Cannabinoid-mediated modulation of neuropathic pain and microglial accumulation in a model of murine type I diabetic peripheral neuropathic pain

Abstract

Background: Despite the frequency of diabetes mellitus and its relationship to diabetic peripheral neuropathy (DPN) and neuropathic pain (NeP), our understanding of underlying mechanisms leading to chronic pain in diabetes remains poor. Recent evidence has demonstated a prominent role of microglial cells in neuropathic pain states. One potential therapeutic option gaining clinical acceptance is the cannabinoids, for which cannabinoid receptors (CB) are expressed on neurons and microglia. We studied the accumulation and activation of spinal and thalamic microglia in streptozotocin (STZ)-diabetic CD1 mice and the impact of cannabinoid receptor agonism/antagonism during the development of a chronic NeP state. We provided either intranasal or intraperitoneal cannabinoid agonists/antagonists at multiple doses both at the initiation of diabetes as well as after establishment of diabetes and its related NeP state.

Results: Tactile allodynia and thermal hypersensitivity were observed over 8 months in diabetic mice without intervention. Microglial density increases were seen in the dorsal spinal cord and in thalamic nuclei and were accompanied by elevation of phosphorylated p38 MAPK, a marker of microglial activation. When initiated coincidentally with diabetes, moderate-high doses of intranasal cannabidiol (cannaboid receptor 2 agonist) and intraperitoneal cannabidiol attenuated the development of an NeP state, even after their discontinuation and without modification of the diabetic state. Cannabidiol was also associated with restriction in elevation of microglial density in the dorsal spinal cord and elevation in phosphorylated p38 MAPK. When initiated in an established DPN NeP state, both CB1 and CB2 agonists demonstrated an antinociceptive effect until their discontinuation. There were no pronociceptive effects demonstated for either CB1 or CB2 antagonists.

Conclusions: The prevention of microglial accumulation and activation in the dorsal spinal cord was associated with limited development of a neuropathic pain state. Cannabinoids demonstrated antinociceptive effects in this mouse model of DPN. These results suggest that such interventions may also benefit humans with DPN, and their early introduction may also modify the development of the NeP state.

Figure 1

Timelines for experiments performed. Experiment 1 examined the natural history of nociception and microglial presence over 8 months of diabetes. Experiment 2 examined the intranasal and intraperitonel delivery of CB2 agonists/antagonists either at diabetes initiation or after diabetic peripheral neuropathy-mediated neuropathic pain has initiated. Experiment 3 examined the intranasal and intraperitonel delivery of CB1 agonists/antagonists (as well as one CB1 and CB2 agonist) either at diabetes initiation or after diabetic peripheral neuropathy-mediated neuropathic pain has initiated.

Figure 2

Tactile (A) and thermal (B) sensory testing data mice with or without diabetes. Significant differences were detected between the diabetic mouse and non-diabetic mouse groups (non-matched ANOVA tests, F-values range between 6.18-9.72 for indicated groups and time points, n ≥ 5, p < 0.05). Area under the curve (AUC) measurements were also significantly different between for the first 21 weeks of study (p < 0.05) [n = 5-10 mice in each mouse cohort for each time point]. Diabetic mice have a significant loss of sensory nerve action potential (SNAP) amplitudes (C) and sensory nerve conduction velocity (SNCV) (D) when compared to the non-diabetic mouse cohorts (non-matched ANOVA tests, F-values range between 2.13-5.88 for indicated groups and time points, n ≥ 5, p < 0.05) after 2-3 months of diabetes [n = 4-6 mice in each mouse cohort for each time point].

Figure 3

During 8 months of diabetes, Western blotting (A) identified mild increasing levels of Iba1 (B), beginning at 3 months of diabetes, when compared to non-diabetic littermates. As well, phosphorylated p38 MAPK elevates during diabetes until 5 months of time (C). Sample protein blots are demonstrated in A from a total of 3 sample blots identified for each marker and each time point. Multiple ANOVA tests were performed in each case, with * indicating significant difference (p < 0.05) between diabetic and non-diabetic cohorts.

Figure 4

Fluroescent labeled cannabidiol (A) was visible within diabetic mouse thalamus 6 hours after delivery, with saline intervention leading to no comparable fluorescence (B). Fluroescent labeled nabilone (C) was also visible within diabetic dorsal spinal cord 6 hours after delivery, with saline intervention leading to no comparable fluorescence (D). Bar = 10 μm

Figure 5

Tactile (A) and thermal (B) sensory testing data for diabetic mice receiving either intranasal or intraperitoneal cannabidiol at low, medium, or high dose, with comparison to non-diabetic mice and saline delivery. Diabetic mice receiving medium or high doses of intranasal or intraperitoneal cannabidiol had amelioration of both tactile allodynia and thermal hyperalgesia after 7 weeks when nociceptive behaviors began. This protection against the development of the neuropathic pain state was also noted continually after the stoppage of cannabidiol at week 14. For both tactile (A) and thermal (B) testing, significant differences were detected between the diabetic mouse group receiving medium (γ) or high doses (θ) of intranasal cannabidiol or high (Ψ) doses of intraperiteonal cannabidiol when compared to the diabetic mouse group receiving low dose intranasal or intraperitoneal cannabidiol respectively (non-matched ANOVA tests, F-values range between 0.88-4.13 for indicated groups and time points, n ≥ 4, p < 0.05). Area under the curve (AUC) measurements were also significantly different between the same comparison groups in each case (p < 0.05). [n = 4-10 mice in each mouse cohort for each time point]

Figure 6

Tactile (A) and thermal (B) sensory testing data for diabetic mice receiving either intranasal or intraperitoneal SR144528 at low, medium, or high dose, with comparison to non-diabetic mice and saline delivery. There were no effects upon either tactile allodynia or thermal hyperalgesia development or maintenance in any group at any dose (non-matched ANOVA tests, p NS). [n = 4-10 mice in each mouse cohort for each time point].

