWL (r = −0.46, p < 0.05; Fig. 3C).
3.4. TNF-˛ and IL-1ˇ production TNF-␣ and IL-1 are two downstream proinflammatory cytokines of TLR4 signaling pathway [1,11,20], their productions by spinal cord from the rats in the control and DM groups were
Fig. 3. Correlation between TLR4 expression and hyperalgesia in DM rats. (A) Time course of relative TLR4 mRNA expression in spinal cord (L4–L6) of DM (open symbols) and control (closed symbols) animals. Data are presented as mean ± SD (n = 6 animals per time point), *p < 0.05, **p < 0.01, ***p < 0.001 by two-way ANOVA analysis. (B) Plot of linear regression analysis showing the relation between TLR4 expression and paw-pressure withdrawal threshold to mechanical stimuli in DM rats (n = 30, r = −0.55, p < 0.01 by t-test). (C) Plot of linear regression analysis demonstrating the relation between TLR4 expression and paw withdrawal latency to thermal stimuli in DM animals (n = 30, r = −0.46, p < 0.05 by t-test).
J.-e. Yan et al. / Neuroscience Letters 526 (2012) 54–58
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4. Discussion
Fig. 4. Correlation between TNF-␣/IL-1 protein levels and hyperalgesia in DM rats. (A and B) Time course of TNF-␣ and IL-1 productions in spinal cord (L4–L6) of DM (open symbols) and control (closed symbols) animals. Data are presented as mean ± SD (n = 6 animals per time point), ***p < 0.001 by two-way ANOVA analysis. (C and D) Plot of linear regression analysis showing the relation between TLR4 expression and production of TNF-␣ (n = 30, r = 0.57, p < 0.01 by t-test) or IL-1 (n = 30, r = 0.44, p < 0.05 by t-test) in DM animals. (E and F) Plot of linear regression analysis showing the relation between paw-pressure withdrawal threshold to mechanical stimuli and production of TNF-␣ (n = 30, r = −0.76, p < 0.001 by t-test) or IL-1 (n = 30, r = −0.92, p < 0.001 by t-test). (G and H) Plot of linear regression analysis showing the relation between paw withdrawal latency to thermal stimuli and production of TNF-␣ (n = 30, r = −0.62, p < 0.001 by t-test) or IL-1 (n = 30, r = −0.77, p < 0.001 by t-test).
similar before treatment. Both levels of TNF-␣ (Fig. 4A) and IL-1 (Fig. 4B) elevated significantly in the DM rat since the induction of diabetes. For DM animals, further studies demonstrated that (1) TLR4 expression was linear correlated with production of TNF-␣ (r = 0.57, p < 0.01; Fig. 4C) and IL-1 (r = 0.44, p < 0.05; Fig. 4D); (2) PPWT was negatively linear correlated with production of TNF-␣ (r = −0.76, p < 0.001; Fig. 4E) and IL-1 (r = −0.92, p < 0.001; Fig. 4F); and (3) PWL was negatively linear correlated with TNF-␣ level (r = −0.62, p < 0.001; Fig. 4G) and IL-1 (r = −0.77, p < 0.001; Fig. 4H).
