Spinal astrocytic activation contributes to mechanical allodynia in a mouse model of type 2 diabetes

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Research Report

Spinal astrocytic activation contributes to mechanical allodynia in a mouse model of type 2 diabetes Yong-Hui Liaoa,1 , Gui-He Zhangb,1 , Dong Jiac,1 , Peng Wangc,1 , Nian-Song Qiand,1 , Fei Hee , Xiang-Tian Zeng f , Yong Hea , Yan-Ling Yanga , Da-Yong Caoa , Yi Zhanga , De-Sheng Wanga , Kai-Shan Taoa,⁎, Chang-Jun Gaob,⁎, Ke-Feng Doua,⁎ a

Department of Hepatobiliary Surgery, Xijing Hospital, The Fourth Military Medical University, Xi'an 710032, PR China Department of Anesthesiology, Tangdu Hospital, The Fourth Military Medical University, Xi'an 710038, PR China c Department of Neurosurgery, Tangdu Hospital, The Fourth Military Medical University, Xi'an 710038, PR China d Department of Hepatobiliary Surgery, Chinese People's Liberation Army General Hospital, Beijing 100853, PR China e Department of Medical Genetics and Developmental Biology, The Fourth Military Medical University, Xi'an 710032, PR China f Intensive Care Unit of Shaoxing People's Hospital, Zhejiang, PR China b

A R T I C LE I N FO

AB S T R A C T

Article history:

Diabetic neuropathic pain (DNP) plays a major role in decreased life quality of type 2 diabetes

Accepted 14 October 2010

patients, however, the molecular mechanisms underlying DNP remain unclear. Emerging

Available online 12 November 2010

research implicates the participation of spinal glial cells in some neuropathic pain models. However, it remains unknown whether spinal glial cells are activated under type 2 diabetic

Keywords:

conditions and whether they contribute to diabetes-induced neuropathic pain. In the present

Diabetes

study, using a db/db type 2 diabetes mouse model that displayed obvious mechanical

Neuropathic pain

allodynia, we found that spinal astrocyte but not microglia was dramatically activated. The

Glia

mechanical allodynia was significantly attenuated by intrathecally administrated

Cytokine

L-α-aminoadipate

(astrocytic specific inhibitor) whereas minocycline (microglial specific

inhibitor) did not have any effect on mechanical allodynia, which indicated that spinal astrocytic activation contributed to allodynia in db/db mice. Further study aimed to identify the detailed mechanism of astrocyte-incudced allodynia in db/db mice. Results showed that spinal activated astrocytes dramatically increased interleukin (IL)-1β expression which may induce N-methyl-D-aspartic acid receptor (NMDAR) phosphorylation in spinal dorsal horn neurons to enhance pain transmission. Together, these results suggest that spinal activated astrocytes may be a crucial component of mechanical allodynia in type 2 diabetes and “Astrocyte-IL-1β-NMDAR-Neuron” pathway may be the detailed mechanism of astrocyte-incudced allodynia. Thus, inhibiting astrocytic activation in the spinal dorsal horn may represent a novel therapeutic strategy for treating DNP. © 2010 Elsevier B.V. All rights reserved.

⁎ Corresponding authors. K.-S. Tao is to be contacted at fax: +86 29 84771094. C.-J. Gao, fax: +86 29 84777439. K.-F. Dou, fax: +86 29 84775260. E-mail addresses: [email protected] (K.-S. Tao), [email protected] (C.-J. Gao), [email protected] (K.-F. Dou). 1 These authors contributed equally to this work and are co-first authors. 0006-8993/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2010.10.044

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1.

Introduction

Diabetic neuropathy occurs in 50% of patients with either type 1 or type 2 diabetes, and diabetic neuropathic pain (DNP) is the most devastating complication of diabetic neuropathy (Boulton et al., 2005; Edwards et al., 2008). Similar to other types of neuropathic pain, DNP is manifested as allodynia, that is, when normally nonpainful stimuli become painful, and hyperalgesia, that is, an increased sensitivity to normally painful stimuli (Barrett et al., 2007). Although peripheral nerve degeneration, ischemia, activation of the polyol pathway, deficits in neurotrophic support or hyperactivity of dorsal root ganglion neurons have been hypothesized for the development and pathophysiology of DNP, current treatments for DNP are usually ineffective and only a few DNP patients could obtain effective pain relief (Barrett et al., 2007; Boulton, 2006; Tesfaye and Kempler, 2005; Vinik, 1999). DNP plays a major role in decreased life quality for diabetes patients; it often subsides over time, sometimes several years (Dworkin et al., 2007; Jensen et al., 2006). It is predicted that there will be more than 200 million type 2 diabetes patients worldwide by 2025 (Narayan et al., 2000). DNP is more prevalent in type 2 than type 1 diabetes (Barrett et al., 2007). Furthermore, DNP is a persistent symptom in patients with type 2 diabetes, but it is less frequently persistent in type 1 diabetes (Barrett et al., 2007; Currie et al., 2007). Understanding the mechanisms of DNP of type 2 diabetes could lead to effective treatments to this devastating disease. DNP has extensively been explored in animal models of type 1 diabetes and, in particular, in streptozotocin (STZ)-induced diabetic rats (Daulhac et al., 2006; Malcangio and Tomlinson, 1998; Tsuda et al., 2008; Wodarski et al., 2009). In contrast, insufficient information is available on pathogenetic mechanisms of DNP in type 2 diabetic models despite the fact that the vast majority of diabetes patients have type 2 (non-insulin dependent) diabetes. According to classic pain research, the pain pathway has been described simply as a serial chain of neuronal elements. It has been generally accepted that activation of spinal N-methyl-Daspartate receptor (NMDAR), a glutamate receptor localized on neurons, plays an important role in neuropathic pain (Christoph et al., 2006; Dickenson, 1990; Woolf and Salter, 2000). NMDAR activation is mainly modulated by post translational modifications including phosphorylation, and this is proposed to underlie its involvement in the production of central sensitization in the spinal level (Brenner et al., 2004; Ultenius et al., 2006). However, recent researches have strongly identified spinal cord glia and proinflammatory cytokines, such as interleukin (IL)-1β and tumor necrosis factor-α, as key factors in the induction and maintenance of neuropathic pain (Watkins et al., 2001b; Watkins and Maier, 2005). After inflammation or nerve injury, glia can be activated and release cytokines that modulate neuronal activity and synaptic plasticity. Several recent studies showed that IL-1β signaling may facilitate NMDAR to enhance neuronal activity (Viviani et al., 2003; Wei et al., 2007). With regard to the research of glia and DNP, two previous studies showed that spinal microglia but not astrocyte activation was involved in mechanical allodynia in STZ-treated rat model of type 1 diabetes (Tsuda et al., 2008; Wodarski et al., 2009). Daulhac et al. (2006) showed that spinal mitogen-activated protein kinase phosphorylation was correlated with mechanical allodynia in

