Mechanism Of Spinal Astrocytic ALDH2 Regulating Alcohol Metabolism And Alcohol Analgesia

Background:Alcohol is one of the oldest and the most widely used drugs in the world.Like opioids and cannabinoids,alcohol has been used for pain relief since ancient times.1 out of every 4 people experiencing chronic pain nowadays turns to alcohol to relieve the suffering.Unfortunately,reaching the maximum pain relief always requires binge drinking.It is notable that the relationship between alcohol and pain is complex which alcohol reduces pain,in turn,pain promotes alcohol intake.Prolonged and excessive use of alcohol to stop pain leads to a number of harmful health consequences in human.The most notable one is increased risk for alcohol dependence.On the other hand,chronic alcohol drinking causes peripheral neuropathy,and alcohol withdrawal often results in hyperalgesia in rodents and in human.Up to now,the precise mechanisms underlying alcohol-induced analgesic effects remain elusive,it is of great significance to clarify the interaction and mechanism between alcohol and pain.Alcohol metabolism is controlled through various genetic factors in humans.The metabolites of ethanol（EtOH）can be more active than EtOH alone in the human body.The key enzyme that converts acetaldehyde（AcH）to acetate is acetaldehyde dehydrogenase type 2（ALDH2）.One major genetic deficiency in ALDH2 is found in nearly one third of the Asian population.This naturally occurring point mutation disrupts ALDH2 activity,increases blood AcH contents and produces various adverse reactions to alcohol.ALDH2 deficiency in human and rodents are associated with an increased pain sensitivity to various noxious stimuli.The deficiency of ALDH2results in serum and tissue AcH elevation and acetate ablation.While the elevated AcH is found to produce peripheral neuronal inflammation and pain hypersensitivity,little is known about the role of acetate in pain modulation.There is strong evidence suggesting that acetate is utilized and metabolized exclusively by astrocytes in the brain.Acetate metabolized by ethanol is not only an important source of body energy,but also mimics or mediates ethanol-induced sedation,motor and discoordination etc.Brain acetate has been,until recently,though to derive from EtOH metabolism in liver.The in vivo consequence of brain alcohol metabolism remains elusive because the levels of alcohol metabolizing enzymes including ALDH2 are very low.Unfortunately,the cell-type specific distribution of brain ALDH2 and how ALDH2mediates alcohol metabolism and alcohol induced behavioral effects is unknown because of lacking specific approaches in vivo.Unlike opioids and cannabinoids,EtOH does not bind to single specific protein target in the central nervous system（CNS）.EtOH produces motor impairment,sedative and anxiolytic effects through enhancement of GABA_A receptor function.Several positive modulators of GABA_B receptors are used to treat alcohol preference and dependence in animals and human.Both GABA_A and GABA_B receptors are found to play a major role in pain control in rodents and in human.While emerging evidence have suggested that both ALDH1a1 and ALDH2 can promote GABA synthesis through putrescine degradation and utilization pathways in neurons and astrocytes.The relationship between EtOH and GABA synthesis remains to be controversial and the metabolic pathways from EtOH to GABA are also less clear.Although spinal cord is an important gateway for transmitting pain signal to the brain,the role of spinal cord in EtOH-induced behavioral effects has not been established.A recent study has suggested that spinal ALDH2 contributes to neuroprotection and recovery after spinal cord injury.Nevertheless,little is known about the roles of spinal ALDH2 in alcohol metabolism and alcohol-induced behavioral changes because of lacking in vivo approaches that can separate peripheral