In the current study, we demonstrate that the upregulated expression of TLR4 is correlated with neuropathic pain in a rat model of STZ-induced diabetes. Furthermore, the spinal release of TNF-␣ and IL-1, two downstream inflammatory cytokines of TLR4 signaling pathway, are also correlated with the behavioral responses toward mechanical and thermal stimuli. Autoimmune diabetes (Type I diabetes) is considered to be a multi-stage T cell mediated auto-immune disease involving slow and gradual islet  cell destruction and complete loss of insulin secretion [26]. The available data about the auto-immune process leading to diabetes, its complications and the nature of the inflammatory response is limited [18]. STZ is an antibiotic extracted from streptomyces acromogenes and is diabetogenic due to a selective cytotoxic action upon pancreatic  cell [24]. In recent years STZinjected rodents is considered to be a useful animal model for study diabetes and its complications, especially PDN [21], largely due to the convenience of the single treatment. In this study, the glucose levels in STZ-treated animals were over 250 mg/dl, the daily food and water consumptions elevated while the body weights decreased significantly. All of these suggested the diabetic animal model was successfully established. Several lines of evidence demonstrate that both mechanical and thermal hyperalgesia occur in the mild PDN, and are suggested to be an indicator of early PDN [6,9]. Here, robust mechanical/thermal hyperalgesia were found and repetitive testing did not result in any signs of habituation. Consistent with previous reports [6,15], PPWT decreased progressively but significantly after STZ injection and reduced over 50% at week 4; meanwhile PWL decreased gradually and slowly for three weeks after treatment, but at week 4, a robust change occurred. This may suggest that more than one patho-physiological mechanism is involved in diabetic pain. Previous studies show a significant upregulation of TLRs in the central nervous system from nerve injury-induced neuropathic pain models [2,8,29]. It is also found that TLR4-deficient mice displayed attenuated behavioral hypersensitivity and decreased expression of spinal glial activation and proinflammatory cytokines [5]. TLR4 is highly expressed by microglial cells in the mammalian nervous system, while several reports indicate that TLR4 can be identified in astrocytes, neurons and neural progenitor cells [4,16,25]. TLR4 can recognize a variety of microbial and host structures including lipopolysaccharide, peptidoglycan, high mobility group proteins, etc. [1,20]. After binding to its ligands, TLR4 lead to activation of two downstream pathways: mitogen-activated protein kinase (MAPK) and NF-B pathway. The activation of NFB pathway causes the synthesis and release of proinflammatory cytokines, including TNF-␣ and IL-1 while the activation of MAPK pathway plays an important role in the regulation of neuronal plasticity [27]. To date, no study has been conducted to investigate the role of TLR4 in peripheral diabetic neuropathy. Our results showed that, at mRNA level, the expression of TLR4 elevated gradually after STZ injection and increased over 50% at week 4 in the spinal cord where is critical to the pain signal transduction. Compare to pretreatment, the protein levels of TNF-␣ and IL-1 elevated over 50% and 500%, respectively. This suggests that TNF-␣ and IL-1 do not contribute equally to neuro-inflammatory and IL-1 may play a more important role in diabetic neuropathy. Further statistical analysis demonstrated that TLR4, TNF-␣ and IL-1 levels are linear correlated with mechanical and thermal hyperalgesia. It is noteworthy that the association between TLR4 and diabetic pain is mild, while proinflammatory cytokines show robust correlation with hyperalgesia. This may suggest that, besides TLR4, some other substances (such as TLR2 and TLR3) are activated in diabetes [4,17] and involved in the pathogenesis of neuropathic pain [14,22]. These