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STZ-treated rat model, and mitogen-activated protein kinase was localized in neurons and microglia but not in astrocyte in the superficial spinal dorsal horn. These results seemed to indicate that spinal microglial activation is a key mechanism underlying DNP of type 1 diabetes. However, related report on the involvement of spinal glia in DNP of type 2 diabetes is still lacking. Therefore, we investigated the role of spinal cord glia in the pathophysiology and development of DNP in a mouse model of type 2 diabetes. The db/db mouse carries a homozygous null mutation of the leptin receptor and is a well-characterized animal model of type 2 diabetes. Diabetic phenotypes such as obesity, hyperglycemia, hyperinsulinemia, and hyperlipidemia are present at 4 to 5 weeks of age (Chua et al., 1996; Sullivan et al., 2007). Recent studies with db/db mice indicated that db/db mice were a useful model for studying DNP, and this model could mimic the chronic pain states that occur in humans (Cheng et al., 2009, 2010). In this study, L-α-aminoadipate (LAA) and minocycline were used to inactivate astrocyte and microglia, respectively, to identify the roles of these spinal components in the development of mechanical allodynia in db/db mice. In addition, the mediating role of inflammatory cytokines on NMDAR activation was investigated.

2.

Results

2.1.

Mechanical allodynia occurred in type 2 diabetic mice

Blood glucose and body weight were detected in db/db, db/+ and C57 mice from postnatal 4 weeks to 20 weeks (P4 to P20) to monitor the development of type 2 diabetes (n = 10/group). No difference in blood glucose was observed between db/+ mice (5.6±0.8 mmol/L; P6) and C57 mice (5.9 ± 0.7 mmol/L; P6), and the blood glucose of these two groups maintained at normal level through the period tested. Compared with db/+ and C57 mice (P6), the blood glucose of db/db mice reached a significant increase at postnatal 6 weeks (P6) (16.2 ± 2.8 mmol/L), peaked at P8 (26.4 ± 5.6 mmol/L), and thereafter maintained at high level (P < 0.05; Fig. 1A). The body weight was also significantly higher in db/db mice beginning at P4 (26 ± 5.2 g), and was persistently elevated through the period tested. By P16, the weights of db/db mice (65 ± 7.2 g) were almost twice those of db/+ (30 ± 3.6 g) and C57 mice (33 ± 2.9 g) (P < 0.05; Fig. 1B). No difference in paw withdrawal threshold was observed between db/+ mice (20± 1.6 g; P6) and C57 mice (20 ± 1.1 g; P6), and the paw withdrawal threshold of these two groups maintained at normal level. The paw withdrawal threshold was significantly lower for db/db mice beginning at P6 (12.3± 2.8 g), suggesting increased sensitivity to mechanical stimuli. Then the mechanical allodynia peaked at P8 (7.4± 1.9 g) and continued thereafter (P < 0.05; Fig. 1C). Thus, these data indicated that db/db mice developed features of type 2 diabetes, and significant mechanical allodynia in db/db mice coincided with hyperglycemia.

2.2. Mechanical allodynia was induced by spinal astrocytic activation which depended on diabetes-induced oxidative stress In order to test our hypothesis that spinal glial activation was involved in mechanical allodynia in db/db mice, we observed

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the expression of astrocytic marker GFAP and microglial marker OX42 in db/db, db/+ and C57 mice at postnatal different time points (n = 10/group/week). Immunohistochemistry indicated that compared with db/+ and C57 mice (P8), GFAP staining was significantly increased in the spinal cord of db/db mice at P8, and continued thereafter. This enhanced staining concentrated in superficial dorsal horn. Activated astrocytes had hypertrophied cell bodies and thickened processes with enhanced GFAP-immunoreactivity (Fig. 2A–D).

Using Western blot, we detected that no significant difference in GFAP expression was observed between db/+ mice (0.55±0.07; P6) and C57 mice (0.53±0.08; P6), and GFAP expression of these two groups maintained at the same level through the period tested. However, compared to db/+ mice and C57 mice (P6), GFAP expression was significantly increased in db/db mice (1.7±0.3) at P6 (P<0.05). GFAP upregulation peaked at P8 (2.0±0.35) and persisted thereafter, which correlated with the changing course of mechanical allodynia (Fig. 2E). With regard to OX42 expression in spinal cord, Western blot and immunostaining showed that there was no difference between db/db mice, db/+ mice and C57 mice at postnatal any week. In all the mice, OX42 expression was unchanged through the period tested (Fig. 3A). We injected LAA or minocycline intrathecally and observed their effects on mechanical allodynia in db/db mice (P10). The astrocytic specific toxin LAA significantly attenuated the allodynia. However, the microglial specific inhibitor minocycline did not influence mechanical allodynia (Fig. 3B). In addition, a systemic treatment with PBN (reactive oxygen species scavenger) significantly reduced GFAP overexpression, which indicated that diabetes-induced oxidative stress may mediate the development of astracytic activation in db/db mice (Fig. 2E).

