Article Figures & Data
Figures
- Fig. 1.
Histological evidence for cannabinoid-mediated protection of CA1 hippocampal neurons in global cerebral ischemia. Rats underwent sham surgery or 15 min of cerebral ischemia induced by four-vessel occlusion, with or without the administration of drugs 40 min before the onset of ischemia. Animals were killed 3 d later and paraffin-embedded brain sections were stained with cresyl violet. Sections are shown at low (left, 25×) and high (right, 400×) power, after sham surgery (Sham) or after the induction of ischemia without (Isch) or with previous administration ofR(+)-WIN 55212-2 [R(+)],S(−)-WIN 55212-3 [S(−)], or SR 141716A (SR) at the indicated doses (milligrams per kilogram, i.p.). Asterisks at leftindicate the region within CA1 from which the fields shown atright were taken.
- Fig. 2.
Neuron counts in CA1 after global cerebral ischemia. Animals were treated as described in the legend to Figure 1, undergoing sham surgery (Sham) or global cerebral ischemia without drugs (Isch) or with previous administration of R(+)-WIN 55212-2 [R(+)], S(−)-WIN 55212-3 [S(−)], or SR 141716A (SR) at the indicated doses (milligrams per kilogram, i.p.). Neurons were counted in cresyl violet-stained sections. Data shown are mean values ± SEs from six to eight animals per condition. *p < 0.05 (ANOVA with post hoc t tests) relative to neuron counts in animals subjected to ischemia without drug treatment (Isch).
- Fig. 3.
Histological evidence for cannabinoid-mediated reduction in infarct size after permanent MCA occlusion. The left MCA (on the viewer’s right) was occluded with a nylon suture as described in Materials and Methods. Animals were given DMSO vehicle 30 min before occlusion (Veh); 1 mg/kgR(+)-WIN 55212-2 30 min before [R(+) −30], or 30 [R(+) 30], 60 [R(+) 60], or 120 [R(+) 120] min after occlusion; or 1mg/kg R(+)-WIN 55212-2 and 1 mg/kg SR141716A [R(+) & SR], 1 mg/kg SR141716A alone (SR), or 1 mg/kg S(−)-WIN 55212-3 [S(−)], all 30 min before occlusion. Drugs were administered intraperitoneally. Animals were killed 24 hr after the onset of occlusion, and brain sections were treated with TTC (red), which stains viable tissue red but does not stain infarcted tissue. The brains shown are representative of six animals per condition. S(−)-WIN 55212-3 [S(−)], or 10 μm THC. After an additional 16 hr under control conditions, cell viability was measured by Alamar blue fluorescence and expressed as a percentage of fluorescence above background in control cultures. Data shown are mean values ± SEs from 4–12 cultures per condition. *p < 0.05 (ANOVA with post hoc t tests) relative to fluorescence in cultures exposed to hypoxia and glucose deprivation in the presence of 10 nmR(+)-WIN 55212-2. C, Concentration dependence of neuroprotection induced by R(+)-WIN 55212-2. Experiments were performed as described in Babove, except that different concentrations of R(+)-WIN 55212-2 were used, and results were expressed as a percentage of the maximal drug-induced increase in viability above that in cultures exposed to hypoxia and glucose deprivation without drugs. Data shown are mean values ± SEs from 4–12 cultures per condition.
- Fig. 4.
Infarct volumes after focal cerebral ischemia. Animals were treated as described in the legend to Figure 3, undergoing permanent MCA occlusion without drugs (Veh) or with intraperitoneal administration of 1 mg/kg R(+)-WIN 55212-2 given 30 min before (−30) or 30, 60, or 120 min after the onset of ischemia; 1 mg/kg R(+)-WIN 55212-2 and 1 mg/kg SR141716A, both given 30 min before ischemia; 1 mg/kg SR141716A alone given 30 min before ischemia; or 1 mg/kgS(−)-WIN 55212-3. Data shown are mean values ± SEs from six animals per condition. *p < 0.05 (ANOVA with post hoc t tests) relative to infarct volumes in animals subjected to ischemia and treated with vehicle only (Veh).
- Fig. 5.
Pharmacological characterization of cannabinoid-mediated neuroprotection from hypoxia and glucose deprivation in vitro. Cultured cerebral cortical neurons were exposed for 24 hr to normoxia and glucose (control), or to 8 hr of hypoxia and glucose deprivation followed by 16 hr of recovery (hypoxic). A, Western analysis of control (lane 1) and hypoxic (lane 2) cultures probed with an antibody against the CB1 cannabinoid receptor (arrow). B, Pharmacological features of cannabinoid-mediated neuroprotection. Cultures were exposed to hypoxia and glucose deprivation in the absence (Hypoxia) or presence of 10 μm MK-801 (MK), 100 nm anandamide (Anand), 10 nmR(+)-WIN 55212-2 (alone [R(+)] or together with the CB1 antagonist SR141617A [R(+) & CB1] or the CB2 antagonist SR144528 [R(+) & CB2], each at 300 nm–1 μm), 10 nm
- Fig. 6.
PANT staining for DNA single-strand breaks afterin vitro hypoxia and glucose deprivation. Cultures were maintained for 8 hr under control conditions (A) or exposed for 8 hr to hypoxia and glucose deprivation in the absence (B) or presence of 10 μm MK-801 (C) or 10 nmR(+)-WIN 55212-2 (D). After an additional 16 hr under control conditions, cultures were fixed and processed for PANT staining as described in Materials and Methods. Cells with dense nuclear staining were considered PANT-positive and were counted at 200× magnification in at least three randomly selected fields per well.E, Quantitative data were expressed as a percentage of all cells in the same fields and are shown are mean values ± SEs from 6–12 cultures per condition. *p < 0.05 (ANOVA with post hoc t tests) relative to values in cultures exposed to hypoxia and glucose deprivation in the absence of drugs (Hypoxia).