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substances may contribute to the elevated levels of TNF-␣ or/and IL-1. In conclusion, we found that TLR4, TNF-␣ and IL-1 levels were correlated with hyperalgesia in a diabetic rat model. These findings imply the TLR4 may become a novel target in the treatment of neuropathic pain. Conflict of interest None of the authors have any conflicts of interest to declare. Acknowledgments We thank Dr. Bin Zhao for his suggestions and editorial assistance. References [1] S. Akira, S. Uematsu, O. Takeuchi, Pathogen recognition and innate immunity, Cell 124 (2006) 783–801. [2] I. Bettoni, F. Comelli, C. Rossini, F. Granucci, G. Giagnoni, F. Peri, B. Costa, Glial TLR4 receptor as new target to treat neuropathic pain: efficacy of a new receptor antagonist in a model of peripheral nerve injury in mice, Glia 56 (2008) 1312–1319. [3] A.J. Boulton, The diabetic foot: from art to science. The 18th Camillo Golgi lecture, Diabetologia 47 (2004) 1343–1353. [4] M. Bsibsi, R. Ravid, D. Gveric, J.M. van Noort, Broad expression of Toll-like receptors in the human central nervous system, Journal of Neuropathology and Experimental Neurology 61 (2002) 1013–1021. [5] L. Cao, F.Y. Tanga, J.A. Deleo, The contributing role of CD14 in toll-like receptor 4 dependent neuropathic pain, Neuroscience 158 (2009) 896–903. [6] C. Courteix, A. Eschalier, J. Lavarenne, Streptozocin-induced diabetic rats: behavioural evidence for a model of chronic pain, Pain 53 (1993) 81–88. [7] M.R. Dasu, S. Ramirez, R.R. Isseroff, Toll-like receptors and diabetes: a therapeutic perspective, Clinical Science (London) 122 (2012) 203–214. [8] E. Dominguez, A. Mauborgne, J. Mallet, M. Desclaux, M. Pohl, SOCS3-mediated blockade of JAK/STAT3 signaling pathway reveals its major contribution to spinal cord neuroinflammation and mechanical allodynia after peripheral nerve injury, The Journal of Neuroscience 30 (2010) 5754–5766. [9] P.J. Dyck, T.S. Larson, P.C. O’Brien, J.A. Velosa, Patterns of quantitative sensation testing of hypoesthesia and hyperalgesia are predictive of diabetic polyneuropathy: a study of three cohorts. Nerve growth factor study group, Diabetes Care 23 (2000) 510–517. [10] M. Fresno, R. Alvarez, N. Cuesta, Toll-like receptors, inflammation, metabolism and obesity, Archives of Physiology and Biochemistry 117 (2011) 151–164. [11] M.L. Hanke, T. Kielian, Toll-like receptors in health and disease in the brain: mechanisms and therapeutic potential, Clinical Science (London) 121 (2011) 367–387.
[12] T.S. Jensen, M.M. Backonja, S. Hernandez Jimenez, S. Tesfaye, P. Valensi, D. Ziegler, New perspectives on the management of diabetic peripheral neuropathic pain, Diabetes & Vascular Disease Research 3 (2006) 108–119. [13] T. Kielian, Toll-like receptors in central nervous system glial inflammation and homeostasis, Journal of Neuroscience Research 83 (2006) 711–730. [14] D. Kim, M.A. Kim, I.H. Cho, M.S. Kim, S. Lee, E.K. Jo, S.Y. Choi, K. Park, J.S. Kim, S. Akira, H.S. Na, S.B. Oh, S.J. Lee, A critical role of toll-like receptor 2 in nerve injury-induced spinal cord glial cell activation and pain hypersensitivity, The Journal of Biological Chemistry 282 (2007) 14975–14983. [15] J.H. Lee, R. McCarty, Pain threshold in diabetic rats: effects of good versus poor diabetic control, Pain 50 (1992) 231–236. [16] S. Lehnardt, L. Massillon, P. Follett, F.E. Jensen, R. Ratan, P.A. Rosenberg, J.J. Volpe, T. Vartanian, Activation of innate immunity in the CNS triggers neurodegeneration through a Toll-like receptor 4-dependent pathway, Proceedings of the National Academy of Sciences of the United States of America 100 (2003) 8514–8519. [17] F. Li, N. Yang, L. Zhang, H. Tan, B. Huang, Y. Liang, M. Chen, X. Yu, Increased expression of toll-like receptor 2 in rat diabetic nephropathy, American Journal of Nephrology 32 (2010) 179–186. [18] E. Lien, D. Zipris, The role of Toll-like receptor pathways in the mechanism of type 1 diabetes, Current Molecular Medicine 9 (2009) 52–68. [19] K.H. Mills, TLR-dependent T cell activation in autoimmunity, Nature Reviews: Immunology 11 (2011) 807–822. [20] K. Miyake, Innate immune sensing of pathogens and danger signals by cell surface Toll-like receptors, Seminars in Immunology 19 (2007) 3–10. [21] T.J. Morrow, Animal models of painful diabetic neuropathy: the STZ rat model, Current Protocols in Neuroscience (2004) (Chapter 9, 9.18.1–9.18.11). [22] K. Obata, H. Katsura, K. Miyoshi, T. Kondo, H. Yamanaka, K. Kobayashi, Y. Dai, T. Fukuoka, S. Akira, K. Noguchi, Toll-like receptor 3 contributes to spinal glial activation and tactile allodynia after nerve injury, Journal of Neurochemistry 105 (2008) 2249–2259. [23] I.G. Obrosova, Diabetic painful and insensate neuropathy: pathogenesis and potential treatments, Neurotherapeutics 6 (2009) 638–647. [24] N. Rakieten, M.L. Rakieten, M.R. Nadkarni, Studies on the diabetogenic action of streptozotocin (NSC-37917), Cancer Chemotherapy Reports 29 (1963) 