2.3. Spinal astrocytes dramatically increased the expression of IL-1β which is related to mechanical allodynia Western blot analysis showed that no significant difference in IL-1β expression in spinal cord was observed between db/+ mice (0.028 ± 0.007; P6) and C57 mice (0.022 ± 0.005; P6), and IL-1β expression of these two groups maintained at the same level through the period tested. However, compared to db/+ mice and C57 mice, IL-1β expression was significantly increased in db/db mice (0.13 ± 0.03) at P6 (P < 0.05). IL-1β upregulation peaked at P8 (0.2 ± 0.04), and persisted thereafter, which was similar to the time course of GFAP expression (Fig. 4A). At P10 in db/db mice, we injected pentoxifylline (cytokine inhibitor) or IL-1ra (interleukin-1 receptor antagonist)

Fig. 1 – Mechanical allodynia occurred in type 2 diabetic db/db mice. (A) Blood glucose in db/db, db/+ and C57 mice. Compared with db/+ and C57 mice (P6), the blood glucose of db/db mice reached a significant increase at postnatal 6 weeks (P6), peaked at P8, and thereafter maintained at high level. (B) Body weight in db/db, db/+ and C57 mice. Compared with db/+ and C57 mice (P4), the body weight of db/db mice was significantly higher beginning at P4, and was persistently elevated through the period tested. (C) Compared with db/+ and C57 mice (P6), the paw withdrawal threshold of db/db mice was significantly lower beginning at P6, suggesting increased sensitivity to mechanical stimuli. The mechanical allodynia peaked at P8 and continued thereafter. All data were calculated as mean ± SEM (n = 10/group/week). *P < 0.05, **P < 0.01 vs. db/+ and C57 mice. db/db: homozygous type 2 diabetic db/db mice; db/+: heterozygous nondiabetic littermates; C57: C57BL6 control mice.

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intrathecally and observed their effects on mechanical allodynia in db/db mice. Both pentoxifylline and IL-1ra could significantly attenuate the allodynia (Fig. 4B). At P10 in db/db mice, intrathecally administered LAA could significantly down-regulate IL-1β expression (0.05 ± 0.009) (Fig. 4A). Subsequent double immunofluorescent staining showed that IL-1β-immunoreactivity was only localized in GFAP-immunopositive cells (Fig. 4C–E) but not in NeuNimmunopositive cells or OX42-immunopositive cells (data not shown).

2.4. IL-1β induces NMDA receptor phosphorylation in spinal dorsal horn neurons Western blot analysis showed that no significant difference in phosphorylated NR1 subunit of NMDA receptor (P-NR1) expression in spinal cord was observed between db/+ mice (0.04 ± 0.01; P6) and C57 mice (0.043 ± 0.012; P6), and P-NR1 expression of these two groups maintained at the same level through the period tested. However, compared to db/+ mice

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and C57 mice, P-NR1 expression was significantly increased in db/db mice (0.38 ± 0.06) at P6 (P < 0.05). P-NR1 upregulation peaked at P8 (0.58 ± 0.09), and persisted thereafter, which was similar to the time course of IL-1β and GFAP expression (Fig. 5A). At P10 in db/db mice, intrathecally administered LAA, pentoxifylline or IL-1ra could significantly down-regulate P-NR1 expression (Fig. 5A). Subsequent double immunofluorescent staining showed that P-NR1-immunoreactivity and IL-1RIimmunoreactivity were only localized in NeuN-immunopositive cells (data not shown), and P-NR1-immunoreactivity and IL-1RIimmunoreactivity were totally double-labeled (Fig. 5B–D).

3.

Discussion

3.1. Spinal astrocyte but not microglia was activated in db/db mice, which contributed to mechanical allodynia The epidemic of obesity worldwide is driving a dramatical increase in type 2 diabetes patients and consequentially setting the scene for an impending wave of morbidity and mortality correlated with diabetes complications including diabetic neuropathic pain (DNP) (Boulton et al., 2005; Narayan et al., 2000). Diabetic neuropathy occurs in 50% of type 2 diabetes patients, and approximately half of patients with diabetic neuropathy experience DNP (Boulton et al., 2005; Edwards et al., 2008). DNP cannot yet be treated effectively, as it often remains refractory to classical analgesics such as opioid analgesics (Barrett et al., 2007). Currently, the most commonly used medications include tricyclic antidepressants (Goodnick, 2001), serotonin reuptake blocker (Owens et al., 2000) and anticonvulsants (Rosenberg et al., 1997), alone or in

Fig. 2 – Spinal astrocyte was activated in db/db mice, which depended on oxidative stress. (A–D) GFAP-like immunoreactivity (−LI) in spinal dorsal horn in db/db, db/+ and C57 mice. Compared with db/+ and C57 mice (P8), GFAP-LI of db/db mice was significantly increased at postnatal 8 weeks (P8), and continued thereafter. This enhanced staining concentrated in superficial dorsal horn. Bar = 200 μm. (E) Representative Western blot bands of GFAP in spinal dorsal horn in db/db, db/+ and C57 mice at postnatal different week. The summary histogram showed the expression ratio of GFAP protein level normalized to β-actin in the same sample. Compared to db/+ mice and C57 mice (P6), GFAP expression was significantly increased in db/db mice at P6. GFAP upregulation peaked at P8, and persisted thereafter. A systemic treatment with PBN (scavenger for reactive oxygen species) significantly reduced GFAP overexpression in db/db mice (P10), which indicated that diabetes-induced oxidative stress may mediate the development of astracytic activation. All data were calculated as mean ± SEM (n = 10/group/week). *P < 0.05 vs. db/+ and C57 mice; ΔP < 0.05 vs. P10 db/db mice. db/db: homozygous type 2 diabetic db/db mice; db/+: heterozygous nondiabetic littermates; C57: C57BL6 control mice; PBN: phenyl N-tert-butylnitrone; w: weeks.