Figure 7

Tactile (A) and thermal (B) sensory testing data for mice with established diabetes receiving either intranasal or intraperitoneal cannabidiol or SR144528 at low high dose, with comparison to non-diabetic mice receiving intranasal or intraperitoneal saline. There were no effects upon either tactile allodynia or thermal hyperalgesia development or maintenance in any group at any dose (non-matched ANOVA tests, p NS). [n = 4-10 mice in each mouse cohort for each time point] despite early intervention studies for cannabidiol demonstrating protection against development and maintenance of neuropathic pain.

Figure 8

After several months of diabetes, microglia accumulation can be noted within dorsal spinal cord and to a lesser degree within thalamic nuclei. Compared to non-diabetic age-matched mice (A), immunohistochemistry identified a mild but significant accumulation of microglia within the thalamus after 5 months of diabetes (B). Microglial accumulation and activation (evaluated qualitatively) was more substantial in the dorsal spinal cord of diabetic mice. Treatment with intranasal high dose cannabidiol (C) attenuated the microglial accumulation identified in age-matched diabetic mice reciving no intervention (D) or receiving intranasal (E) or intraperitoneal (F) WIN55212-2 (see Tables 2 and 3). Bar = 50 μm

Figure 9

Tactile (A) and thermal (B) sensory testing data for diabetic mice receiving either intranasal or intraperitoneal nabilone at low, medium, or high dose, with comparison to non-diabetic mice and saline delivery is shown. Those diabetic mice receiving medium or high doses of intranasal or intraperitoneal nabilone had amelioration of both tactile allodynia and thermal hyperalgesia beginning at week 7 when nociceptive behaviors began. This protection against the development of the neuropathic pain state disappeared after discontinuation of nabilone at week 14. Tactile (C) and thermal (D) sensory testing data for mice with diabetes receiving either intranasal or intraperitoneal WIN55212-2 at low, medium, or high dose, with comparison to non-diabetic mice receiving intranasal or intraperitoneal saline is also demonstrated. Those diabetic mice receiving high doses of intranasal or intraperitoneal WIN55212-2 had amelioration of tactile allodynia beginning at week 7 when nociceptive behaviors began, while thermal hyperalgesia was modulated by either medium or high doses of intranasal or intraperitoneal WIN55212-2. This protection against the development of the neuropathic pain state disappeared after discontinuation of nabilone at week 14, after which all diabetic mouse cohorts had comparable levels of tactile allodynia and thermal hyperalgesia. For both tactile (A, C) and thermal (B, D) testing, significant differences were detected between the diabetic mouse group receiving medium (γ) or high doses (θ) of intranasal nabilone/WIN55212-2 or medium (φ) high (Ψ) doses of intraperiteonal nabilone/WIN55212-2 when compared to the diabetic mouse group receiving low dose intranasal or intraperitoneal nabilone/WIN55212-2 respectively (non-matched ANOVA tests, F-values range between 0.76-3.07 for indicated groups and time points, n ≥ 4, p < 0.05). Area under the curve (AUC) measurements were also significantly different in each case (p < 0.05) for weeks 1-13, but not for assessment of tactile allodynia in mice receiving any dose/route for WIN55212-2 (C). [n = 4-10 mice in each mouse cohort for each time point]

Figure 10

Tactile (A) and thermal (B) sensory testing data for mice with established diabetes receiving either intranasal or intraperitoneal cannabinoid agents, with comparison to non-diabetic mice and saline delivery. Diabetic mice receiving medium and high doses of intranasal nabilone or WIN55212-2 had improvement of thermal hyperalgesia +/- tactile allodynia beginning at week 13 when interventions began. Stoppage of cannabinoid agents saw resumption of the full neuropathic pain state at week 21. For both tactile (A) and thermal (B) testing, significant differences were detected between the diabetic mouse group receiving moderate (γ) or high doses (θ) of intranasal cannabidiol or medium (χ) or high (τ) doses of intraperiteonal cannabidiol when compared to the diabetic mouse group receiving low dose intranasal or intraperitoneal cannabidiol at outset respectively (non-matched ANOVA tests, F-values range between 0.95-2.85 for indicated groups and time points, n ≥ 4, p < 0.05). Area under the curve (AUC) measurements were also significantly different between the same comparison groups in each case (p < 0.05). [n = 4-10 mice in each mouse cohort for each time point]

Figure 11

During 5 months of diabetes, Western blotting (A) identified mild increased levels of Iba1 (B) in diabetic mice. As well, phosphorylated p38 MAPK was elevated during diabetes at 5 months of time (C). Sample protein blots are demonstrated in A (total of 3 sample blots identified for each marker and each intervention at the final time point). Only intranasal or intraperitoneal cannabidiol administered at the onset of diabetes was associated with suppression of Iba1 and phosphorylated p38 MAPK levels at endpoint (B, C). Multiple ANOVA tests were performed in each case, with * indicating significant difference (p < 0.05) between diabetic cohorts receiving intranasal cannabidiol and no intervention after 5 months of diabetes.