and central ALDH2-mediated effects.In this study,we asked if spinal ALDH2 regulates alcohol metabolites and EtOH-induced analgesic effect in mice.We tested this hypothesis by performing various in vitro and in vivo experiments in cell-type specific ALDH2 deficient mice.We also explored the possible mechanism underlying astrocytic ALDH2 control of EtOH analgesia including GABA and GABA receptor in spinal cord.Methods:1.Spinal cord virus injectionFor spinal cord astrocyte-specific Aldh2 gene deficiency,AVV5-GFAPCre and AVV5-GFP lentivirus were injected into the bilateral dorsal horn of spinal cord（L3-4）with the depth of 300μm in Aldh2 flox male mice.After 4 weeks of the virus injection,mice were tested with tail flick reflex（TFR）test,Complete Freund’s adjuvant（CFA）induced inflammatory pain with electronic von Frey and paw thermal stimulating test,locomotion activity and rotarod performance under the intraperitoneal（i.p.）administration with 1.2 g/kg or 2 g/kg EtOH.2.Detection of ALDH2 protein,mRNA and enzyme activitiesALDH2 protein,mRNA and enzyme activities were detected by western blot,immunofluorescence,RNA in situ hybridization（ISH）,qRT-PCR and ALDH2enzyme activity kit.3.Intrathecal delivery of GABA receptor antagonistC57BL/6J male mice were used for intrathecal injections.Either saline,GABA_Areceptor antagonist bicuculline（200 ng,10μl）or GABA_B receptor antagonist CGP55845（200 ng,10μl）was delivered via a 50μl Hamilton micro-syringe with a30-gauge needle into the cerebral spinal fluid at the L5-6 levels.After 30 min of the intrathecal injection,tail flick reflex and rotarod test were performed as the following procedure.4.Inflammatory pain modelAstrocytic and hepatocytic ALDH2 deficient male mice and spinal cord virus injected male mice were used for Complete Freund’s adjuvant（CFA）induced inflammation.Briefly,the left hind paw was injected intra-plantar with 20μl CFA using a 50μl Hamilton micro-syringe and a 30-gauge needle under brief anesthesia with isoflurane.The paw to mechanical hypersensitivity and noxious heat were measured by electronic von Frey and thermal pain hypersensitivity system.5.Behavioral testsC57BL/6J and spinal cord virus injected male mice,as well as astrocytic-specific Aldh2 deficient,hepatocytic-specific Aldh2-deficient male mice and their wild type littermates were used in behavior tests.TFR,electronic von Frey,paw thermal stimulating,hotplate,locomotion activity and rotarod tests were performed in the study to evaluate EtOH or acetate induced antinociception.All animals were habituated to procedure room for at least 1 hours before the testing.6.Measurement of spinal cord tissue and serum EtOH,AcH by GC-MS/MSAldh2^Gfap-/-and their wild-type littermates were used for EtOH and AcH measurement in spinal cord tissue and blood.Mice were injected with EtOH（2 g/kg）or an equal volume of saline intraperitoneally after at least 1 h acclimation.Lumbar spinal cord tissue and blood were harvested at baseline,10,30,60 min after i.p.EtOH.The concentrations of EtOH and AcH in plasma and tissue were detected by gas chromatography-mass spectrometry（GC/MS）and calculated by comparing the integrated areas of EtOH and AcH peaks on the gas chromatograms with that of internal standards added in each sample.7.Measurement of spinal cord GABA content by LC-MS/MSAldh2^Gfap+/+and Aldh2^Gfap-/-mice were injected with 2 g/kg EtOH,or 1.5 g/kg acetate or an equal volume of saline i.p.The mice were anesthetized with isoflurane and decapitated to get the spinal cord tissue at baseline,30 and 60 min after i.p.EtOH or acetate.LC-MS/MS analyses were conducted on an Agilent 6470 triple quadrupole mass spectrometer coupled to an Agilent 1260 LC system.Levels of GABA were determined against a standard curve,using GABA as standard.Values are expressed as nmol/mg wet tissue.8.Measurement of serum and spinal cord tissue acetate by colorimetric