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Fig. 3 – Spinal microglia was not activated in db/db mice, astrocytic specific inhibitor LAA but not microglial specific inhibitor minocycline could attenuate mechanical allodynia in db/db mice. (A) Representative Western blot bands of OX42 in spinal dorsal horn in db/db, db/+ and C57 mice at postnatal different week. The summary histogram showed the expression ratio of OX42 protein level normalized to β-actin in the same sample. With regard to OX42 expression in spinal cord, there was no difference between db/db mice, db/+ mice and C57 mice at postnatal any week. In all the mice, OX42 expression was unchanged through the period tested. (B) LAA or minocycline were injected intrathecally and observed their effects on mechanical allodynia in db/db mice (P10). The astrocytic specific toxin LAA significantly attenuated the allodynia. However, minocycline did not influence mechanical allodynia. All data were calculated as mean ± SEM (n = 10/group). *P < 0.05, **P < 0.01 vs. db/+ and C57 mice. db/db: homozygous type 2 diabetic db/db mice; db/+: heterozygous nondiabetic littermates; C57: C57BL6 control mice; LAA: L-α-aminoadipate; w: weeks.

combination. Nevertheless, these drugs have a rather limited efficacy and often produce undesirable side effects. The current situation dictates a necessity for detailed studies of DNP in type 2 diabetes animal models. Astrocytic activation has been observed in the spinal cord in some models of persistent pain, including neuropathic pain (Scholz and Woolf, 2007), inflammatory pain (Raghavendra et al., 2004) and bone cancer pain (Zhang et al., 2005). Here we examined whether spinal astrocytes contribute to the development of mechanical allodynia in a db/db mouse model of type 2 diabetes. To the best of our knowledge, we are the first to report that this glia cell plays an important role in DNP in db/db mice. Our results showed that GFAP (astrocytic activation marker) was significantly increased in the spinal cord of db/db mice at postnatal 8 weeks, and continued thereafter. The changing course of astrocytic activation was in parallel with that of hyperglycemia and mechanical allodynia. Astrocytic specific toxin LAA significantly attenuated the allodynia, which elucidated that astrocytic activation surely contributed to allodynia in db/db mice. In addition to supporting roles in normal neuronal function, glial cells in central nervous system are increasingly known to be important regulators of synaptic activity and key functional units of the nervous system (Fields and StevensGraham, 2002). Peripheral tissue or nerve injury induces glial activation in central nervous system, mainly involving astrocytes and microglia (Watkins et al., 2001b). Previous studies have clarified that compared with microglia, astrocytes contribute more to the maintenance of chronic pain (Watkins et al., 2001a; Zhuang et al., 2005). The results of this study demonstrated for the first time that a persistent increase of spinal astrocytic activation but not microglial activation occurred in type 2 diabetic mice. So astrocytic activation is probably one of the most important factors in the pathophysiology of DNP in type 2 diabetes. To examine whether spinal astrocytic activation is important for mechanical allodynia in db/db mice, we administrated LAA (astrocytic specific inhibitor) and minocycline (microglial specific inhibitor) intrathecally to db/db mice and detected mechanical withdrawal threshold. We observed that mechanical allodynia was significantly attenuated by LAA, whereas minocycline did not have any effect on mechanical allodynia. With regard to the relationship between glia and DNP, a recent study showed that spinal microglial but not astrocytic activation was involved in mechanical allodynia in STZ-treated rat model of type 1 diabetes (Wodarski et al., 2009). Also, Tsuda et al. (2008) indicated that spinal microglial activation played an important role in mechanical allodynia in STZ-treated rat model of type 1 diabetes. However, Tsuda's research group did not investigate the role of spinal astrocytic activation. Daulhac et al. (2006) showed that spinal mitogen-activated protein kinase localized in microglia was correlated with mechanical allodynia in STZ-treated type 1 diabetes rat model. These results seemed to indicate that activated dorsal horn microglia may be a crucial component of type 1 diabetes-induced neuropathic pain. In this study, by using a db/db type 2 diabetes mouse model that displayed obvious mechanical allodynia, we found that spinal astrocyte but not microglia was dramatically activated. The mechanical allodynia was significantly attenuated by intrathecally administrated LAA (astrocytic specific inhibitor) whereas minocycline (microglial

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Fig. 4 – IL-1β overexpression in spinal cord was related to mechanical allodynia in db/db mice, and activated astrocyte is the only source of IL-1β release. (A) Representative Western blot bands of IL-1β in spinal dorsal horn in db/db, db/+ and C57 mice at postnatal different week. The summary histogram showed the expression ratio of GFAP protein level normalized to β-actin in the same sample. IL-1β expression was significantly increased in db/db mice compared to db/+ mice and C57 mice (P6). An intrathecal treatment with LAA (astrocytic specific toxin) significantly reduced IL-1β overexpression in db/db mice (P10). (B) Pentoxifylline (cytokine inhibitor) or IL-1ra (interleukin-1 receptor antagonist) were injected intrathecally and observed their effects on mechanical allodynia in db/db mice (P10). Both pentoxifylline and IL-1ra could significantly attenuated the allodynia. (C-E) Double immunofluorescent staining showed that IL-1β-immunoreactivity was only localized in GFAP-immunopositive cells. Bar = 20 μm. All data were calculated as mean ± SEM (n = 10/group). *P < 0.05, **P < 0.01 vs. db/+ and C57 mice; ΔP < 0.05 vs. P10 db/db mice. db/db: homozygous type 2 diabetic db/db mice; db/+: heterozygous nondiabetic littermates; C57: C57BL6 control mice; IL-1β: interleukin-1β; LAA: L-α-aminoadipate; IL-1ra: interleukin-1 receptor antagonist; w: weeks.

