Differential Efficacy of Ketamine in the Acute versus Chronic Stages of Complex Regional Pain Syndrome in Mice

Abstract

Background: Complex regional pain syndrome (CRPS) is a painful, disabling, and often chronic condition, where many patients transition from an acute phase with prominent peripheral neurogenic inflammation to a chronic phase with evident central nervous system changes. Ketamine is a centrally acting agent believed to work through blockade of N-methyl-D- aspartate receptors and is being increasingly used for the treatment of refractory CRPS, although the basis for the drug's effects and efficacy at different stages of the syndrome remains unclear.

Methods: The authors used a mouse model of CRPS (n = 8 to 12/group) involving tibia fracture/cast immobilization to test the efficacy of ketamine (2 mg kg day; 7 days) or vehicle infusion during acute (3 weeks after fracture) and chronic (7 weeks after fracture) stages.

Results: Acute-phase fracture mice displayed increased limb temperature, edema, and nociceptive sensitization that were not reduced by ketamine. Fracture mice treated with ketamine during the chronic phase showed reduced nociceptive sensitization that persisted beyond completion of the infusion. During this chronic phase, ketamine also reduced latent nociceptive sensitization and improved motor function at 18 weeks after fracture. No side effects of the infusions were identified. These behavioral changes were associated with altered spinal astrocyte activation and expression of pain-related proteins including N-methyl-D-aspartate receptor 2b, Ca/calmodulin-dependent protein kinase II, and brain-derived neurotrophic factor.

Conclusions: Collectively, these results demonstrate that ketamine is efficacious in the chronic, but not acute, stage of CRPS, suggesting that the centrally acting drug is relatively ineffective in early CRPS when peripheral mechanisms are more critical for supporting nociceptive sensitization.

Fig. 1. Summary of experimental design

Timeline of surgical/pharmacological interventions and behavioral/biochemical measurements when ketamine was administered at 3 weeks (1 cohort of mice, A) or 7 weeks (4 cohorts, B) post fracture. Fx=fracture; C=control; W=week; K=ketamine; S=saline.

Fig. 2. Physiological and behavioral changes in complex regional pain syndrome (CRPS) mice

CRPS mice display increased edema (A) and temperature (B) on the affected hindpaw at 3, but not 7, weeks post-fracture. In addition, they show signs of mechanical allodynia in the ipsilateral (Ipsi, 3- and 7-week timepoints, C) and contralateral (Contra, 3-week timepoint, D) hindpaw. *p<0.05, *** p<0.001. n=16 mice for each of the 4 groups.

Fig. 3. Ketamine is efficacious when administered at the late, but not early, phase of complex regional pain syndrome

Ketamine shows no efficacy in reversing mechanical allodynia when administered 3 weeks after fracture (A, B). In contrast, ketamine reverses mechanical allodynia on the ipsilateral (Ipsi) hindpaw when administered at the 7-week timepoint (C, D). No changes were observed in the contralateral (Contra) hindpaw. *p<0.05, ** p<0.005, *** p<0.001 compared to the Fracture+Saline group. #p<0.05, ## p<0.005, ### p<0.001 compared to the Control+Saline group. Panels A and B: n= 6 mice for Control+Saline, n= 6 mice for Contol+Ketamine, n= 8 mice for Fracture+Saline, and n= 11mice for Fracture+Ketamine. Panels C and D: n= 8 mice for Control+Saline, n= 8 mice for Contol+Ketamine, n= 6 mice for Fracture+Saline, and n= 9 mice for Fracture+Ketamine. The red lines indicate the duration of ketamine administration.

Fig. 4. Ketamine exposure improves rotarod performance 18 weeks after fracture

3 different cohorts were used to measure initial rotarod performance at 3 different timepoints. Fracture animals show no obvious impairment in the rotarod assay at the 7- (A) and 8- (B) week timepoints, however, they do show impairment at the 18-week timepoint, which can be ameliorated by ketamine administration (C). Subsequent training in fracture/cast mice showed no statistically-significant differences between the ketamine- and saline-treated groups (D–F). *p<0.05, *** p<0.001. Panels A and D: n= 10 mice for Control+Saline, n= 8 mice for Contol+Ketamine, n= 8 mice for Fracture+Saline, and n= 8mice for Fracture+Ketamine. Panels B and E: n= 6 mice for each of the 4 groups. Panels C and F: n= 8 mice for Control+Saline, n= 8 mice for Contol+Ketamine, n= 6 mice for Fracture+Saline, and n= 12 mice for Fracture+Ketamine.

