| Background and PurposeAcute limb ischemia (acute limb ischemia, ALI) is one of the most common peripheral artery disease caused by arterial emboli, thrombosis or a sudden interruption of arterial blood flow to distal extremity due to vessels injury. Although the diagnosis and treatment for ALI have significantly improved in recent years, it still results in high amputation and mortality rate, up to10%-30%and15%within a month, respectively. Muscle cell necrosis, microcirculation disturbance, loss of sensation and motor initiate after3hours of ischemia, while irreversible muscle necrosis complete within6hours of ischemia. Therefore, it is a principle treatment for ALI to restore blood flow as soon as possible to avoid amputation, best within6hours, and usually within24hours. However, restoration of blood flow often results in limb ischemia/reperfusion (I/R) injury, including inflammatory injury to muscles, vessels and distant organs such as the heart, lungs, kidneys, intestines etc. Severe I/R injury even cause life-threatening complications, such as metabolic acidosis, hyperkalemia, acute renal failure, multiple organ dysfunction syndrome (MODS). And many ALI patients can’t tolerate vascular reconstruction surgery, because of complications including hypertension, coronary heart disease, rheumatic heart disease and cerebral infarction. Therefore it is really an important issue to extend surgical treatment "window" by protecting vascular structure and function, which will facilitate blood recovery of ischemia limb. In conclusion, further research on hemodynamics, pathology and molecular biology during the process of acute limb ischemia/reperfusion is particularly important, which will provide theoretical basis and new clues for the surgical and drug treatment and prognosis of ALI.Ischemia and I/R can induce inflammatory cytokine mediated inflammation cascade, which is responsible for ischemic injury and I/R injury. Inflammation in artery vessels during acute limb I/R results in inflammatory injury of endothelial cells (EC) and smooth muscle cells (SMC) by promoting secretion of tumor necrosis factor-a (TNF-a), interleukin-6(IL-6), up-regulating ICAM and VCAM and attracting white cells. Phenotype switch of SMC and degradation of extracellular matrix (ECM) lead to vascular remodeling and dysfunction. The inflammatory injury of ECs and SMCs combined with vascular remodeling and dysfunction influence the hemodynamics of ischemic and reperfusion limb. Therefore, we presume that the elimination of inflammation of vessels is helpful to protect vascular structure and function, attenuate inflammatory injury after reperfusion and facilitate blood recovery.High mobility group box protein1(HMGB1) is a highly conserved DNA-binding non-histone proteins and presents in the eukaryotic cell nucleus. HMGB1helps maintaining nucleosome structure, facilitating gene transcription and modulating steroid hormone receptor within nucleus. When released extracellularly, HMGB1participates in I/R injury, repair after tissue injury, atherosclerosis, inflammation as an early inflammatory cytokines. HMGB1initiates inflammatory cascade during I/R injury by up-regulating intercellular adhesion molecule, vascular cell adhesion molecule, stimulating secretion of TNF-α and IL-6from macrophages and monocytes, promoting leukocyte and platelet aggregation, and recruiting macrophages and smooth muscle cell. Previous studies have testified that HMGB1participates in I/R injury of heart, brain, lung, liver, kidney, intestine and skeletal muscle tissue, and treatment targeting HMGB1can significantly attenuate inflammatory injury. But the expression of HMGB1and its role during arterial vessel inflammation when acute ischemia and reperfusion attack lower limbs have not been clarified. We presume that HMGB1participates in disruption of vascular structure and function and hemodynamics fluctuation by inducing and maintaining inflammation cascade.Heparin is used clinically as anticoagulant for ischemic diseases. And recent studies have testified its anti-inflammation activity for pancreatitis, rheumatoid arthritis, ulcerative colitis, allergies by reducing leukocyte aggregation, inhibiting platelet activation and release of elastase and cathepsin G. It also inhibits lipopolysaccharide (LPS)-induced secretion of TNF-α and IL from monocyte in vitro. Furthermore, heparin changes HMGB1configuration, and thus attenuates its affinity to RAGE. Ethyl pyruvate (EP) decreases