specific inhibitor) did not have any effect on mechanical allodynia. All the above results indicated that spinal microglial activation contributed to type 1 diabetes-induced neuropathic pain and spinal astrocytic activation contributed to type 2 diabetes-induced neuropathic pain in animal experiment. The discrepancy between previous study and our study may be attributed to various factors which may include internal biological variability within rat and mice, different pathogenetic mechanism within type 1 diabetes and type 2 diabetes, and so on. In future studies, we will elucidate the role of glia cell in DNP by employing rat models of type 2 diabetes, including BBZDR/Wor rats and Zucker fatty rats. Nevertheless, our promising findings regarding LAA-induced alleviation of mechanical allodynia in db/db mice suggest that pharmaco-

logical antagonism of astrocytic activation in spinal cord may offer a great advantage in the treatment of type 2 diabetesinduced neuropathic pain.

3.2. Diabetes-induced oxidative stress may contribute to spinal astrocytic activation in db/db mice Improved blood glucose control dramatically reduces the risk of diabetic neuropathy, thereby implicating hyperglycemia as a leading causative factor (Boulton, 2001). Hyperglycemia was reported to not only directly induce oxidative stress (Low et al., 1989; Russell et al., 1999), but also contribute to increased aldose reductase activity, increased advanced glycation end products, nerve hypoxia/ischemia, activation of protein kinase C, and insulin-like growth factor deficiency (Greene et al., 1992). These

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Fig. 5 – IL-1β released from astrocyte induced NMDA receptor phosphorylation in spinal dorsal horn neurons in db/db mice. (A) Representative Western blot bands of P-NR1 in spinal dorsal horn in db/db, db/+ and C57 mice at postnatal different week. The summary histogram showed the expression ratio of GFAP protein level normalized to β-actin in the same sample. P-NR1 expression was significantly increased in db/db mice compared to db/+ mice and C57 mice (P6). Intrathecal treatment with LAA (astrocytic specific toxin), pentoxifylline (cytokine inhibitor) or IL-1ra (interleukin-1 receptor antagonist) could significantly reduce IL-1β overexpression in db/db mice (P10). (B) Double immunofluorescent staining showed that P-NR1-immunoreactivity and IL-1RI-immunoreactivity were totally double-labeled. Bar = 50 μm. All data were calculated as mean ± SEM (n = 10/group). *P < 0.05, **P < 0.01 vs. db/+ and C57 mice; ΔP < 0.05 vs. P10 db/db mice. db/db: homozygous type 2 diabetic db/db mice; db/+: heterozygous nondiabetic littermates; C57: C57BL6 control mice; LAA: L-α-aminoadipate; PF: pentoxifylline; IL-1ra: interleukin-1 receptor antagonist; P- NR1: P-ser896 NR1; IL-1RI: interleukin-1 receptor1; w: weeks.

pathological pathways all in turn converge in producing dramatical oxidative stress (Yasuda et al., 2003). Here we hypothesize that hyperglycemia-induced oxidative stress may be a key mechanism for the development of spinal astrocytic activation in db/db mice. In the present study, a systemic treatment with PBN (antioxidant for scavenging reactive oxygen species) significantly reduced GFAP overexpression, which suggested that oxidative stress may function as an initiator of astrocyte activation in db/db mice. In our future studies, we will also use blockers for these pathological pathways to further observe GFAP expression and mechanical allodynia. Clinical reports regarding the severity of DNP suggest a direct correlation between hyperglycemia and neuropathic pain, recommending strict glycemic control could become the mainstay of therapy (Bansal et al., 2006). Our finding showed that mechanical allodynia in db/db mice coincide with hyperglycemia, and a previous study elucidated that mechanical allodynia in ob/ob mice was readily reversed with insulin

therapy. In our future studies, we will also use hypoglycemic agents to elucidate the role of hyperglycemia in astrocytic activation in spinal dorsal horn of db/db mice.

3.3. “Astrocyte-IL-1β-NMDAR-neuron” pathway may be the detailed mechanism of astrocyte incudced allodynia While under physiological condition, astrocytes are primarily involved in providing energy sources and neurotransmitter precursors to neurons, cleaning up debris, regulating extracellular levels of ions and neurotransmitters (Volterra and Meldolesi, 2005). Astrocytes in this state only release very low levels of proinflammatory cytokines that is not sufficient to activate the neurons (Volterra and Meldolesi, 2005). However, under pathological condition, activated astrocytes could release numerous neuroexcitatory substances, including prostaglandins, IL-1, IL-6, and NO. As a sort of proinflammatory cytokines, interleukin-1 beta (IL-1β) is one of the most