91–98. [25] A. Rolls, R. Shechter, A. London, Y. Ziv, A. Ronen, R. Levy, M. Schwartz, Tolllike receptors modulate adult hippocampal neurogenesis, Nature Cell Biology 9 (2007) 1081–1088. [26] D.B. Schranz, A. Lernmark, Immunology in diabetes: an update, Diabetes/Metabolism Reviews 14 (1998) 3–29. [27] C. Sommer, Painful neuropathies, Current Opinion in Neurology 16 (2003) 623–628. [28] R. Spirig, J. Tsui, S. Shaw, The emerging role of TLR and innate immunity in cardiovascular disease, Cardiology Research and Practice 2012 (2012) 181394. [29] F.Y. Tanga, N. Nutile-McMenemy, J.A. DeLeo, The CNS role of Toll-like receptor 4 in innate neuroimmunity and painful neuropathy, Proceedings of the National Academy of Sciences of the United States of America 102 (2005) 5856–5861. [30] A. Veves, M. Backonja, R.A. Malik, Painful diabetic neuropathy: epidemiology, natural history, early diagnosis, and treatment options, Pain Medicine 9 (2008) 660–674.
Contents lists available at SciVerse ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Streptozotocin-induced diabetic hyperalgesia in rats is associated with upregulation of toll-like receptor 4 expression Jian-e Yan a , Wei Yuan b , Xiaoli Lou c , Tao Zhu d,∗ a
Department of Anesthesia, Guiyang Medical College, Guiyang, China Department of gynecology and obstetrics, Songjiang Branch, Shanghai First People’s Hospital Affiliated to Shanghai Jiaotong University, Shanghai, China Central Laboratory, Songjiang Branch, Shanghai First People’s Hospital Affiliated to Shanghai Jiaotong University, Shanghai, China d Department of Anesthesia, Songjiang Branch, Shanghai First People’s Hospital Affiliated to Shanghai Jiaotong University, Shanghai, China b c
h i g h l i g h t s
Role of TLR4 in diabetes-induced hyperalgesia in rats was evaluated. Streptozotocin-induced diabetic rats show robust hyperalgesia. Association between upregulated TLR4 mRNA expression and neuropathic pain. Protein levels of TNF-␣ and IL-1 are also increased along with hypersensitivity. In summary, TLR4 and its signaling pathway are associated with STZ-induced diabetic hyperalgesia in rats.
a r t i c l e
i n f o
Article history: Received 11 June 2012 Received in revised form 24 July 2012 Accepted 8 August 2012 Keywords: Toll-like receptor Tumor necrosis factor-alpha Interleukin-1 beta Diabetes Neuropathic pain
a b s t r a c t Neuropathic pain is one of the common complications of diabetes mellitus, and current treatments often do not meet medical needs. Toll-like receptor 4 (TLR4) has been implicated as a potential therapeutic target in neuropathic and other pain models. In this study, we investigated whether TLR4 expression in spinal cord would be altered in streptozotocin-induced diabetic rat model, which had persistent mechanical and thermal hypersensitivity. The results showed that the mRNA expression of TLR4 was upregulated in streptozotocin-treated animals. Furthermore, TLR4 expression was associated with both paw-pressure withdrawal threshold toward mechanical stimulus and paw withdrawal latency toward thermal stimulus. The protein levels of TNF-␣ and IL-1, two downstream proinflammatory cytokines of TLR4 signaling pathway, were also significantly raised and correlated with mechanical/thermal hypersensitivity in diabetic rats. Together, these data have demonstrated that TLR4 and its signaling pathway are associated with neuropathic pain in a diabetic model. It may imply that TLR4 could be a novel target for treating diabetic neuropathy. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Peripheral diabetic neuropathy (PDN) is one of the most common complications of diabetes mellitus (DM), affects more than 50% of diabetic patients in the United States, and is the leading cause of foot amputation [3,12]. In clinic, symptoms encountered in PDN include abnormal sensations such as paresthesias, hyperalgesia, allodynia and spontaneous pain. Although it is thought to be associated with peripheral demyelination, decrease inperipheral nerve
Abbreviations: DM, diabetes mellitus; IL-1, interleukin-1 beta; PDN, peripheral diabetic neuropathy; PPWT, paw-pressure withdrawal threshold; PWL, paw withdrawal latency; STZ, streptozotocin; TLR, toll-like receptor; TNF-␣, tumor necrosis factor-alpha. ∗ Corresponding author. Tel.: +86 18918289046; fax: +86 02167722251. E-mail address: [email protected] (T. Zhu). 0304-3940/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2012.08.012