the expression of HMGB1and eliminates inflammation intensity after reperfusion. Therefore, we will administrate heparin and EP to observe the change of vascular inflammation and limb hemodynamics.Laser Doppler perfusion imaging (LDPI) displays blood images based on the Doppler effect of blood flow. Perfusion unit (PU) reveals the blood perfusion within local tissue, depending on the number and movement speed of red blood cell. Mean PU directly reveals the blood perfusion of the area within a specific region, while Max PU value reveals the blood flow velocity within vascular vessels. Therefore, we apply LDPI to evaluate the rat model and carry on qualitative and quantitative research on limb hemodynamics.We mean to investigate the limb hemodynamics, vascular inflammation, the correlation and the role of HMGB1by establishing a rat model of acute limb I/R. This study will help clarify the limb hemodynamics, vascular pathology and the correlation, and provide experiment support and new clues for the therapy of ALI.Study Contents1. Evaluation and analysis of limb hemodynamics in a rat model of acute limb ischemia/reperfusion2. Investigation of the expression profile of HMGB1protein and mRNA in artery vessel during acute limb ischemia and post-ischemia reperfusion 3. Research on the influence and mechanism of HMGB1during acute limb ischemia/reperfusion.MethodsPart Ⅰ1. Animal model:to establish acute ischemia model in hind limb of rat by using vascular clamp and tourniquet. The reperfusion model was established by removing clamp and tourniquet after3hours of ischemia. All rats were divided into sham group, acute ischemia group, treated ischemia group, reperfusion group, treated reperfusion group. Heparin was subcutaneously injected in treated group immediately after surgery.2. Gross signs of hind limbs were observed at1,3,6,9,12,18,24hours after acute limb ischemia and reperfusion, respectively.3. Limb hemodynamics:the rats were anesthetized and placed in the prone position under room temperature, and positioned under the laser Doppler imaging machine (Moor Instruments). LDPI parameter was set as follows: image scan mode,15cm scanning distance,20X15cm scanning field,256X64scanning resolution and media speed. Limb blood images were acquired at1,3,6,9,12,18,24hours after acute limb ischemia and reperfusion, respectively.4. Vessel samples:femoral arteries were collected at1,3,6,9,12,18,24hours after acute limb ischemia and reperfusion, respectively.Part Ⅱ1. Animal model establishment and collection of vessel samples were performed as described previously.2. Immunofluorescence was performed to observe HMGB1protein expression and distribution in arterial tissue.3. Western blot was performed to investigate HMGB1protein level in arterial tissue and the effect of heparin on protein expression.4. Syber Green real-time PCR was performed to investigate HMGB1mRNA level in arterial tissue and the effect of heparin on mRNA expression.Part Ⅲ1. Animal model:all rats were divided into sham group, ischemia group, heparin treated ischemia group, heparin+EP treated ischemia group and sham group, reperfusion group, heparin treated reperfusion group, heparin+EP treated reperfusion group. Surgical procedure was performed as described previously. Heparin and EP were injected in treated group immediately after surgery.2. Evaluation of limb hemodynamics and collection of vessel samples were performed at the peak time of HMGB1based on the research of Part II.3. HE and EVG staining was performed to observe the pathological change of vascular vessels.4. Immunohistochemical staining was performed to observe HMGB1and related inflammation cytokines protein expression and distribution in arterial tissue.3. Western blot was performed to investigate HMGB1protein level in arterial tissue.4. Syber Green real-time PCR was performed to investigate HMGB1and related inflammation cytokines mRNA level in arterial tissue.ResultPart Ⅰ1. Ischemia/reperfusion model in hind limb of rat was successfully established by using vascular clamp and tourniquet.In ischemia group, it was paleness that firstly presented after surgery, followed by cyanosis after9hours of ischemia, which gradually worsened until24hours of ischemia. Heparin improved limb ischemia and delayed the occurrence of cyanosis until12hours after ischemia.In reperfusion group, red and swelling paws were observed immediately after reperfusion. The symptoms began to ease until18hours after reperfusion. Heparin