Δ9-tetrahydrocannabinol prevents methamphetamine-induced neurotoxicity

Abstract

Methamphetamine (METH) is a potent psychostimulant with neurotoxic properties. Heavy use increases the activation of neuronal nitric oxide synthase (nNOS), production of peroxynitrites, microglia stimulation, and induces hyperthermia and anorectic effects. Most METH recreational users also consume cannabis. Preclinical studies have shown that natural (Δ9-tetrahydrocannabinol, Δ9-THC) and synthetic cannabinoid CB1 and CB2 receptor agonists exert neuroprotective effects on different models of cerebral damage. Here, we investigated the neuroprotective effect of Δ9-THC on METH-induced neurotoxicity by examining its ability to reduce astrocyte activation and nNOS overexpression in selected brain areas. Rats exposed to a METH neurotoxic regimen (4 × 10 mg/kg, 2 hours apart) were pre- or post-treated with Δ9-THC (1 or 3 mg/kg) and sacrificed 3 days after the last METH administration. Semi-quantitative immunohistochemistry was performed using antibodies against nNOS and Glial Fibrillary Acidic Protein (GFAP). Results showed that, as compared to corresponding controls (i) METH-induced nNOS overexpression in the caudate-putamen (CPu) was significantly attenuated by pre- and post-treatment with both doses of Δ9-THC (-19% and -28% for 1 mg/kg pre- and post-treated animals; -25% and -21% for 3 mg/kg pre- and post-treated animals); (ii) METH-induced GFAP-immunoreactivity (IR) was significantly reduced in the CPu by post-treatment with 1 mg/kg Δ9-THC1 (-50%) and by pre-treatment with 3 mg/kg Δ9-THC (-53%); (iii) METH-induced GFAP-IR was significantly decreased in the prefrontal cortex (PFC) by pre- and post-treatment with both doses of Δ9-THC (-34% and -47% for 1 mg/kg pre- and post-treated animals; -37% and -29% for 3 mg/kg pre- and post-treated animals). The cannabinoid CB1 receptor antagonist SR141716A attenuated METH-induced nNOS overexpression in the CPu, but failed to counteract the Δ9-THC-mediated reduction of METH-induced GFAP-IR both in the PFC and CPu. Our results indicate that Δ9-THC reduces METH-induced brain damage via inhibition of nNOS expression and astrocyte activation through CB1-dependent and independent mechanisms, respectively.

Figure 1. Synopsis of the experimental design, including treatment schedule and IHC assays.

A. Pre-treatment: rats received injections of Δ9-THC (1 or 3 mg/kg) or vehicle (VEH) 30 min before each METH or SAL injection, and 3 days (3d) after the last METH or SAL injection were perfused and used for IHC analysis. B. Post-treatment: rats received injections of Δ9-THC (1 or 3 mg/kg) or vehicle (VEH) 0.5, 12, 24, 36 and 48 h after the last METH or SAL administration, and 3 days (3d) after the last METH injection were perfused and used for IHC analysis. C. Post-treatment + SR treatment: rats received injection of SR (1 mg/kg, i.p.) or VEH 15 min prior each Δ9-THC (1 mg/kg) or VEH post-treatment injection, and 3 days (3d) after the last METH or SAL injection were perfused and used for IHC analysis. 0, 2 h, 4 h, 6 h: 1st, 2nd, 3rd and 4th injection of METH (10 mg/kg, s.c.) or SAL; IHC: immunohistochemistry; SR: SR141716A; VEH: vehicle.

Figure 2. Core body temperature: effect of methamphetamine (METH) in the presence and absence of Δ9-THC (1 and 3 mg/kg).

Rats were given SAL (1 mL/kg) or METH (4×10 mg/kg s.c., every 2 h) with and without Δ9-THC (1 and 3 mg/kg) pre-treatment. Body temperature was measured prior to and 1 h after each METH injection. Values are expressed as means ± SEM. Arrows indicate each injection of METH or SAL. No difference in baseline temperature was detected among groups. METH administration resulted in a significant increase in rectal temperature over time in comparison with SAL-treated rats. Both doses of Δ9-THC did not significantly change rectal temperature in METH-administered rats at any time point. METH: *p<0.05, **p<0.01 and ***p<0.001 vs corresponding SAL group at each time point. Δ9-THC1-METH: ^##p<0.01 and ^###p<0.001 vs corresponding Δ9-THC1-SAL group at each time point; Δ9-THC3-METH: ⁺p<0.05 and ⁺⁺⁺p<0.001 vs corresponding Δ9-THC 3-SAL group at each time point.

Figure 3. METH increases the number of neuronal nitric oxide synthase (nNOS) neurons and GFAP-immunoreactivity (IR).