conduction, impaired cutaneous endothelium-related vasodilation, and degeneration of myelinated sensory fibers, the exact cause and mechanism of PDN still remain unclear [23]. Therefore, the treatment of diabetic neuropathic pain often has only limited efficacy and/or are associated with intolerable side effects [30]. In rodents, symptoms of PDN such as mechanical allodynia and thermal hyperalgesia can be studied after induction of diabetes with streptozotocin (STZ) [21]. Assessment of behavioral responses to external stimuli in this diabetic model has led to identification of a number of mechanisms of abnormal sensation and pain in diabetes [23]. Evidence from both animal models and humans indicates that systemic inflammation is involved in the pathophysiological processes of DM and is facilitated by innate immune responses [7]. Toll-like receptors (TLRs) are a class of proteins that play a key role in the innate immune response. Interactions between TLRs and their ligands expressed by microbial pathogens can induce a
J.-e. Yan et al. / Neuroscience Letters 526 (2012) 54–58
cascade of intracellular signaling events, culminating in the upregulation of proinflammatory pathways [20]. TLRs are expressed in various cell types in the nervous system and contribute to infectious as well as non-infectious disorders [10,11,13]. After tissue insult and nerve injury, TLRs can induce the activation of microglia and the production of the proinflammatory cytokines in the spinal cord, leading to the development of inflammatory pain and neuropathic pain [14,22,29]. Although TLR4 expression is increased in many inflammatory disorders [10,11,19,28], the effects of metabolic aberrations on TLR4 and its role in diabetes and its complications are still poorly understood. In current study, by establishing the timeline of TLR4 and two pro-inflammatory cytokines (TNF-␣ and IL-1) levels in the rat spinal cord following streptozotocin injection, we aimed to characterize the role of TLR4 in peripheral diabetic neuropathy. 2. Materials and methods 2.1. Animal Two-month old male Sprague-Dawley rats weighing 180–200 g were obtained from the Animal Care Facility of Shanghai First people’s hospital and housed in light-controlled (12-h light–dark cycle) and temperature-controlled (20 ± 2 ◦ C) room with food and water available ad libitum. Diabetes was induced by a single intraperitoneal injection of 1% STZ (Sigma–Aldrich, USA) at 65 mg/kg body weight after 14 h of food deprivation. STZ was dissolved in 10 mM citrate buffer, pH 4.38. Rats injected with citrate buffer alone without STZ served as the normal control. Establishment of diabetes was confirmed at one week after STZ induction and at weekly intervals by measuring fasting (>8 h) blood glucose levels (>250 mg/dl) with a glucometer (Roche, Swiss). All procedures were approved by the Ethical Committee of Animal Research of the hospital and performed in accordance with the NIH’s Guiding Principles in the Care and Use of Laboratory Animals. 2.2. Mechanical hyperalgesia and thermal hyperalgesia Pain responses were evaluated in pre- and post-diabetic rats. Tests were conducted in the morning between 9:00 and 12:00 AM in a noise-free environment. For mechanical testing, animals were placed in a hanging cage with metal mesh floor and allowed to acclimate for at least 30 min. Mechanical hyperalgesia was assessed by measuring the paw-pressure withdrawal threshold (PPWT) when exposed to increasing mechanical stimulation with an analgesiometer (Sunny Instruments, China). The computer converted the applied stimulus intensity into grams, and the pressure at which a paw withdrawal occurred was recorded. On a given test, the same procedure was repeated three times at 5 min intervals, the mean withdrawal pressure was calculated by averaging the three measurements. To quantify thermal sensitivity, rats were placed on a clear Plexiglas chamber and given 5–10 min to acclimate. The radiant heat source in II T336 paw-flick instrument (life science, USA) delivered a heat stimulus to the plantar surface of the hind paw. The reflective photocell sensor detected when the rat moved or lifted its paw. The time between onset of the stimulus and paw flick was defined as the paw withdrawal latency (PWL). On a given test and for each hind paw, the same procedure was repeated three times at 5 min intervals, the mean withdrawal pressure was computed by averaging the six measurements. 2.3. Spinal cord transection Rats were deeply anesthetized with 10% chloral hydrate (300 mg/kg, intraperitoneal injection) and sacrificed rapidly.