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important factors which is also the focus for our literature searching (DeLeo and Yezierski, 2001; Watkins et al., 2003). Our results showed that spinal IL-1β expression was significantly increased in db/db mice compared to control mice. Intrathecally injected pentoxifylline (cytokine inhibitor) or IL-1ra (interleukin-1 receptor antagonist) could significantly attenuated the allodynia. On the other hand, the time course of IL-1β upregulation was similar to that of GFAP expression. IL-1β was selectively localized in astrocytes and intrathecally administered LAA could significantly down-regulate IL-1β expression. All these results strongly suggest that activated astrocytes are the only source of IL-1β release which contributed to mechanical allodynia in db/db mice. In support of our findings, previous studies reported selective localization of IL-1β in astrocytes in the spinal cord in inflammatory pain model and bone cancer pain model (Raghavendra et al., 2004; Zhang et al., 2005). Spinal NMDA receptor activation is prominently involved in induction and maintenance of persistent pain (Christoph et al., 2006; Woolf and Salter, 2000). Recent studies indicate that NMDA receptor activation mainly involves phosphorylation of the NR1 (a subunit of NMDA receptor) in the spinal dorsal horn, which is strongly correlated with behavioral hyperalgesia (Brenner et al., 2004; Ultenius et al., 2006). The present study showed that spinal P-NR1 expression was significantly increased in db/db mice compared to control mice. The time course of P-NR1 upregulation was similar to that of IL-1β and GFAP expression. Immunofluorescent staining showed that P-NR1-immunoreactivity was only localized in spinal dorsal horn neurons. Therefore, we hypothesized that IL-1β mediated allodynia may be through binding its receptor on the neurons, possibly via intracellular signaling pathways leading to the phosphorylation of NMDA receptor NR1 subunit. IL-1R, a subfamily of the Toll/IL-1 receptor superfamily, is the firstly binding receptor for IL-1β signaling. IL-1R contains two subtypes: the type I IL-1R (IL-1RI) is a transmembrane molecule and responsible for IL-1 signaling and the type II IL-1R lacks an intracellular domain and is incapable of signal transduction (Dayer, 2003). The present study showed that IL-1RI-immunoreactivity was only localized in spinal dorsal horn neurons. In support of our findings, a previous study also reported selective localization of IL-1RI in spinal dorsal horn neurons (Samad et al., 2001). Next, double immunofluorescent staining showed that IL-1RI-immunoreactivity and P-NR1-immunoreactivity were totally double-labeled, which supported a close interaction of inflammatory cytokine signaling with neuronal NMDA receptor. In subsequent investigations, data showed that intrathecally administered LAA, pentoxifylline or IL-1ra could block the upregulation of P-NR1. Thus, all the present results indicated that spinal activated astrocytes dramatically increased the expression of IL-1β which directly binds to its receptor IL-1RI to induce NMDA receptor phosphorylation in spinal dorsal horn neurons, and finally neuronal activity and pain transmission were enhanced. In conclusion, we were the first to provide evidence that spinal astrocytic activation contributed to mechanical allodynia in db/db mice of type 2 diabetes. The detailed mechanism in astrocyte induced allodynia may be that spinal activated astrocytes dramatically increased the expression of IL-1β which may induce NMDA receptor phosphorylation in spinal

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dorsal horn neurons to enhance neuronal activity and pain transmission (Fig. 6). IL-1β not only passively acts as the product of the upstream astrocytic activation, but also can enhance the downstream neuronal activation. Thus, spinal astrocytes and IL-1β signaling play significant roles in central hyperexcitability induced by type 2 diabetes. These findings will help to identify potential novel targets for clinical management of diabetic neuropathic pain.

4.

Experimental procedures

4.1.

Animals

Homozygous diabetic mice (db/db) and heterozygous nondiabetic littermates (db/+) of C57BLKS strain were purchased from Jackson Laboratory (stock number 000662; Bar Harbor, Maine, USA). Age-matched C57BL6 mice were obtained from the Laboratory Animal Center of Fourth Military Medical University (Xi'an, PR China). The db/db mice were used as a model of type 2 diabetes, while both db/+ mice and C57BL6 mice served as nondiabetic controls. Mice were housed under standard conditions (temperature 22 ± 2 °C, 12 h-light/dark cycle) with food and water available ad libitum. All procedures of our experiments were approved by the Committee of Animal Use for Research and Education of the Fourth Military Medical University, and all efforts were made to minimize the number of animals used and their suffering (Zimmermann, 1983).

4.2.

Blood glucose and body weight

Blood sample was obtained by tail prick and blood glucose was measured by using a glucose-oxidase test strip and reflectance

Fig. 6 – Schematic drawing showed the development of mechanical allodynia in type 2 diabetic db/db mice. In the situation of type 2 diabetes, various etiological factors such as hyperglycemia may induce oxidative stress which dramatically activates astrocytes in spinal cord. Cytokines like IL-1β are then released from astrocyte and act on spinal dorsal horn neurons to mediate pain facilitation.

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meter (Roche Diagnostics, Mannheim, Germany). Body weight was measured by an electronic weighing scale.

4.3.

Antibodies

Primary antibodies: mouse anti-GFAP IgG (astrocytic marker; Chemicon, Temecula, CA, USA), mouse anti-NeuN IgG (neuronal marker; Chemicon, Temecula, CA, USA), mouse anti-OX42 IgG (microglial marker; Chemicon, Temecula, CA, USA), rabbit anti-IL1β (Endogen, Rockford, IL, USA), rabbit anti-P-ser896 NR1 (Millipore, Bedford, MA, USA) and rat anti-IL-1RI IgG (Santa Cruz Biotechnology; Santa Cruz, CA, USA). Secondary antibodies: FITC-labeled donkey anti-mouse IgG (Chemicon, Temecula, CA, USA), cyanin 3 (Cy3)-labeled donkey anti-rabbit IgG (Chemicon, Temecula, CA, USA), Cy3labeled donkey anti-rat IgG (Chemicon, Temecula, CA, USA) and FITC-labeled donkey anti-rat IgG (Chemicon, Temecula, CA, USA).

4.4.

Drugs

Chemicals and their sources were as follows: L-α-aminoadipate (LAA, astrocytic specific inhibitor; Sigma, St. Louis, MO, USA), minocycline (microglial specific inhibitor; Sigma, St. Louis, MO, USA), pentoxifylline (cytokine inhibitor; Polfilin, Polfarma, Poland), interleukin-1 receptor antagonist (IL-1ra; Amgen, Thousand Oaks, CA, USA) and phenyl N-tert-butylnitrone (PBN, scavenger for reactive oxygen species; Sigma, St. Louis, MO, USA).

