| Diabetic neuropathic pain (DNP) plays a major role in decreased life quality of type 2 diabetes patients, however, the molecular mechanisms underlying DNP remain unclear. Emerging 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 conditions and whether they contribute to diabetes-induced neuropathic pain. In the present study, 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 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)-1βexpression 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.Objective: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. As the incidence of type 2 diabetes increasing, the morbidity and mortality rates of DNP and other complications of diabetes are more and more higher。DNP cannot yet be treated effectively, as it often remains refractory to classical analgesics such as opioid analgesics. And 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. In this study, type 2 diabetic mice as a model to study the mechanism of mechanical hyperalgesia, for the treatment of DNP to find a new breakthrough.Methods:1. 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, mice from each group were anesthetized and perfused transcardially with paraformaldehyde. The L4–L5 spinal cord segments were removed and cut into 20μm 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, IL-1βor P-NR1.2. 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 0.5μl. The intrathecal catheter placement was acute. Under halothane anesthesia, a small incision wasmade 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 catheterwas flushedwith 1μl of saline and thewound was closed with sutures. Although it only took us about 5 min to complete the above procedure, therewere injuries in the back of 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 3μl of saline and administered intrathecally. After intrathecal drug injection, mechanical withdrawal threshold was immediately measured (n=10/group).3. 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 pentoxifyllinewas dissolved in 3μl of saline and administered intrathecally in db/db mice, and mechanical withdrawal threshold was immediately measured (n=10/group). Secondly, LAA was dissolved in 3μl 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-1βwas performed in the sections.4. 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 3μl 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. Results:1. Mechanical allodynia occurred in type 2 diabetic mice Blood glucose and bodyweightwere 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±0.8mmol/L; P6) and C57 mice (5.9±0.7mmol/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±2.8mmol/L), peaked at P8 (26.4±5.6mmol/L), and thereafter maintained at high level (P<0.05). The body weight was also significantly higher in db/db mice beginning at P4 (26±5.2g), and was persistently elevated through the period tested. By P16, the weights of db/db mice (65±7.2g) were almost twice those of db/+ (30±3.6g) and C57 mice (33±2.9g) (P<0.05). No difference in paw withdrawal threshold was observed between db/+ mice (20±1.6g; P6) and C57 mice (20±1.1g; P6), and the paw withdrawal thresholdof these two groupsmaintained atnormal level. The pawwithdrawal thresholdwas significantly lower for db/db mice beginning at P6 (12.3±2.8g), suggesting increased sensitivity to mechanical stimuli. Then the mechanical allodynia peaked at P8 (7.4±1.9g) and continued thereafter (P<0.05). Thus, these data indicated that db/db mice developed features of type 2 diabetes, and significantmechanical allodynia in db/db mice coincided with hyperglycemia.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 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/dbmice 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. UsingWestern 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 andC57mice (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. 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. 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. 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. 3. Spinal astrocytes dramatically increased the expression of IL-1βwhich is related to mechanical allodynia Western blot analysis showed that no significant difference in IL-1βexpression in spinal cord was observed between db/+ mice (0.028±0.007; P6) and C57 mice (0.022±0.005; P6), and IL-1βexpression of these two groups maintained at the same level through the period tested. However, compared to db/+ mice and C57 mice, IL-1βexpression was significantly increased in db/db mice (0.13±0.03) at P6 (P<0.05). IL-1βupregulation peaked at P8 (0.2±0.04), and persisted thereafter, which was similar to the time course of GFAP expression. At P10 in db/db mice, we injected pentoxifylline (cytokine inhibitor) or IL-1ra (interleukin-1 receptor antagonist) intrathecally and observed their effects on mechanical allodynia in db/db mice. Both pentoxifylline and IL-1ra could significantly attenuate the allodynia. At P10 in db/db mice, intrathecally administered LAA could significantly down-regulate IL-1βexpression (0.05±0.009). Subsequent double immunofluorescent staining showed that IL-1β-immunoreactivity was only localized in GFAP-immunopositive cells.4. IL-1βinduces 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 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-1βand GFAP expression. At P10 in db/db mice, intrathecally administered LAA, pentoxifylline or IL-1ra could significantly down-regulate P-NR1 expression. Subsequent double immunofluorescent staining showed that P-NR1-immunoreactivity and IL-1RI immunoreactivity were totally double-labeled.Conclusion: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-1βwhich may induce NMDA receptor phosphorylation in spinal dorsal horn neurons to enhance neuronal activity and pain transmission. IL-1βnot only passively acts as the product of the upstream astrocytic activation, but also can enhance the downstream neuronal activation. Thus, spinal astrocytes and IL-1βsignaling 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.
|
PDF Full Text Request