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A dorsal midline incision was made from L2–S2. Once exposed, L4–L6 spinal cord were harvested and placed in the liquid nitrogen immediately. 2.4. RT-PCR Total RNA was extracted from spinal tissue with Trizol reagent (Invitrogen, USA) according to the manufacturer’s recommendations. Complementary DNA sequences of -actin and TLR4 were obtained from the database of NCBI. The following PCR primers (Sangon Biotech, China) were used: TLR4 (forward: 5 -GCCGGAAAGTTATTGTGGTGGT-3 ; reverse: 5 ATGGGTTTTAGGCGCAGAGTTT-3 ), the anticipated length was 356 bp; -actin (forward: 5 -CACGATGGAGGGGCCGGACTCATC-3 ; reverse: 5 -GTAGCCCACGTCGTAGCAAA-3 ), and the anticipated length was 240 bp. The amplification protocol was: 10 min at 95 ◦ C; followed by 30 cycles 30 s at 95 ◦ C, 50 s at 62 ◦ C and 50 s at 72 ◦ C; then 10 min at 72 ◦ C for extension. Amplification products were separated by agarose gel electrophoresis and photographed. 2.5. ELISA The spinal cord was collected and homogenized followed by centrifugation. TNF-␣ and IL-1 concentrations were quantified by ELISA (Neobioscience, China). According to the manufacturer’s instructions, the absorbance (A) was detected at 450 nm (A450 ). The content of each sample was obtained according to the standard curve. 2.6. Statistics All the data are presented as mean ± standard deviation (SD). To assess the significance of difference between groups, summed effects of STZ over the course of an experiment were used to compare treatment by analysis of variance (ANOVA) followed by Fisher’s least significant difference. Two-way ANOVA was used to evaluate the interaction between different groups. p < 0.05 was considered statistically significant. The regression and correlation analysis was based on least squares method. Once a regression model has been constructed, the goodness of the fit was checked by R-squared, analyses of the pattern of residuals and hypothesis testing. Statistical significance was checked by an F-test of the overall fit, followed by t-test of individual parameters.
3. Results 3.1. Induction of diabetes with STZ Since the primary indicator for the successful induction of diabetes using animal model was the development of hyperglycemia, it was necessary to measure the levels of glucose in blood circulation. As shown in Fig. 1A, one week after STZ injections, treated animals developed clear diabetes. Glucose concentrations in all STZ-treated diabetic rats (DM group) were greater than 250 mg/dl, a standard level defined as diabetic hyperglycemia, at week 1, week 2, week 3 and week 4 (n = 6 animals per time point). Meanwhile the glycemia levels in citrate-treated non-diabetic rats (control group) remained relatively stable and typically ranged from 100 to 110 mg/dl. The body weights of the DM animals did not show any differences from the controls before treatment (Fig. 1B). After STZ injections, the rats in DM group lost their weights
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Fig. 1. Time course of blood glucose concentration (A) and body weight (B) after streptozotocin (DM) or citrate buffer (control) treatment. Data are presented as mean ± SD (n = 6 animals per time point), ***p < 0.001 by two-way ANOVA analysis.
