Astrocyte Is Involved In The Long-lasting Inflammation Hyperalgesia After Subcutaneous Formalin Injection Into The Rat Hindpaw

AMINO ACID TRANSPORT SYSTEM A IS INVOLVED IN INFLAMMATORY NOCICEPTION IN RATS (Part1)Objective:The current study investigated the role of amino acid transport system A in central sensitization and hyperalgesia induced by intraplantar injection of formalin in rats.Methods:The rat was removed from this chamber and5%formalin (50μ1) was administered subcutaneously (s.c.) into the right hindpaw pad. The rat was immediately replaced in the chamber. Spontaneous nociceptive behaviors were assessed by measuring the duration of licking/biting and the number of flinches every5min in the injected hindpaw over a period of1h after formalin injection by two experimenters, as reported previously. After the completion of behavioral testing (i.e.,1h after formalin injection), all rats were sacrificed for GFAP staining using immunohistochemistry. The sections were used for immunohistochemical staining of GFAP using the avidin-biotin-peroxidase (ABC) method for GFAP staining.Results:Formalin (5%,50μ1) injected subcutaneously into the unilateral hindpaw pad induced typical biphase nociceptive behaviors, including licking/biting and flinching of the injected paw and an increase of glial fibrillary acid protein (GFAP, an activated astrocyte marker) expression in spinal dorsal horn, and these effects could be attenuated by intrathecal injection of the competitive inhibitor of amino acid system A transporter, methylaminoisobutyric acid (MeAIB,0.1,0.3,0.5, and0.7mmol). in a dose-dependent manner. Intrathecal injection of vehicle (PBS) had no effect on the formalin-induced nociceptive behaviors and increase of the GFAP.Conclusion:These findings suggest that amino acid transport system A is involved in inflammation-induced nociception, and inhibition of this transporter system results in inhibition of the central sensitization and hyperalgesia. INVOLVEMENT OF ENDOCANNABINOID IN THE CONTINUOUS INFLAMMATORY PAIN INDUCED BY FORMALIN THROUGH MODULATING ASTROCYTES (Part2)Objective:To investigate the roles of endocannabionoid and astrocytes in continuous inflammatory pain induced by formalin through blockade of endocannabionoid receptors (CB).Materials and methods:Continuous inflammatory pain model was established through intraplantar injection of formalin (5%,50μ1). The distribution of CB subtypes (CB1, CB2) in glial cells (microglia and astrocyte) and neurons of spinal dorsal horn and the MAPK pathway were observed through immunohistochemistry. CB antagonists (AM281, AM630,1mg/kg in500μ1, intraperitoneal) were administrated to observe the changes of formalin-evoked nociceptive responses at acute stage (licking/biting paws and twitching reflex) and allodynia at chronic stage (50%paw withdrawal threshold, PWT), and the distribution of CB1and CB2in glial cells neurons.Results:Blockade of CB1and CB2facilitated the spontaneous pain at acute stage (licking/biting paws and twitching reflex) and allodynia at chronic stage (50%paw withdrawal threshold. PWT) evoked by formalin stimulation, which was significant different from that of formalin injection alone (p<0.05). Blockade of CB1and CB2promoted the incurrence of mirror pain in formalin test. Morphological results showed that blockade of CB receptor significantly increased the expression of astrocytes in lumbar dorsal horn than formalin injection alone, that the expression of CB1continuously increased following the time elapsed and was colocalized with astrocytes while the CB2expression was not observed in blank control group but was continuously induced by formalin injection within7days and was further increased after blockade of CB1and CB2. The pJNK expression in astrocyte induced by formalin was not affected by blockade of CB1and CB2.Conclusion:The results indicated that endocannabinoid is involved in the formalin induced-continuous inflammatory hyperalgesia through JNK pathway by activating astrocytes, suggesting that enhancement of endocannabinoid level can play analgesic effect under continuous inflammatory pain.