The Use Of [¹⁸F]FDG MicroPET In Acute Lung Injury In Rats & Protective Effect Of Raloxifene On It

Acute lung injury(ALI)and acute respiratory distress syndrome(ARDS)are a process of acute inflammatory lung injury resulted from a variety of direct or indirect injuries to the lungs,such as severe pneumonia,sepsis,massive transfusion,trauma, aspiration,pancreatitis,drug overdose,ALI/ARDS are the manifestation in the lungs of systemic inflammatory response syndrome(SIRS).Extensive neutrophil influx into the lungs,the production of pro-inflammatory mediators,and damage to lung epithelial and endothelial surfaces are the main characteristics of ALl.ARDS was defined as more severe form of ALl,the criteria to diagnose both ALl and ARDS included an acute onset,bilateral infiltrates on frontal chest radiographs,and either a pulmonary capillary wedge pressure of＜18mmHg or the absence of clinical evidence of elevated left atrial pressure,and in ALI the criteria of PaO2/FiO2 was≤300mmHg,while in ARDS PaO2/FiO2 was≤200mmHg.ARDS and ALI often result in multiple organ dysfunction syndrome(MODS),with an acute onset,the mortality rate is still high,around 30-70%, it is a puzzle leave to be solved by all clinical doctors.To date,despite the strategies for treating ALI/ARDS being extensively investigated,treatment options are limited and most of them are supportive;the only intervention that can reduce the mortality rate is the use of low tidal volume mechanical ventilation.While.drug therapy for ARDS/ALI is disappointing,no pharmacological intervention has been proven to be effective to the survival rate of ARDS/ALI patients clinically.Base on reason above,we pulled out investigation on the protective effect of raloxifene on acute lung injury via an animal model.Although numerous previor animal models have shown that lipopolysaccharide(LPS) or hydrochloric acid(HCL)alone can induce ALI.Clinicly,the development of ALI/ARDS is complex and only one single instigating factor rarely causes these syndromes,the occurrence of ALI increases with multiple risk factors,direct pulmonary disorders such as pneumonia,aspiration or pulmonary contusion and indirect pulmonary risk factors such as sepsis or multiple trauma are well-known risk constellations.Animal models of acute lung injury are numerous,and to date,there is not a model which is regarded as "classic" or "golden standard".There is uncertainty as to which model best reflects the real situation in humans,but many workshop participants suggest that two-hit models might be more appropriate to reflect common comorbidities and risk factors commonly present in ALI patients.However,others do not believe so, they argue that "one-hit" is better than "two-hit",the reproducibility,rapid onset of clinical signs and lack of expense make "one-hit" models extremely attractive and similarly,several articles have reported that the introduction of a second-hit had no impact on inflammation or increased lung injury.Based on reseans above,the first part of this article was aimed to investigate which is better? "one-hit" or "two-hit" ? and we used[¹⁸F]FDG microPET to evaluate the inflammatory response in the lungs during these models.Raloxifene,a selective estrogen receptor modulator(SERM),can selectively activate or inhibit ERαor ERβ,thus via different signal transduction and produce different biological effects in different tissue.Raloxifene has antiestrogenic properties in breast tissues,a neutral effect on the endometrium,and potentially beneficial estrogen-like effects in non-reproductive tissues,such as bone.Clinically,raloxifene has now been widely used in the prevention and treatment of osteoporosis in postmenopausal women. Raloxifene can upregulate the expresstion of ERα,suppress the activation of interleukin-6(IL-6)and NF-κB,and prohibit the differentiation of macrophage colony stimulating factor(M-CSF),thus exert protective effect in osteoporosis.Raloxifene can also elevate the level of high density lipoprotein(HDL)in blood,lower the level of low-density lipoprotein(LDL)and cholesterol total(CT)and thus produce some protective effect in cardiovascular system.Raloxifene was also shown to have an anti-inflammatory role in acute inflammation,raloxifene has been reported to inhibit IL-6 production as well as having an anti-inflammatory role in a murine model induced by LPS.It was also found to have protective effects on the development of carrageenan-induced edema and pleurisy,this effect may be caused by reducing inducible pro-inflammatory enzymes(COX-2 and iNOS)and by decreasing the infiltration of neutrophils into the inflamed paw and pleural cavity,and raloxifene was also found to have the effect of suppressing the activation of NF-κB via ERαin myeloma cell.Clinically,raloxifene can be quickly absorbed after orally administration, and