4.5.

the neck and atlanto-occipital membrane. Therefore, the mice were allowed to recover for 3 days before being used experimentally. Subsequently, saline, LAA or minocycline was dissolved in 3 μl of saline and administered intrathecally. After intrathecal drug injection, mechanical withdrawal threshold was immediately measured (n = 10/group). In the third series of experiments, db/db mice at postnatal 10 weeks were used. After intrathecal catheterization, the mice were allowed to recover for 3 days before being used experimentally. Firstly, saline, IL-1ra or pentoxifylline was dissolved in 3 μl of saline and administered intrathecally in db/db mice, and mechanical withdrawal threshold was immediately measured (n = 10/group). Secondly, LAA was dissolved in 3 μl of saline and administrated intrathecally in db/db mice, and 1 h later the fresh spinal cords were harvested for Western blot analysis of IL-1β (n = 10). Thirdly, db/db mice were perfused transcardially with paraformaldehyde and the L4–L5 spinal cord were cut into sections. Then double-labeling immunofluorescence of GFAP and IL-1β was performed in the sections. In the fourth series of experiments, db/db mice at postnatal 10 weeks were used. After intrathecal catheterization, the mice were allowed to recover for 3 days before being used experimentally. Firstly, LAA, pentoxifylline or IL-1ra was dissolved in 3 μl of saline and administered intrathecally in db/db mice, and 1 h later the fresh spinal cords were harvested for Western blot analysis of P-NR1 (n = 10/group). Secondly, db/db mice were perfused transcardially with paraformaldehyde and the L4–L5 spinal cord were cut into sections. Then double-labeling immunofluorescence of IL-1RI and P-NR1 was performed in the sections.

Experimental design 4.6.

In the first series of experiments, mice were divided into C57 group, db/+ group, db/db group and db/db + PBN (1.5 mg/kg/ day, i.p.) group. At postnatal different time points (per week), blood glucose, body weight and mechanical allodynia were detected in each group. At postnatal different week for immunostaining study of GFAP or OX42, mice from each group were anesthetized and perfused transcardially with paraformaldehyde. The L4–L5 spinal cord segments were removed and cut into 20 μm thick sections. Then the sections were incubated with corresponding antibodies. At postnatal different week for Western blot study, mice from each group were anesthetized and the L4–L5 spinal cord segments were rapidly removed. The collected tissue was mechanically homogenized and then centrifuged. The supernatant was collected and used for Western blot analysis of GFAP, OX42, IL1β or P-NR1. In the second series of experiments, intrathecal catheters were placed in db/db mice at postnatal 10 weeks, when the mechanical allodynia reached the highest level. The length of the catheter used in this study was 2.0 cm and the dead volume inside the catheter was about 0.5 μl. The intrathecal catheter placement was acute. Under halothane anesthesia, a small incision was made at the back of the neck and a small puncture was made in the atlanto-occipital membrane of the cisterna magna. The catheter was immediately intrathecally inserted. The catheter was flushed with 1 μl of saline and the wound was closed with sutures. Although it only took us about 5 min to complete the above procedure, there were injuries in the back of

Intrathecal catheter insertion and drug administration

The procedure of intrathecal catheterization in this study was based on modifications of previous methods (Storkson et al., 1996; Wu et al., 2004). The length of the catheter used in this study was 2.0 cm and the dead volume inside the catheter was about 0.5 μl. Polyethylene tube, PE 10 (Clay Adams, Becton Dickinson Co., MD, USA) with outer diameter 0.6 mm is widely used in intrathecal catheterization for rats. In this study, the PE 10 catheter was slowly stretched to about 0.2 mm in outer diameter after heating. Saline was injected through the thin catheter to check that there was no occlusion and leakage. This thin catheter was used to make intrathecal catheterization for mice so as to avoid compression injury to the spinal cord of mice. Under halothane anesthesia, mice were placed prone in a stereotaxic frame and a small incision was made at the back of the neck. A small puncture was made in the atlanto-occipital membrane of the cisterna magna and the catheter was intrathecally inserted so that the caudal tip reached subarachnoid space of the lumbar enlargement of the spinal cord. The catheter was flushed with 1 μl of saline and the wound was closed with sutures. The mice were allowed to recover for 3 days before being used experimentally. At the experimental day, only the mice judged as neurologically normal and that showed complete paralysis of the bilateral hind legs after intrathecal administration of 2% lidocaine (1 μl) were used for the drug administration. After intrathecal injection of lidocaine, motor paralysis of the hindlimbs occured within 15 s and lasted for no more than 20 min. Four hours later, we performed the drug

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administration. LAA, minocycline, pentoxifylline and IL-1ra were dissolved in 3 μl of saline. Treatment group received intrathecal injection of LAA (10 nmol), minocycline (10 nmol), pentoxifylline (15 nmol) or IL-1ra (20 nmol), while the same volume of saline was injected in control group. For each injection, the solution was slowly injected over 3 min. Finally, the injection was followed by a flush of 1 μl of saline.

4.7.

Pain behavioral test

For the behavioral tests, the same room in which the mice were routinely housed was used. The mice were adapted to the testing situation for at least 15 min before stimulation was initiated. The observers of the behaviors were blind to the treatment of the mice. As previously described (Hao et al., 1999), a set of von Frey monofilaments (Stoelting, Chicago, IL, USA) was used to test the mechanical withdrawal threshold of the hindpaws. The monofilaments were applied with increasing force until the mice withdrew the paw. Lifting of the paw due to normal locomotory behavior was ignored. Each monofilament was applied five times. The threshold was taken as the lowest force that evoked a brisk withdrawal response to one of the five repetitive stimuli. The withdrawal thresholds were measured three or four times in order to obtain two consecutive values that differed not more than 10%. The average of these two values was taken for each mouse. To observe how different drug treatments affected the allodynia, behavioral tests were performed 12 h before the drugs administration to provide baseline scores.