assay kitSpinal cord tissue and serum were used to measure acetate using the spectrophotometric assay.Tissue and serum were harvested and processed as same with the measurement of EtOH by GC-MS.The aliquots of 50μl diluted samples were incubated at room temperature in a 96 well plate with the addition of 50μl reaction mix as a substrate.Finally,the absorbance of the solutions was measured at450 nm in a 96 well plate using the Microplate Absorbance Reader.The acetate content was normalized to that of Aldh2^Gfap+/+mice,and the value in the baseline was assigned as 1.Results:1.Astrocytic expression of ALDH2 in spinal cordA strong signal of ALHD2（green）was detected in spinal cells evenly distributed in both white and gray matters.ALDH2 mRNA was expressed in 36.38%of total gray matter cells.While ALDH1L1 mRNA were mainly expressed in dorsal horn cells（27.12%of total cells）,MAP2 mRNA was primarily detected in the large cells of anterior gray horn（6.55%of total cells）.ALDH2 mRNA was detected in 76.5%of ALDH1L1 positive cells and 82.6%of MAP2 positive cells.We examined the expression of ALDH2 mRNA in spinal slices from astrocytic ALDH2 conditional knockout mice,Aldh2^Gfap-/-mice（target for astrocytes）.In these slices,the signal of ALDH2 mRNA seemed weak,and there was a substantial reduction in ALDH2colocalization with ALDH1L1 as compared to slices shown in their littermates.In contrast,only slight decrease was observed in the colocalization between ALDH2 and MAP2 positive cells in spinal anterior horn of astrocytic ALDH2 knockout mice.Consistent with this,the expression levels of ALDH2 mRNA,protein and enzymatic activity were significantly reduced in Aldh2^Gfap-/-mice.Similarly,astrocytic ALDH2deficiency disrupted the colocalization of ALDH2 with GFAP immunoactivity in spinal slices.2.Astrocytic ALDH2 mediates EtOH analgesia Systemic administration of EtOH,in a dose-dependent manner（0.5-3 g/kg,i.p.）,produced an analgesic effect in the TFR test in wildtype(Aldh2^Gfap+/+)mice.The magnitude of EtOH analgesia reached the maximum 15-30 min after systemic EtOH.EtOH-induced analgesia magnitude was significantly decreased in astrocytic ALDH2deficient(Aldh2^Gfap-/-)mice.For instance,astrocytic ALDH2 deficiency decreased%MPE induced by EtOH from 23.92%to 8.25%（EtOH at 1 g/kg）and from47.85%to 27.60%（EtOH at 2 g/kg）.EtOH increased the response latency in the hot plate test,respectively.However,astrocytic ALDH2 deficiency did not significantly alter the analgesic effect of EtOH in the hot plate test.Unlike astrocytic ALDH2deficiency,hepatocytic ALDH2 deficiency did not significantly affect the analgesic effect of EtOH in TFR test.Mechanical allodynia was evaluated using electronic von Frey filaments before and after injection of CFA.Systemic administration of EtOH significantly inhibited the level of mechanical allodynia by enhancing pain threshold from 0.6675（0.049）g to 5.083（0.224）g.Astrocytic ALDH2 deficiency significantly inhibited EtOH enhancement of pain threshold by nearly 42%.In contrast to astrocytic ALDH2 deficient mice,hepatocytic ALDH2 deficient mice did not significantly alter EtOH-induced analgesic effect on CFA-induced pain hypersensitivity in mice.The levels of locomotor activity were reduced after systemic EtOH at both 1.2 g/kg and 2 g/kg in mice,respectively.There was no significant difference in the levels of locomotion after EtOH between astrocytic ALDH2deficient mice and their wildtype littermates.3.Astrocytic ALDH2 mediates EtOH conversion to acetate in the spinal cordTo examine the impact of spinal ALDH2 on alcohol metabolism,we assessed serum and spinal cord EtOH and AcH in Aldh2^Gfap+/+and Aldh2^Gfap-/-mice using gas chromatography-mass spectrometry（GC-MS）.While serum and spinal EtOH and AcH contents rapidly rose after systemic administration of EtOH,there was no difference observed in serum and spinal EtOH metabolite contents