extensively distributed in liver,lung,kidney,and blood serum,and estrogen receptor are widely distributed in human body,ERαare highly expressed both in lung parenchyma and pneumoangiogram,thus,we conclude that raloxifene can also suppress the activity of NF-κB via ERαand thus produce the protective effect on ALI.Presently,[¹⁸F]FDG PET has been widely used in infection and inflammatory diseases,[¹⁸F]FDG PET can locate fever of undetermined origin(FUO).The inflammatory reaction in the lungs and kinetics of neutrophils can also be evaluated during ALI by using[¹⁸F]FDG.In this article,we first used[¹⁸F]FDG microPET domesticly to evaluate the inflammatory reaction during ALI,we also used[¹⁸F]FDG microPET to evaluate the therapeutic efficacy of drug therapy on ALI.A profound research on the index which quantilate neutrophils during ALI was also made.Briefly,in this article,there are two different parts:1):using[¹⁸F]FDG microPET to evaluate a two-hit acute lung injury model in rats which was induced first by LPS IP and second by HCL IT;2):to evaluate the protective effect of raloxifene on ALI in rats.Section one:[¹⁸F]FDG microPET to evaluate a two-hit acute lung injury model induced first by LPS IP and second by HCL IT in rats Thirty three,adult,male Sprague-Dawley rats each weighing 180-210 g were used and divided into 4 groups,rats in LPS group(n=10)and LPS-HCL group(n=10)were challenged with intraperitoneal administration of LPS,at the dose of 5 mg/kg,while rats in NS group(n=3)and HCL group(n=10)received normal saline solution intraperitoneally at the dose of 1 ml/kg,after 16 hours,all animals were anesthetized with an intraperitoneal injection of sodium pentobarbital(40 mg/kg)and placed in a 60°inclined position,the femoral artery was cannulated and connected to a pressure transducer to record the arterial pressure and heart rate on a polygraph recorder,the trachea was surgically exposed,rats in HCL group and LPS-HCL group received direct intratracheal injection of HCL(PH=1.2)at the dose of 0.5ml/kg while rats in NS group and LPS group received the same volume of normal saline solution.Blood gas samples (each 0.3 ml)were obtained at 30,90,240 min after the instillation and replaced by the same volume of saline solution,the samples were analysed using a blood gas analyzer. 240 minutes after HCL or NS IT,the rats underwent a microPET scan,then,all the rats were sacrificed and the lungs were obtained for histology and W/D use.Results showed that 1)blood gas analysis:the PO₂ in NS group and LPS group remained normal throughout this experiment.As expected,the PO₂ showed an initial decline in HCL group and LPS-HCL group after acid instillation,the total mean PO₂ in LPS-HCL group was 58.67±7.77mmHg while it was 70.68±8.67mmHg in HCL group,at the end of this experiment the mean PO₂ in HCL group reached 77.29±7.51mmHg,but in LPS-HCL group it was 58.81±10.27mmHg,and there were significant difference between these two groups,P＜0.05;PCO₂ The PCO₂ values in LPS-HCL group was 52.14±13.86mmHg,and was much higher than that in HCL group 41.17±9.18mmHg(P＜0.05)and LPS group 38.25±11.24mmHg(P＜0.05),PCO₂ was 33.32±3.02 in NS control group;pH The pH value in LPS-HCL group was under the normal range during this experiment,significantly lower than HCL group(P＜0.01)and NS group(P＜0.01),but when compared with LPS group,there were no significant difference(P＞0.05);2)MAP Basal measurements of MAP were not different.MAP decreased markedly in LPS-HCL group,and there were significant difference compared with other groups,while MAP in other groups remained stable;3)microPET The ratio of ROI between the right lung and the muscle tissue of the right arm were compared, that ratio in LPS-HCL group was 9.00±1.41,and was significantly higher than that in LPS group(4.01±0.60)and HCL group(3.33±0.55);4)W/D The W/D ratio in LPS-HCL group was 5.84±0.27,much higher than HCL group(5.19±0.08)and LPS group (5.11±0.07),it was 5.07±0.04 in NS control group.There were no significant difference between HCL group and NS group,P＞0.05,there were also no significant difference between LPS group and NS group,P＞0.05;5)Histology Histologic examination revealed lung injury of varying degree in all animals received LPS IP or HCL IT or both, neutrophils infiltrates and hemorrhage were present in all animals of these three groups, but were more prominent in LPS-HCL group.Hyaline membranes were common in LPS group and LPS-HCL group but were rare in HCL group.Alveolar edema and airway epithelial necrosis were common and prominent in HCL group and LPS-HCL group while that were inconsistent findings in LPS group.Briefly,the mean lung injury score in LPS-HCL group was 12.7±0.95,while it was 8.4±1.26 in HCL group and 7.0±0.82 in LPS group,and there were significant differences(compared with the HCL