The Experimental Study On Effect Of NMDA Receptor Which Involved In The Pathogenesis Of OVA-induced Asthma And Mechanisms Of Ketamine In The Pretreatment With Bronchial Asthma

Considerable advances have been made towards understanding thephysiology and pathophysiology of central glutamate receptors, and theirimportance in neuronal plasticity and in acute and chronic neurologicaldisorders. The existence or importance of peripheral glutamate receptorsin the tissues and cells has becoming a focus issue in recent years.Studies demonstrated the expression of a variety ofN-methyl-D-aspartic acid (NMDA) glutamate receptor subtypes in thelung and airways. Stimulation of the NMDA receptor in the airwayresults in airway constriction and it seems likely that over-activation ofNMDA receptors in lungs may be a pathogenetic mechanism of injuryand inflammation in those sites. As in central neuronal glutamate toxicity,the lung injury was NMDA receptor-mediated and NO-dependent.Excessive stimulation of NMDA receptor induced excitotoxicity in lungsmight be an important pathogenesis of the airway inflammation andhyper-reactivity found in bronchial asthma.Anesthetic ketamine has very complicated pharmacological effects. As an analogue to phencyclidine(PCP), ketamine noncompetitivelyinhibited the NMDA receptor by binds to the phencyclidine binding site,this might possibly explain its anesthetic and analgesic properties, andneuroprotective effects on the glutamate-mediated neuroexcitatory events.As a potent broncodilator, it is recommended as optimizing anaestheticfor the asthmatic patient and has been clinically administered to treatbronchospasm, asthma exacerbation and status asthmaticus. In recentyears, protective role of ketamine in the lung injury through itsanti-inflammatory properties has become highlights of the anestheticstudy.This study was designed to explore the pathophysiology ofglutamate and glutamate receptors in the lungs which involved in theinjury of tissues or cells elicited by inflammation and oxidative stress,and explore anti-inflammatory and anti-oxidative effects of ketamine inthe treatment of asthma through NMDA receptor-mediated pathway.These may provide new clues for clinical therapy in lung dieases andvaluable laboratory data for understanding of the mechanism of ketamineaction on treatment of asthma. Part OneProtective Effects of Ketamine on Allergen-induced AirwayInflammatory Injure and High Airway Reactivity in AsthmaBrown-Norway RatsObjectiveTo observe the effects of ketamine on bronchial hyperresponsiveness andairway inflammation in a Brown-Norway rat model of ovalbumin(OVA)-induced allergic asthma.Methods52 rats were randomly assigned to seven groups: those are phosphatebuffered solution (PBS) control group(n=8); OVA control group(n=8);12.5 mg/ml Inhalation Test Group(n=8), 25 mg/ml Inhalation Test Group(n=8), 50 mg/ml Inhalation Test Group(n=8); 50μg/kg InjectedComparison Group(n=6), 100μg/kg Injected Comparison Group(n=6);The rats were sensitized by injection of OVA together with aluminumhydroxide and Bordetella pertussis as adjuvants and challenged byrepeated intermittent (thrice weekly) exposure to aerosolized OVA fortwo weeks. Before challenge, the sensitized rats were exposed to anaerosol of PBS or ketamine at concentration of 12.5 mg/ml, 25 mg/ml and50 mg/ml or were intraperitoneally injected ketamine at dose of 50μg/kgor 100μg/kg respectively. Airway reactivity to acetylcholine (ACh) wasassessed in vivo 18 hr after the last OVA challenge, then lungs were lavaged and total and differential cell count of leukocytes in thebronchoalveolar lavage fluid (BALF) were determined, the left lungswere removed for histopathologic examination. The effects of nebulizedketamine at different concentrations on the normal lung structure and theplasma levels were evaluated by exposure non-sensitized rats to aerosolof ketamine at 12.5 mg/ml or 25 mg/ml or 50 mg/ml respectively for 30min once every 2 d for two weeks in 12.5 mg/ml ketamine-inhaledcontrol group (n=6), 25 mg/ml ketamine-inhaled control group (n=6) and50 mg/ml ketamine-inhaled control group (n-6).Results(1) When the dose of acetylcholine reached 25 or 50μg/kg, the increase ofairway pressure(P), the decrease of tidal volume(Vt) and the decrease ofdynamic lung compliance(Cldyn) in OVA-sensitized and-challenged ratswere much more significant than those in PBS control rats. Thedose-response curve of Re shifted to the upper-left ward, in addition, theprovocation doses required to