Fig. 2. Time course of paw-pressure withdrawal threshold to mechanical stimuli (A) and paw withdrawal latency to thermal stimuli (B) in DM (open symbols) and control (closed symbols) animals. Data are presented as mean ± SD (n = 6 animals per time point), *p < 0.05, **p < 0.01, ***p < 0.001 by two-way ANOVA analysis.
gradually while the body weights of the control animals elevated progressively.
3.2. Assessment of mechanical hyperalgesia and thermal hyperalgesia Pain reactions were characterized in both DM rats and their respective controls for diabetes-induced polyneuropathic pain symptoms. The PPWT toward mechanical stimulus and the PWL toward thermal stimulus in control animals were relatively stable and typically ranged around 11–13 g (Fig. 2A) and 10–12 s (Fig. 2B), respectively. On the contrary, in the DM rats PPWT and PWL decreased gradually after STZ injection and showed significant difference since the second week after treatment. Both of them reached their minima at week 4 (Fig. 2).
3.3. TLR4 expression The expressions of TLR4 in L4–L6 spinal cord from DM rats were upregulated gradually but significantly in week 1–4 and the peak was observed four weeks after STZ injection while the TLR4 levels in control group remained relatively stable (Fig. 3A). Further analysis showed that TLR4 expressions in these DM animals (n = 30) were negatively linear correlated with PPWT (r = −0.55, p < 0.01; Fig. 3B) and PWL (r = −0.46, p < 0.05; Fig. 3C).
3.4. TNF-˛ and IL-1ˇ production TNF-␣ and IL-1 are two downstream proinflammatory cytokines of TLR4 signaling pathway [1,11,20], their productions by spinal cord from the rats in the control and DM groups were
Fig. 3. Correlation between TLR4 expression and hyperalgesia in DM rats. (A) Time course of relative TLR4 mRNA expression in spinal cord (L4–L6) of DM (open symbols) and control (closed symbols) animals. Data are presented as mean ± SD (n = 6 animals per time point), *p < 0.05, **p < 0.01, ***p < 0.001 by two-way ANOVA analysis. (B) Plot of linear regression analysis showing the relation between TLR4 expression and paw-pressure withdrawal threshold to mechanical stimuli in DM rats (n = 30, r = −0.55, p < 0.01 by t-test). (C) Plot of linear regression analysis demonstrating the relation between TLR4 expression and paw withdrawal latency to thermal stimuli in DM animals (n = 30, r = −0.46, p < 0.05 by t-test).
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4. Discussion
Fig. 4. Correlation between TNF-␣/IL-1 protein levels and hyperalgesia in DM rats. (A and B) Time course of TNF-␣ and IL-1 productions in spinal cord (L4–L6) of DM (open symbols) and control (closed symbols) animals. Data are presented as mean ± SD (n = 6 animals per time point), ***p < 0.001 by two-way ANOVA analysis. (C and D) Plot of linear regression analysis showing the relation between TLR4 expression and production of TNF-␣ (n = 30, r = 0.57, p < 0.01 by t-test) or IL-1 (n = 30, r = 0.44, p < 0.05 by t-test) in DM animals. (E and F) Plot of linear regression analysis showing the relation between paw-pressure withdrawal threshold to mechanical stimuli and production of TNF-␣ (n = 30, r = −0.76, p < 0.001 by t-test) or IL-1 (n = 30, r = −0.92, p < 0.001 by t-test). (G and H) Plot of linear regression analysis showing the relation between paw withdrawal latency to thermal stimuli and production of TNF-␣ (n = 30, r = −0.62, p < 0.001 by t-test) or IL-1 (n = 30, r = −0.77, p < 0.001 by t-test).
similar before treatment. Both levels of TNF-␣ (Fig. 4A) and IL-1 (Fig. 4B) elevated significantly in the DM rat since the induction of diabetes. For DM animals, further studies demonstrated that (1) TLR4 expression was linear correlated with production of TNF-␣ (r = 0.57, p < 0.01; Fig. 4C) and IL-1 (r = 0.44, p < 0.05; Fig. 4D); (2) PPWT was negatively linear correlated with production of TNF-␣ (r = −0.76, p < 0.001; Fig. 4E) and IL-1 (r = −0.92, p < 0.001; Fig. 4F); and (3) PWL was negatively linear correlated with TNF-␣ level (r = −0.62, p < 0.001; Fig. 4G) and IL-1 (r = −0.77, p < 0.001; Fig. 4H).