group,P±0.01,compared with the LPS group,P＜0.01).Section two:Protective effect of raloxifene on acute lung injury in ratsThirty adult,male Sprague-Dawley rats each weighing 180-210 g were used and divided into 3 groups:the Raloxifene-LPS-HC1 group(n=10),the LPS-Raloxifene-HCl group(n=10),and the placebo group(n=10).All the rats were injected intraperitoneally (IP)with 5 mg/kg LPS,and raloxifene(30 mg/kg)was orally administered 1 h before and 14 h after LPS injection into the Raloxifene-LPS-HCl and the LPS-Raloxifene-HCl groups,respectively;the placebo group received nothing.Sixteen hours after LPS injection,all the animals were anesthetized and the femoral artery was cannulated.All the rats received a direct intratracheal injection of HCI(pH 1.2;0.5 mL/kg).MAP and blood gas concentrations were measured.Fifteen rats(5 in each group,respectively) underwent microPET scan of the thorax 4 h after HCl instillation.The expression of CD11b in the blood were measured by flow cytometry.The wet/dry(W/D),weight ratio determination,MPO and histopathological examination were also performed.Results showed that:1)PO₂ The PO₂ remained normal and there were no differences between the 3 groups immediately prior to tracheal acid instillation.As expected,the PO₂ showed an initial decline in all animals 30 min after acid instillation,but the PO₂ in the placebo group decreased more markedly and there were significant differences compared with that in the LPS-Raloxifene-HCl group(P＜0.01).As time passed,the PO₂ in the LPS-Raloxifene-HCl group slowly returned to normal ranges,while that of the placebo group at the end of the experiment was 58.81±10.27 mmHg;2)pH The pH values differed between the LPS-Raloxifene-HCl group and the placebo group(P＜0.05). In the placebo group,the pH value remained below the normal range,although it reached 7.27±0.05 at 90 min after instillation,while that of the LPS-Raloxifene-HCl group gradually returned to the normal range after acid instillation and finally reached 7.38±0.06(7.24±0.11 in the placebo group).However,there were no significant differences between the Raloxifene-LPS-HC1 group and the placebo group;3)PCO₂ In the LPS-Raloxifene-HCl group,the average value of the PCO₂ was 40.07±7.79 mmHg, while in the placebo group that value was 50.75±13.03 mmHg,much higher than that in LPS-Raloxifene-HCl group(P＜0.05).There were also no significant differences between the Raloxifene-LPS-HCl group and the placebo group(P＞0.05);4)MAP There were no differences between the three groups in initial measurements of MAP. However,after HCl instillation,MAP in the LPS-Raioxifene-HCl group remained stable,while MAP in placebo-treated group and Raloxifene-LPS-HCl group showed a marked decrease.There were significant differences between the LPS-Raloxifene-HCl group and the placebo group(P＜0.01);5)mieroPET The ratio of ROI in the right lung to the muscle was 9.014±1.58 in the placebo group,significantly higher than that (4.674±1.33)of the LPS-Raloxifene-HCl group(P＜0.01).There were no significant differences between the Raloxifene-LPS-HCl group and the placebo group(P＞0.05);6) W/D The W/D weight ratio was 5.894±0.26 in the placebo group,5.34±0.20 in the LPS-Raloxifene-HCl group,and 5.77±0.20 in the Raloxifene-LPS-HCl group.There were significant differences between the placebo group and the LPS-Raloxifene-HCl group(P＜0.01);7)Histology An histological examination revealed lung injury of varying degrees in all animals in the 3 groups,but were more prominent in the placebo group.Briefly,the mean lung injury score in the placebo group was 12.6±0.97,much higher than that(8.20±1.23)of the LPS-Raloxifene-HCl group(P＜0.01).That score in the Raloxifene-LPS-HCl group was 11.4±1.65,no significant difference when compared with the placebo group(P＞0.05);8)Correlation The ratio of the ROI between the right lung and the muscle correlated significantly with the histological lung injury score(R=0.824,P＜0.001);9)MPO The MPO value in LPS-Raloxifene-HCL group was(2.22±0.52)U/g,significantly lower than that in placebo group(3.31±0.52) U/g(P＜0.01);10)CD11b The CD11b value in LPS-Raloxifene-HCL group was 251.71±24.73,significantly lower than that in placebo group 482.41±125.45(P＜0.01).Conclusion1.LPS pretreatment significantly magnified and prolonged the inflammatory response to the subsequent acid instillation in both lungs.When compared with "one-hit", "two-hit" can more easily induce the animals to ALI,and more appropriate to reflect common comorbidities and risk factors commonly present in ALI patients clinically;2.[¹⁸F]FDG microPET is a useful method to evaluate the inflammatory reaction during ALI,and the results correlate well with histologic lung injury score;3.Raloxifene exert significant protective effects on acute lung injury induced first by LPS IP and second by HCL IT in rats.