increase Re by 100ï¼…, 200ï¼…and 400ï¼…inrats from OVA control group were significantly lower than those of PBScontrol rats(14.65±1.19 versus 32.28±1.43,; 15.17±1.19 versus38.91±1.39 and 16.28±1.18 versus 56.53±1.38, P＜0.01 respectively).OVA-sensitized rats treated with ketamine before OVA challengedemonstrated a significant decrease in AHR by the significantly lowerincrease of P, decrease of Vt and Cldyn, a rightward shift of the dose-response curves to Ach and significantly high provocation doses,which compared with that of OVA control rats(P＜0.05). (2) FollowingOVA challenge, the BALF of sensitized rats showed significantly highertotal cell numbers compared with PBS-exposed rats (5.97±0.72×10⁶versus 2.51±0.49×10⁶, P＜0.001). Differential cell counting revealed anincrease in the percentages of eosinophils and lymphocytes and adecrease in the percentage of macrophages in OVA-exposed animalscompared with these parameters in PBS control animals; ketaminetreatment significantly decreased the total cell numbers and thepercentages of eosinophils and lymphocytes, but did not change thepercentage of neutrophils and macrophages in OVA-challenged rats. (3)Marked inflammatory changes in the airways of OVA control group werepresent, while obviously lessen inflammatory cells infiltration inperibronchial and perialveolar and improved lung edema were observedin rats treated with ketamine. (4) The plasma peak levels of ketaminewere 890.27, 1313.48 and 2805.97μg/L respectively in non-sensitizedrats receiving 12.5, 25 and 50 mg/ml nebulized ketamine.ConclusionsBoth inhalation and systemic administration of ketamine attenuatedOVA-induced inflammatory lung injure and airway hyperreactivity inasthma model rats. Part TwoThe Effects of Ketamine on NMDA-receptor/NO in Lungs ofExperimental AsthmaObjectiveTo observe the effects of ketamine on the contents of amino acid, theexperession of N-methyl-D-aspartic acid Receptor 1(NMDA-R1)andnitric oxide synthase (NOS) and the levels of guanosine 3′,5′-monophosphate (cGMP) in asthma model rats.Methods(1) The contents of amino acid were assayed by high performanceliquid chromatography (HPLC).(2) The mRNA expression of NMDA-R1, neuronal nitric oxidesynthase (nNOS), endothelial nitric oxide synthase (eNOS) andinducible nitric oxide synthase (iNOS) in lung tissues was detectedby reverse transcription polymerase chain reaction (RT-PCR).(3) The protein expression of NMDA-Rland iNOS was assayed byWestern-Blot.(4) The level of cGMP was measured by non-radioactive enzymeimmunoassay (EIA).Results(1) The contents of glutamate (Glu) and glycine (Gly) were higher inlungs of OVA group than those of PBS group, while there was no difference among ketamine pretreatment groups and OVA group.(2) The mRNA expression of NMDA-R1 and iNOS significantlyincreased in OVA group when compared with that of PBS group,while this higher expression was attenuated by administration ofinhaled ketamine at concentration of 12.5 mg/ml or 25 mg/ml andinjected ketamine at dose of 50μg/kg. These results were confirmedby the protein expression ofNMDA-Rland iNOS.(3) Higher level of cGMP was assayed in OVA group than that in PBSgroup, and lower levels of cGMP were observed in 12.5 mg/mlgroup, 25 mg/ml group, 50μg/kg group and 100μg/kg group, whencompared with those of OVA group.ConclusionsKetamine attenuated OVA-elicited inflammatory lung injure in asthmamodel rats, may be through its inhibition role on over-expression ofNMDA-R1 and iNOS and production of iNO. Part ThreeEffects of Ketamine on OVA-induced the changes of FreeIntracellular Calcium and Free-radical in Alveolar Macrophages inVitroObjectiveTo observe the effect of ketamine on OVA-induced the changes of freeintracellular calcium ([Ca²⁺]_i) and free-radical in alveolar macrophagesfrom sensitized rat in vitro.MethodsMacrophages were collected from Bronchoalveolar Lavage fluid ofOVA-sensitized Sprague-Damley rats and treated with ketamine ofconcentration at 10μM, 100μM and 1000μM before challenge with 100μg/mL OVA. The production of nitric oxide (NO) and hydroxy radical(·OH) in the supernatant of culture was assayed by kits and the [Ca²⁺]_i ofmacrophages was measured with mean fluorescent intensity(MFI) bylaser scanning confocal microscope(LSCM).ResultsOVA challenge induced an elevation of [Ca²⁺]_i and an increase of NOand·OH in sensitized rat macrophages, which were inhibited by ketamineat concentration-related method.ConclusionsKetamine inhibited OVA-induced oxidative stress which involved in calcium.