Values represent means ± SEM of either number of nNOS positive neurons, expressed per mm² (A) or as percentage of GFAP-IR density (B). **p<0.01 and ***p<0.001 compared to SAL.

Figure 4. Δ9-THC reduces METH-induced increase of nNOS neurons in the CPu.

A. Rats received injections of 1 or 3/kg of Δ9-THC either 0.5 h before each METH injection (PRE) or 0.5, 12, 24, 36, and 48 h after the last METH administration (POST), and were sacrificed 3 days after the last METH injection. Pre- and Post-treatment with both doses of Δ9-THC significantly decreased the number of nNOS positive neurons in the CPu. *p<0.05 and **p<0.01 vs PRE METH-VEH; ^# p<0.05 and ^### p<0.001 vs POST METH-VEH (Bonferroni's post-hoc test). Horizontal dot lines represent the values of nNOS positive neurons (31±1.03) in SAL-VEH group. B. Representative images of nNOS immunohistochemical staining 72 h after the last METH or SAL administration in SAL-VEH, METH-VEH, METH-Δ9-THC 1 and 3 mg. Scale bar  =  100 µm.

Figure 5. Δ9-THC reduces METH-induced astrogliosis in the CPu.

A. Rats were treated as described in the legend of Figure 4. Two-way ANOVA revealed a significant effect of treatment (F_(2,32) = 16.28, p<0.0001) as well as a significant interaction between time of treatment and treatment (F_(2,32) = 8.12, p = 0.0014). Post-hoc comparisons showed that GFAP-IR was lower in the CPu of Post Δ9-THC (1 mg/kg) and Pre Δ9-THC (3 mg/kg) treated rats than in controls (METH-VEH). ***p<0.001 vs PRE METH-VEH and ^### p<0.001 vs POST METH-VEH (Bonferroni's post-hoc test). Horizontal dot lines represent the values of percentage of GFAP-IR density (0.75±0.07) in SAL-VEH group. B. Representative images of GFAP immunostaining in the CPu 72 h after the last METH or SAL administration in SAL-VEH, METH-VEH, METH-Δ9-THC 1 and 3 mg. Scale bar  =  100 µm.

Figure 6. Δ9-THC reduces METH-induced astrogliosis in the PFC.

A. Rats were treated as described in the legend of Figure 4. Pre- and Post-administration of Δ9-THC attenuated the astrogliosis induced by METH (Pre: −34% and −37%, Post: −47% and −29%, for 1 and 3 mg/kg, respectively) compared to control groups. **p<0.01 vs PRE METH-VEH and ^# p<0.05, and ^### p<0.001 vs POST METH-VEH (Bonferroni's post-hoc test). Horizontal dot lines represent the values of percentage of GFAP-IR density (1.31±0.10) in SAL-VEH group. B. Representative images of GFAP immunostaining in the PFC 72 h after the last METH or SAL administration in SAL-VEH, METH-VEH, METH- Δ9-THC 1 and 3 mg. Scale bar  =  100 µm.

Figure 7. Effects of SR on nNOS and GFAP-IR in the CPu.

A. Rats received injections of 1/kg Δ9-THC or VEH at 0.5, 12, 24, 36 and 48 h after the last METH administration (Post-treatment, POST) and were sacrificed 3 days after the last METH injection. SR (1 mg/kg, i.p.) or VEH were administered 15 min before each Δ9-THC or VEH injection. Two-way ANOVA in the CPu (A) showed a significant Δ9-THC x SR interaction (F_(1,40) = 32.45, p<0.0001); the administration of SR blunted the effect of Δ9-THC on METH-induced nNOS over-expression. SR alone decreased nNOS labeled neurons compared to that of control. ***p<0.001 vs METH-VEH (VEH pretreated) and ^##p<0.01 vs METH-VEH-Δ9-THC (VEH pretreated). B. Two-way ANOVA for GFAP-IR revealed a significant interaction between Δ9-THC and SR in the CPu (F_(1,35) = 19.86, p<0001). Δ9-THC and SR, alone or in combination, attenuated the METH-induced increase of GFAP-IR in the CPu. ***p<0.001 vs METH-VEH (VEH pretreated).

Figure 8. Effects of SR on GFAP-IR in the PFC.

Two-way ANOVA for GFAP-IR revealed a significant interaction between Δ9-THC and SR in the CPu (F_(1,33) = 45.91, p<0001). METH-Δ9-THC significantly reduced METH-induced GFAP-IR. Moreover, GFAP-IR was lower in METH-SR-VEH and METH-SR-THC groups as compared to METH-VEH treated rats. ***p<0.001 vs METH-VEH (VEH pretreated).