between astrocytic ALDH2 deficient mice and their WT littermates.The acetate contents in both serum and spinal tissues were elevated after systemic EtOH in mice,respectively.Astrocytic ALDH2 deficiency did not significantly alter serum acetate derived from EtOH,however,the deficiency in astrocytic ALDH2 completely prevented EtOH-induced elevation of acetate in spinal tissues.Systemic EtOH significantly increased spinal cord GABA contents by 21%after 30 min.No such spinal GABA increase by EtOH was observed in astrocytic ALDH2 deficient mice.The concentrations of spinal GABA were slightly lower from that of baseline GABA 30 min and 60 min after EtOH in astrocytic ALDH2 deficient mice.There was a significant difference in spinal GABA contents following systemic EtOH between Aldh2^Gfap+/+and Aldh2^Gfap-/-mice.Consistent with this,GABA immunoactivity was increased in the dorsal horn of spinal slices from WT mice but astrocytic ALDH2 deficient mice after systemic EtOH.4.Acetate mediates EtOH analgesia and GABA synthesisThe observations above suggest that astrocytic ALDH2 mediates EtOH analgesia and EtOH-derived acetate in the brain.To test if acetate can mimic EtOH analgesia,we examined the analgesic effect of systemic acetate in acute and chronic pain models.Systemic administration of acetate dose-dependently increased the latency of the pain threshold in TFR test.The analgesic effect reached the maximum at 30 min and lasted for 90 min after systemic acetate.The acetate also alleviated the mechanical allodynia induced by CFA in mice.The time courses of acetate-induced analgesia on both TFR and CFA-induced pain were nearly identical to EtOH analgesia,suggesting that acetate mediates EtOH-induced analgesic effect.Like EtOH,systemic acetate significantly enhanced GABA concentrations from 7.21（0.32）nmol/mg to 8.69（0.39）nmol/mg of lumbar spinal cord tissues.Consistent with this,the signal of GABA immunoactivity was enhanced in the dorsal horn area of the spinal slices from mice60 min after systemic acetate.5.Spinal ALDH2 mediates EtOH analgesiaThe signals of Cre and GFP immunoactivity were detected in the area of spinal dorsal horn 4 weeks after the viral injections.The levels of spinal ALDH2 proteins were significantly decreased by 61.9%（P=0.0005）in mice previously injected with AAV5-GFAPCre as compared with the mice injected with AAV5-GFP.EtOH increased pain threshold in TFR test from the baseline of 3.53（0.62）s to 5.54（0.30）s.in AAV-GFP mice.Spinal astrocytic ALDH2 deficient mice attenuated the peak of EtOH analgesic magnitude by 22%（P=0.0259）.Similarly,spinal astrocytic ALDH2deficiency significantly inhibited the peak magnitudes of EtOH analgesia in both thermal stimulation-induced hyperalgesia and mechanical stimulation-induced allodynia in CFA injected mice.On the other hand,spinal astrocytic ALDH2deficiency did not significantly alter EtOH-induced hypolocomotion,discoordination and hypothermia in mice.6.Spinal GABA receptors mediate EtOH and acetate-induced analgesic effectsTo test if spinal GABA_A and GABA_B receptors are involved in EtOH or acetate-induced antinociception,we delivered a GABA_A receptor antagonist,or a GABA_B receptor antagonist,via intrathecal（i.t.）infusion in mice.Bicuculline（Bic,200 ng）or CGP55845（CGP,200 ng）prevented EtOH-induced analgesia without significantly affecting EtOH-induced discoordination.Similarly,GABA_A and GABA_B receptor antagonists inhibited acetate-induced analgesia but not acetate-induced discoordination.Conclusions:These findings suggest that spinal ALDH2 mediates the production of acetate from ethanol metabolism in an astrocyte-dependent mechanism.Astrocytic ALDH2 regulates EtOH-induced analgesic effects via distinct acetate-GABA synthesis pathways.Spinal astrocytic ALDH2 is an important target not only for the analgesic effect of EtOH but also for pathophysiology of pain at the spinal level.