In the current study, we demonstrate that the upregulated expression of TLR4 is correlated with neuropathic pain in a rat model of STZ-induced diabetes. Furthermore, the spinal release of TNF-␣ and IL-1, two downstream inflammatory cytokines of TLR4 signaling pathway, are also correlated with the behavioral responses toward mechanical and thermal stimuli. Autoimmune diabetes (Type I diabetes) is considered to be a multi-stage T cell mediated auto-immune disease involving slow and gradual islet  cell destruction and complete loss of insulin secretion [26]. The available data about the auto-immune process leading to diabetes, its complications and the nature of the inflammatory response is limited [18]. STZ is an antibiotic extracted from streptomyces acromogenes and is diabetogenic due to a selective cytotoxic action upon pancreatic  cell [24]. In recent years STZinjected rodents is considered to be a useful animal model for study diabetes and its complications, especially PDN [21], largely due to the convenience of the single treatment. In this study, the glucose levels in STZ-treated animals were over 250 mg/dl, the daily food and water consumptions elevated while the body weights decreased significantly. All of these suggested the diabetic animal model was successfully established. Several lines of evidence demonstrate that both mechanical and thermal hyperalgesia occur in the mild PDN, and are suggested to be an indicator of early PDN [6,9]. Here, robust mechanical/thermal hyperalgesia were found and repetitive testing did not result in any signs of habituation. Consistent with previous reports [6,15], PPWT decreased progressively but significantly after STZ injection and reduced over 50% at week 4; meanwhile PWL decreased gradually and slowly for three weeks after treatment, but at week 4, a robust change occurred. This may suggest that more than one patho-physiological mechanism is involved in diabetic pain. Previous studies show a significant upregulation of TLRs in the central nervous system from nerve injury-induced neuropathic pain models [2,8,29]. It is also found that TLR4-deficient mice displayed attenuated behavioral hypersensitivity and decreased expression of spinal glial activation and proinflammatory cytokines [5]. TLR4 is highly expressed by microglial cells in the mammalian nervous system, while several reports indicate that TLR4 can be identified in astrocytes, neurons and neural progenitor cells [4,16,25]. TLR4 can recognize a variety of microbial and host structures including lipopolysaccharide, peptidoglycan, high mobility group proteins, etc. [1,20]. After binding to its ligands, TLR4 lead to activation of two downstream pathways: mitogen-activated protein kinase (MAPK) and NF-B pathway. The activation of NFB pathway causes the synthesis and release of proinflammatory cytokines, including TNF-␣ and IL-1 while the activation of MAPK pathway plays an important role in the regulation of neuronal plasticity [27]. To date, no study has been conducted to investigate the role of TLR4 in peripheral diabetic neuropathy. Our results showed that, at mRNA level, the expression of TLR4 elevated gradually after STZ injection and increased over 50% at week 4 in the spinal cord where is critical to the pain signal transduction. Compare to pretreatment, the protein levels of TNF-␣ and IL-1 elevated over 50% and 500%, respectively. This suggests that TNF-␣ and IL-1 do not contribute equally to neuro-inflammatory and IL-1 may play a more important role in diabetic neuropathy. Further statistical analysis demonstrated that TLR4, TNF-␣ and IL-1 levels are linear correlated with mechanical and thermal hyperalgesia. It is noteworthy that the association between TLR4 and diabetic pain is mild, while proinflammatory cytokines show robust correlation with hyperalgesia. This may suggest that, besides TLR4, some other substances (such as TLR2 and TLR3) are activated in diabetes [4,17] and involved in the pathogenesis of neuropathic pain [14,22]. These
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