ABSTRACT
In mouse the cannabinoid receptor 2 (CB2) agonists L768242 and (+)-AM1241, at doses of 30 mg/kg i.p. and 1 and 3 mg/kg i.v., respectively, reduced the second phase of nocifensive behaviors elicited by formalin intraplantar injection. This effect was counteracted by the selective CB2 antagonist SR144528 (1 mg/kg i.p.). In rat (+)-AM1241 (3 and 6 mg/kg i.v.) and L768242 (30 mg/kg i.p.) reduced allodynia elicited by L5-L6 spinal nerve ligation. SR144528 reverted these effects, supporting a CB2-mediated action. To clarify the mechanisms underlying these effects we investigated CB2 gene expression and function in the nervous system. CB2 mRNA was expressed in spinal cord and dorsal root ganglia (DRG) of both sham and neuropathic rats and was up-regulated in the ipsilateral spinal cord of neuropathic rats. Expression studies demonstrated the presence of CB2 mRNA in culture of spinal cord microglia. A biomarker, CGRP, was used to investigate modulation of DRG primary afferents by CB2 agonists. Both L768242 and (+)-AM1241 dose dependently (EC50 of 3.6 and 4.5 nM, respectively) reduced capsaicin-induced calcitonin gene-related peptide (CGRP) release. Coadministration of SR144528 resulted in a rightforward shift (pKB 8.1 and 8.2 for (+)-AM1241 and L768242, respectively) of the dose-response curve. Experiments on capsaicin-induced CGRP release in tissue from CB1-/- mice ruled out a CB1-mediated effect. These results confirm that CB2 is present in the central nervous system and suggest that CB2 agonists may elicit their analgesic effect by acting not only at non-neuronal peripheral sites but also at neural level, making CB2 an attractive target for chronic pain treatment.
Subject(s)
Analgesics/pharmacology , Hyperalgesia/physiopathology , Nervous System Physiological Phenomena , Receptor, Cannabinoid, CB2/physiology , Animals , Calcitonin Gene-Related Peptide/metabolism , Capsaicin/antagonists & inhibitors , Capsaicin/pharmacology , Cells, Cultured , DNA Primers , DNA, Complementary/biosynthesis , DNA, Complementary/genetics , Formaldehyde , Ligation , Male , Mice , Microglia/physiology , Pain Measurement/drug effects , RNA/biosynthesis , RNA/isolation & purification , Rats , Rats, Sprague-Dawley , Receptor, Cannabinoid, CB2/agonists , Receptor, Cannabinoid, CB2/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction , Sciatic Nerve/physiology , Spinal Cord/cytology , Spinal Cord/drug effects , Spinal Cord/metabolism , Spinal Nerves/physiology , Transcription, GeneticABSTRACT
The potential neuroprotective effects of the novel nitro-derivate of aspirin (NCX4016) on permanent focal cerebral ischemia in spontaneously hypertensive rats (SHRs) was investigated. Reference compounds were acetylsalicilic acid (ASA) and FK506 (tacrolimus). Ten minutes after surgery, SHRs were randomly divided into four groups of ten, pharmacologically treated and sacrificed 24 h after treatment. Brains were removed and processed to measure infarct volume, 70 kDa heat shock protein (hsp70), glial fibrillary acidic protein (GFAP) and vimentin (Vim) immunoreactivity (IR), and apoptosis using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end-labeling (TUNEL) assay. NCX-4016 significantly reduced total infarct volume compared to ASA (-20%, P < 0.05), FK506 (-18%, P < 0.05) and vehicle treatment (-20%, P < 0.05). Experimental groups did not differ in hsp70-IR and GFAP-IR. Conversely, hyperplastic astrocytes, measured by Vim-IR, were significantly lower in NCX-4016 than in the vehicle group (-36%, P<0.01). TUNEL assay indicated a significantly lower degree of apoptosis in NCX-4016 group than vehicle in both the homolateral (-27%, P < 0.01) and contralateral hemisphere (-29%, P < 0.05). These findings indicate that NO release associated with aspirin confers neuroprotective effects against ischemic injury.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Aspirin/pharmacology , Brain Injuries/drug therapy , Brain Injuries/prevention & control , Brain Ischemia/drug therapy , Brain Ischemia/metabolism , Brain/drug effects , Neuroprotective Agents/pharmacology , Animals , Apoptosis/drug effects , Apoptosis/physiology , Aspirin/analogs & derivatives , Astrocytes/drug effects , Astrocytes/metabolism , Astrocytes/pathology , Brain/metabolism , Brain/pathology , Brain Injuries/pathology , Brain Ischemia/pathology , Cell Survival/drug effects , Cell Survival/physiology , Functional Laterality/drug effects , Functional Laterality/physiology , Glial Fibrillary Acidic Protein/drug effects , Glial Fibrillary Acidic Protein/metabolism , HSP70 Heat-Shock Proteins/drug effects , HSP70 Heat-Shock Proteins/metabolism , Immunohistochemistry , Immunosuppressive Agents/pharmacology , Male , Nerve Degeneration/drug therapy , Nerve Degeneration/pathology , Nerve Degeneration/prevention & control , Neurons/drug effects , Neurons/metabolism , Neurons/pathology , Rats , Rats, Inbred SHR , Tacrolimus/pharmacology , Vimentin/drug effects , Vimentin/metabolismABSTRACT
The time course of 72-kDa heat-shock protein (hsp72) induction was evaluated by immunoblotting in cerebral cortex, striatum, hippocampus, cerebellum, liver, and kidney of rats subjected to 60-min focal cerebral ischemia following proximal unilateral occlusion of the right middle cerebral artery (MCA). Neurological examinations indicated that maximum deficits in reflex and sensorimotor functions occurred 24-48 h after reperfusion (40% lower than baseline), while significant recovery occurred at 72 h (33% higher than 48 h). hsp72 was present in all tissues at 6 h. The regions perfused by the occluded MCA showed a higher induction than the corresponding contralateral ones. hsp72 reached its maximum level in ipsilateral cerebral cortex and striatum at 24 h, whereas in the contralateral cortex and cerebellum the protein reached its maximum expression at 48 h, that is 24 h before functional recovery. This delay suggests a role of the protein in plastic events sustaining neurological recovery.