improved symptoms obviously and the paws recovered to normal within24hours.2. Ischemia group:mean PU and max PU decreased to a particular minimal level and persisted within24h after surgery, testifying successful establishment of rat model. The mean PU and max PU of contralateral limb also decreased after surgery, but gradually recovered since3rd hour after surgery. The max PU correlated mean PU in ischemia limbs, but not significantly (r=0.14,P=0.17).3. Treated ischemia group:there was not significant elevation of mean PU and max PU in ischemia limb after heparin administration (PmeanPU=0.75, PmaxPU=0.69). The ischemia limb remained the same after treatment. The hemodynamics of contralateral limbs still presented the same profile after treatment.4. Reperfusion group:mean PU and max PU presented characteristic profiles within24hours after reperfusion. Both presented a slight elevation followed by decreasing to a valley at12hour and9hour of reperfusion, respectively. Within24hours of reperfusion, none of them recovered to the base line compared with the sham group. The mean PU and max PU of contralateral limb also presented transient incline and recovered since9hour after reperfusion. The max PU correlated mean PU in reperfusion limbs significantly (r=0.68,P<0.001).5. Treated reperfusion group:there was a significant elevation of mean PU and max PU in reperfusion limb after heparin administration (PmeanPU<0.001, PmaxPU<0.001). And both of them presented a gradual increase but could not recovered to the base line compared with the sham group. The mean PU and max PU of contralateral limb also presented the same profile after treatment.6. Sham group:LDPI revealed benign blood perfusion and similar PU in bilateral limbs. Part Ⅱ1. Expression of HMGB1protein and mRNA were elevated in arterial tissues after acute limb ischemia but presented different curves.1.1Immunofluorescence revealed weak staining in nucleus of ECs and SMCs in sham group, which testified a minimal existing of HMGB1. As early as1hour after onset of acute ischemia, strong staining of HMGB1presented both in the nucleus of both ECs and SMCs. The district of cells with strong-stained nucleus and cytoplasm spread gradually to the periphery and finally involved the whole vascular wall within6hours after ischemia. The strong staining in nucleus and cytoplasm lasted at least24hours, but staning in cytoplasm began to decrease slightly since12hours after ischemia.1.2Western blotting analysis revealed a negative result in vessels of the sham rat. The protein expression increased immediately after ischemia, followed by a sharp increase to a peak at9hours, and stayed at a high level until24hours. HMGB1protein expression was positively correlated with ischemic time (P<0.001,r=0.801).1.3PCR analysis revealed a positive result in vessels of the sham rat. The mRNA expression increased immediately after ischemia and reached a peak at6hours, followed by a persistent decline until24hours. HMGB1mRNA expression was negatively correlated with ischemic time (P<0.001, α=-0.847).2. Heparin decreased the level of HMGB1protein and mRNA obviously after ischemia.2.1Immunofluorescence revealed the staining was attenuated by heparin compared with ischemia group. However, strong staining of HMGB1still presented in the nucleus of ECs and SMCs at1hour after ischemia. The whole vascular wall was still involved by strong-staining cells at6hours after ischemia. The staining in nucleus and cytoplasm began to decrease since24hours after ischemia.2.2Western blotting analysis revealed a significant decrease of protein by heparin (P<0.001). The expression curve was similar, but the peak was delayed to18hours after ischemia. The decline scale of protein at the peak was0.526±0.047. HMGB1protein expression was positively correlated with ischemic time (P<0.001, r=0.926).2.3PCR analysis revealed HMGB1mRNA was also reduced by heparin (P=0.003). It also increased early after ischemia and reached a peak at6hours of ischemia, followed by a persistent decline. The decline scale of mRNA at the peak was0.348±0.02. HMGB1mRNA expression was negatively correlated with ischemic time (P<0.001, r=-0.863).3. HMGB1protein and mRNA increased in arterial tissues in post-ischemia reperfusion limb and presented identical curves.3.1Immunofluorescence revealed as early as1hour of reperfusion, strong staining of HMGB1presented both in the nucleus of SMCs and ECs. The district of strong-stained cells involved