Tables
- Table 1.
Blood pressure, arterial blood gases, and serum glucose in transient global cerebral ischemia
Time relative to ischemia Drug treatment MABP PaO2 PaCO2 pH Glucose Before Vehicle 102 ± 3 151 ± 21 38 ± 1 7.42 ± 0.01 116 ± 4 R(+)-WIN (0.03 mg/kg) 102 ± 6 143 ± 10 38 ± 1 7.41 ± 0.01 115 ± 8 R(+)-WIN (0.1 mg/kg) 105 ± 5 142 ± 13 40 ± 2 7.40 ± 0.01 114 ± 12 R(+)-WIN (0.3 mg/kg) 108 ± 5 147 ± 3 38 ± 2 7.42 ± 0.00 111 ± 13 R(+)-WIN (1 mg/kg) 111 ± 8 141 ± 3 38 ± 3 7.41 ± 0.01 105 ± 15 R(+)-WIN (1 mg/kg) + SR (1 mg/kg) 107 ± 8 152 ± 8 37 ± 2 7.41 ± 0.01 119 ± 19 SR (1 mg/kg) 104 ± 10 156 ± 12 39 ± 1 7.41 ± 0.01 114 ± 21 S(−)-WIN (1 mg/kg) 104 ± 6 143 ± 6 38 ± 1 7.41 ± 0.00 117 ± 9 During Vehicle 132 ± 2 150 ± 9 38 ± 2 7.41 ± 0.01 110 ± 2 R(+)-WIN (0.03 mg/kg) 135 ± 6 144 ± 6 39 ± 2 7.42 ± 0.01 116 ± 15 R(+)-WIN (0.1 mg/kg) 135 ± 9 148 ± 20 40 ± 2 7.40 ± 0.01 125 ± 22 R(+)-WIN (0.3 mg/kg) 137 ± 4 156 ± 7 39 ± 0 7.41 ± 0.00 121 ± 15 R(+)-WIN (1 mg/kg) 137 ± 5 155 ± 1 39 ± 0 7.41 ± 0.00 126 ± 23 R(+)-WIN (1 mg/kg) + SR (1 mg/kg) 140 ± 8 136 ± 10 38 ± 1 7.42 ± 0.01 116 ± 7 SR (1 mg/kg) 139 ± 11 141 ± 14 39 ± 1 7.41 ± 0.02 117 ± 18 S(−)-WIN (1 mg/kg) 138 ± 8 146 ± 15 39 ± 1 7.41 ± 0.02 120 ± 17 After Vehicle 121 ± 9 151 ± 17 38 ± 1 7.42 ± 0.01 122 ± 5 R(+)-WIN (0.03 mg/kg) 119 ± 12 149 ± 9 39 ± 1 7.41 ± 0.02 112 ± 17 R(+)-WIN (0.1 mg/kg) 122 ± 8 148 ± 15 39 ± 1 7.40 ± 0.01 114 ± 21 R(+)-WIN (0.3 mg/kg) 116 ± 11 143 ± 8 41 ± 2 7.41 ± 0.00 121 ± 8 R(+)-WIN (1 mg/kg) 124 ± 6 143 ± 7 38 ± 2 7.40 ± 0.02 119 ± 23 R(+)-WIN (1 mg/kg) + SR (1 mg/kg) 113 ± 11 140 ± 17 38 ± 1 7.41 ± 0.01 129 ± 15 SR (1 mg/kg) 122 ± 8 141 ± 20 39 ± 1 7.41 ± 0.02 125 ± 12 S(−)-WIN (1 mg/kg) 116 ± 10 147 ± 19 39 ± 1 7.41 ± 0.01 120 ± 18 Data shown are mean values ± SEs from six to eight animals per condition. MABP, Mean arterial blood pressure (mm Hg); Glucose, plasma concentration of glucose (mg/dl); R(+)-WIN,R(+)-WIN 55212-2; SR, SR141716A; S(−)-WIN,S(−)-WIN 55212-3.
- Table 2.
Blood pressure, arterial blood gases, and serum glucose 10 min after the onset of permanent focal cerebral ischemia
Drug treatment MABP PaO2 PaCO2 pH Glucose Vehicle 120 ± 11 124 ± 9 32 ± 1 7.40 ± 0.01 117 ± 6 R(+)-WIN, −30 min 124 ± 8 118 ± 11 33 ± 1 7.39 ± 0.01 117 ± 6 R(+)-WIN, +30 min 125 ± 8 115 ± 4 37 ± 4 7.38 ± 0.03 113 ± 12 R(+)-WIN, +60 min 122 ± 14 113 ± 3 37 ± 5 7.39 ± 0.02 117 ± 6 R(+)-WIN, +120 min 118 ± 16 112 ± 6 35 ± 4 7.39 ± 0.01 110 ± 10 R(+)-WIN + SR 124 ± 12 122 ± 15 36 ± 9 7.41 ± 0.03 109 ± 14 SR 117 ± 7 115 ± 4 36 ± 2 7.40 ± 0.02 113 ± 13 S(−)-WIN 121 ± 6 115 ± 10 33 ± 3 7.39 ± 0.04 107 ± 6 Data shown are mean values ± SEs from six animals per condition. MABP, Mean arterial blood pressure (mm Hg); Glucose, plasma concentration of glucose (mg/dl); R(+)-WIN,R(+)-WIN 55212-2; SR, SR141716A; S(−)-WIN,S(−)-WIN 55212-3.