Fig. 5. Ketamine exposure improves capsaicin-induced hypersensitivity 18 weeks after fracture

18 weeks after injury, fracture mice spend more time engaging in capsaicin-evoked behaviors (licking, biting, scratching, and shaking) compared to control mice (A, B, red arrows, p<0.001). This is indicative of persistent hindpaw sensitization in the absence of notable differences in mechanical thresholds. Furthermore, the ketamine-treated group displayed less capsaicin-evoked behaviors when compared to the saline-treated group (B). No changes were observed in mechanical thresholds one hour following capsaicin or vehicle treatments in the ipsilateral (Ipsi, C, D) and contralateral (Contra, E, F) hindpaws. *** p<0.001. n= 8 mice for Control+Saline, n= 8 mice for Contol+Ketamine, n= 6 mice for Fracture+Saline, and n= 8 mice for Fracture+Ketamine.

Fig. 6. No side-effects were observed during chronic ketamine administration

No changes in body weight (A), stereotypic behaviors (B) and activity levels in the home cage (C) were observed between ketamine- and saline-treated groups 5 days after ketamine exposure (at 7 weeks post-fracture). n= 7 mice for Control+Saline, n= 8 mice for Contol+Ketamine, n= 6 mice for Fracture+Saline, and n= 12 mice for Fracture+Ketamine.

Fig. 7. Ketamine exposure ameliorates complex regional pain syndrome-related upregulation of spinal astrocytes

Immunohistochemical staining for GFAP shows that, in comparison with control animals, fracture mice exhibit increased numbers of astrocytes in the dorsal horn of the ipsilateral spinal cord 10 weeks after fracture. This increase is absent in the ketamine treated group (A). In contrast, no changes were seen in the number of Iba1⁺ cells at this timepoint (B). Examples of GFAP- and Iba1-stained sections are included in panels C–F. The areas enclosed by the dotted squares are further enlarged in the central panels. Scale bar = 100 μm *p<0.05. Sample sizes are shown in the scatter graph. GFAP= Glial fibrillary acidic protein, Iba1= Ionized calcium binding adaptor molecule 1, Ctl/Sal=Control + Saline, Ctl/Ket= Control + Ketamine, Fx/Sal= Fracture + Saline, Fx/Ket= Fracture + Ketamine.

Fig. 8. Ketamine exposure is accompanied by biochemical changes in the lumbar spinal cord

10 weeks following fracture, our data shows a decrease in the protein levels of NR2b in the fracture group, with ketamine exposure being associated with increased NR2b in both the fracture and control groups (A). Additionally, we show an increase in CAMK2 in the fracture group, with ketamine exposure linked to increased CAMK2 levels in both the fracture and control groups (B). No significant differences in synaptophysin levels were observed among the 4 groups (C). Finally, ketamine administration was accompanied by a decrease in BDNF levels in the fracture group only (D). *p<0.05, ** p<0.005, *** p<0.001. Sample sizes are shown in the scatter graph. NR2b= N-methyl-D-aspartate receptor 2b, CaMK2= Ca2+/calmodulin-dependent protein kinase ii, SYP= Synaptophysin, BDNF= Brain derived neurotrophic factor.

Fig. 9. Summary

Illustration of potential nociceptive mechanisms observed in the acute (A) vs. chronic (B) stages of complex regional pain syndrome in the fracture/cast rodent model. * indicates data shown in the current manuscript. CCL2= chemokine (C-C motif) ligand 2, COX2= Cytochrome c oxidase subunit 2, NALP1= NAcht Leucine-rich repeat Protein 1, TNFα= Tumor necrosis factor alpha, IL1β= Interleukin 1 beta, IL6= Interleukin 6, NGF= Nerve growth factor, IgM= Immunoglobulin M, CaMK2= Ca2+/calmodulin-dependent protein kinase ii, NR2b= N-methyl-D-aspartate receptor 2b, SYP= Synaptophysin, BDNF= Brain derived neurotrophic factor.