Astroglial Glutamate-glutamine Shuttle In Spinal Cord Is Involved In Central Sensitization Induced By Formalin In Rat

BACKGROUND CONTEXT: Central sensitization, a state of increased excitabilityof nociceptive neurons in the spinal cord dorsal horn following peripheral tissueinjury and/or inflammation, is an important mechanism underling hyperalgesia andneuropathic pain, in which glutamate plays a major role. Previous studies haveindicated that the glia-neuronin interactions are involved in the development ofcentral sensitization. However, most studies have focused on the activation ofmicroglia, while the role of astrocytes in the process of central sensitization has notbeen given equivalent consideration. Growing evidence indicates that astrocytes maybe important participants in the process of central sensitization due to their function assources of the neurotransmitter glutamate for neurons. Previous studies have indicatedthat glutamate plays a major role in central sensitization, and that theglutamate-glutamine shuttle is a key function of astrocytes. This shuttle includes theneuronal release of glutamate into the synaptic cleft and its uptake and conversioninto glutamine in astrocytes, followed by release from astrocytes for use as aprecursor of neurotransmitter glutamate in neurons. Astrocytes are the main sourcesof glutamine in the central nervous system (CNS), produced via the glutaminesynthetase (GS) reaction, which is restricted to astrocytes. Studies of the trigeminal subnucleus caudalis (Vc, medullary dorsal hom) have indicated that astrocyte GS isinvolved in trigeminal central sensitization in functionally identified nociceptiveneurons. However, the neurochemical and neurophysiological changes induced byinjury to nerves of the trigeminal system are clearly distinct from those seen followinglesions of other peripheral nerves, and there has been no study on the involvement ofthe astrocyte-neuronal glutamate-glutamine shuttle in central sensitization in thespinal cord.PURPOSE: Methionine sulfoximine (MSO), an inhibitor of astrocyte glutaminesynthetase(GS), was considered to involve in the ghtamate-ghtamine shuttle.Glutamine(Glu), a precursor of neurotransmitter glutamate, may be implicated in thehyperalgesia induced by application of formalin to the rat hindpaw. The aim of thepresent study was to determine whether the astrocyte-neuronal glutamate-ghtamineshuttle in the spinal cord is involved in formalin-induced central sensitization byexamining the effects of methionine sulfoximine (MSO) by intrathecal administration.METHODS: Thirty-six adult male Sprague Dawley (S-D) rats weighting 260-280 gwere provided by the Experimental Medical Animal Center of the Medical College ofLanzhou University. The involved rats were randomly divided into 6 groups as groupPBS+5% formalin, group MSO +5% formalin, group MSO + Gln + 5% formalin,group Gln +5% formalin, group sham +5% formalin, and group blank control +5%formalin, with 6 rats in each group. The drugs observed in the experiment included L-Methioninesulfoximine (MSO) (0.1 mM; Sigma-Aldrich); L-glutamine (Gln)(0.25mM; Sigma-Aldrich); PBS, pH 7.4, as vehicle (Sigma-Aldrich). All reagents (PBS, MSO or MSO+Gln, 50μl each) were intrathecal injection (i.t.) administered 15 minbefore subcutaneous injection (s.c.) of formalin (5%, 50μl) into the left hindpaw pad.Then, the rat was immediately placed into the chamber. Classical manual detectionmethods were used for formalin tests to measure nociceptive behavior, includingbiting/sticking and flinching of the injected hindpaw. The response to formalininjection was monitored by measuring the total duration (seconds) of licking/bitingand the incidence of flinching (Hz) of the injected hindpaw in every 5-min intervalduring the 60-min observation period.All rats were perfused with a 10-min transcardiac infusion of 4% bufferedparaformaldehyde, apart from group Gln +5% formalin. By the immune fluorescencedouble-labeling technique, we observe whether GS or GFAP (glial fibrillary acidicprotein, astrocyte-specific marker) in the lumbar spinal cord decreases the expressionand distribution in the group of intrathecal injection MSO and the co-expression levelof GS and GFAP in the lumbar spinal cord after intrathecal injection MSO+GLN.RESULTS: The first stage occurred immediately after formalin injection and lastedabout 5 min. The second stage began at about 10 min after formalin injection andlasted about 40-50 min. Nociceptive behavior of licking/biting induced by injection offormalin can be significantly attenuated by i.t. administration of MSO (0.1 mM, 50 ul;F(1,143)=15.717, p＜0.05).The duration of licking induced by formalin during a60-min observation period in the MSO group was significant shorter than that in thevehicle group, and there were significant differences at four of eleven time points(P＜0.05,). The duration of licking after MSO administration was significantly shorter than that in the PBS group between 25 and 40 min after administration (P＜0.05).Simultaneous administration of MSO and Gln (50 ul, 0.25 mM) reversedformalin-induced nociceptive behavior. Two-way RM-ANOVA analyses indicatedthat the three time course curves were significantly different among treatments (F(2,215)=8.53, P=0.007), across times (F(11, 215)=7.58, P＜0.001) and for the interactionof time and treatment (F(22, 215)=2.23, P=0.003). Further analyses showed that atsome time-points the duration of licking induced by formalin in the MSO plus Glngroup was significantly longer than that in the MSO group (P＜0.05), whereas at notime-point was the duration different from that in the PBS control group (P＞0.05).Similarly, i.t. application of MSO significantly reduced the incidence of flinching inboth phases, with mean values from 3.825±0.54 Hz in the PBS group to 1.692±0.23Hz in the MSO group (F(1, 143)=25.568, p=0.004) during the 60-min observationperiod. This effect could be blocked by i.t. administration of MSO plus Gln. In theMSO plus Gln group, the mean incidence of flinching (3.355±0.29 Hz) wassignificantly larger than that obtained from the MSO group (1.692±0.23 Hz)(F(1,143)=27.729, t=4.072, P＜0.05). However, there was no significant difference inthe incidence of flinching between the PBS group (3.825±0.54 Hz,) and the MSO plusGln group (3.355±0.29 Hz t=1.15, p=0.83).After intrathecal injection of MSO, the expression of GS and GFAP in the spinalcord reduced by double labeling immunofluorescence experiments (P＜0.05). Afterintrathecal injection of MSO + Gln, the co-expression lever of GS and GFAP in thespinal cord enhanced or restored basic level (P＜0.05). CONCLUSIONS: The results suggest that the astrocyte-neuronalglutamate-glutamine shuttle in the spinal cord may be involved in the centralsensitization induced by formalin in rat.