4.8.

Immunofluorescence histochemical staining

4.8.1.

Tissue preparation

Mice were anesthetized with an overdose of sodium pentobarbital (100 mg/kg body weight, i.p.) and were perfused transcardially with 10 ml of 0.9% (w/v) saline, followed by 50 ml of 4% (w/v) paraformaldehyde and 0.2% (w/v) picric acid in 0.1 M phosphate buffer (PB, pH 7.4). The L4–L5 spinal cord segments were removed and postfixed in the same fixative and then transferred to 30% sucrose in 0.1 M PB for cryoprotection. Spinal cord sections were cut at 20 μm on a cryostat and collected in 0.01 M phosphate-buffered saline (PBS, pH 7.4). After being pre-treated with 10% normal goat serum (NGS), sections were incubated with corresponding antibodies.

4.8.2.

Single immunofluorescence

After washed in PBS containing 0.3% Triton X-100 (PBS-X, pH 7.4), the sections were incubated sequentially with: (1) mouse anti-GFAP IgG (1:500) or mouse anti-OX42 IgG (1:200) in 0.0l M PBS containing 5% (v/v) normal donkey serum (NDS), 0.3% (v/v) Triton X-100, 0.05% (w/v) NaN3 and 0.25% (w/v) carrageenan (PBS-NDS, pH 7.4) for 48 h at 4 °C; (2) FITC-labeled donkey antimouse IgG (1:200) in PBS-NDS for 12 h at 4 °C.

4.8.3.

Double immunofluorescence

After washing in PBS-X, the sections were incubated sequentially with: (1) rabbit anti-IL-1β IgG (1:300), rabbit anti-P-ser896 NR1 IgG (1:500) and rat anti-IL-1RI IgG (1:600) were respectively double labelled with mouse anti-GFAP IgG (1:500), mouse anti-

333

NeuN IgG (1:1000) or mouse anti-OX42 IgG (1:200) in PBS-NDS for 48 h at 4 °C; (2) a mixture of FITC-labeled donkey anti-mouse IgG (1:200) with either cyanin 3 (Cy3)-labeled donkey anti-rabbit IgG (1:200) or Cy3-labeled donkey anti-rat IgG (1:200) in PBS-NDS for 12 h at 4 °C. Also, rabbit anti-P-ser896 NR1 IgG (1:500) was double labelled with rat anti-IL-1RI IgG (1:600). The second antibodies were cyanin 3 (Cy3)-labeled donkey anti-rabbit IgG (1:200) and FITC-labeled donkey anti-rat IgG (1:200). Between each step, the sections were washed with PBS for three times. After staining, the sections were then rinsed in PBS, air dried, coverslipped with a mixture of 50% (v/v) glycerin and 2.5% (w/v) triethylene diamine (anti-fading agent) in PBS, and observed with a confocal laser scanning microscope (Olympus FV1000, Tokyo, Japan) under appropriate filters for greenemitting FITC (excitation 490 nm; emission 520 nm) and for red-emitting Cy3 (excitation 552 nm; emission 565 nm).

4.9.

Western blot analysis

Mice were anesthetized with an overdose of pentobarbital (100 mg/kg), and the dorsal halves of the spinal cord innervated by the L4/5 dorsal roots were rapidly removed. The collected tissue was mechanically homogenized in ice-cold 0.05 M Tris-buffered saline (TBS) containing 40 mM Tris–HCl (pH 7.5), 2% SDS, 2 mg/ml aprotinin, 2 mg/ml antipain, 2 mg/ ml chymostatin, 2 mg/ml bestatin, 2 mg/ml pepstatin A, 2 mg/ ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 1 mM EDTA. Homogenates were centrifuged at 10,000g for 10 min. The supernatant was collected and stored at −80 °C. Protein concentrations of the homogenate were determined using the BCA Protein Assay Kit (Pierce, Rockford, IL, USA). Proteins of interest were separated by SDSPAGE electrophoresis (20 μg of total protein per well), and transferred onto nitrocellulose membranes. The membranes were placed in a blocking solution (TBS with 0.02% Tween and 5% non-fat dry milk powder) for 1 h, and incubated overnight with mouse anti-GFAP IgG (1:500), mouse anti-OX42 IgG (1:200), rabbit anti-IL-1β IgG (1:300) or rabbit anti-P-ser896 NR1 IgG (1:500). After washing, the membranes were incubated in peroxidase-conjugated secondary antibody (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h, and then the membranes were detected by the enhanced chemiluminescence detection method (Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA). The concentrations of β-actin, a housekeeping protein, were also measured by using rabbit anti-β-actin antibody (A2066, 1:5000 dilution; Sigma, St.Louis, MO, USA) as an intra control. The densities of protein blots were analyzed by using Labworks Software (Ultra-Violet Products Ltd., Cambridge, UK) and normalized to β-actin levels.

4.10.

Data analysis

The results were presented as mean ± SEM. Statistical analysis of the data was carried out with a one-way analysis of variance (ANOVA) followed by Bonferroni post hoc analysis. Comparisons between two means were performed by a Student's t-test. Significance level was set at P < 0.05. The statistics software used was SPSS 12.0 for Windows.

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Conflicts of interest statement The authors had no conflicts of interest to declare in relation to this article.

Acknowledgments The study was supported by the National Natural Science Foundation of China (Nos. 30772102, 30772094).

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