the whole vascular wall within3hours. The strong staining in nucleus and cytoplasm lasted at least24hours, but began to decrease since18hours after reperfusion.3.2Western blotting analysis revealed HMGB1protein increased early after reperfusion and reached a peak at12hours, followed by a decreased until24hours. HMGB1protein expression was not correlated with reperfusion time (P=0.176).3.3PCR analysis revealed HMGB1mRNA presented an identical expression curve. It also reached a peak at12hours after reperfusion, followed by a gradual decline until24hours. HMGB1mRNA expression was not correlated with reperfusion time (P=0.12).4. Heparin decreased HMGB1protein and mRNA level in post-ischemic reperfusion artery tissues.4.1Immunofluorescence revealed the staining was attenuated by heparin compared with reperfusion group. Strong staining still presented in the nucleus at1hour after reperfusion, and involved the whole vascular wall within3hours after reperfusion. Strong staining in nucleus and cytoplasm began to decrease since18hours after reperfusion. 4.2Western blotting analysis revealed a significant decrease of protein by heparin (P<0.001). The protein increased early after reperfusion and peaked at6hours. The decline scale of protein at the peak was0.63±0.021. HMGB1protein expression was not correlated with reperfusion time (P=0.839).4.3PCR analysis revealed HMGB1mRNA was also reduced by heparin (P<0.001). It also peaked at6hours of reperfusion, followed by a persistent decline. The decline scale of mRNA at the peak was0.531±0.024. HMGB1mRNA expression was not correlated with reperfusion time (P=0.058).Part Ⅲ1. Blockade of expression of HMGB1could not provide improvement on ischemia limbs. Neither heparin nor heparin+EP could elevate mean PU and max PU in ischemia limbs significantly (PmeanPU=0.12, Pmax PU=0.924). Western blot reveals significant decrease of HMGB1protein in artery vessel (P<0.001). And HMGB1level correlated mean PU and max PU negatively, but not significantly (rmean PU=-0.41, Pmean PU=0.049; rmax PU=-0.39, Pmax PU=0.059).2. Acute limb ischemia promoted expression of inflammation cytokines in artery vessel.HE and EVG staining revealed degradation of collagenous fibers and normal elastic lamellae, ECs and SMCs. Heparin and EP treatment prevented collagenous fiber degradation obviously and maintained the normal structure of elastic lamellae.IH staining reveals that ischemia up-regulated expression of HMGB1、TNF-α、 IL-6、ICAM、VCAM、MMP-2、MMP-9、α-SM actin in ECs and/or SMCs to different levels. Treatment decreased the expression levels significantly (P <0.001). HMGB1correlated the expression levels of related inflammation cytokines positively, testifying blockade of HMGB1might attenuate the inflammation intensity in vascular tissue after ischemia.PCR analysis also revealed the elevation of mRNA of the above cytokines and a significant decrease after treatment (P<0.001). 3. Blockade of expression of HMGB1could improve blood perfusion on reperfusion limbs obviously. Heparin and EP administration could elevate mean PU and max PU in reperfusion limbs significantly (Pmean PU<0.001, Pmax PU<0.001).Western blot reveals significant decrease of HMGB1protein in artery vessel (P<0.001). And HMGB1level correlated mean PU and max PU significantly (rmeanPU=-0.74,Pmean PU<0.001;rmaxPU=-0.66,Pmax PU<0.001).4. Acute limb post-ischemic reperfusion promoted expression of inflammation cytokines and vascular remodeling.HE and EVG staining revealed degradation of collagenous fibers and disruption of elastic lamellae. Heparin and EP treatment prevented collagenous fiber degradation obviously and restored the wavy structure of elastic lamellae. Some ECs were detached and SMCs disappeared in local region of artery vessel after perfusion. Treatment provided significant protection of ECs and SMCs.IH staining reveals intensive vascular inflammatory reaction. The expression of HMGB1、TNF-α、IL-6、ICAM、VCAM、MMP-2、 MMP-9、α-SM actin in ECs and/or SMCs were elevated to different levels, while treatment decreased the expression levels significantly (P<0.001). HMGB1correlated the expression levels of related inflammation cytokines positively, testifying blockade of HMGB1might attenuate the inflammation intensity in vascular tissue after reperfusion.PCR analysis also revealed the elevation of mRNA of the above cytokines and a significant decrease after treatment (P<0.001), which testified the inflammation process in artery tissue from the point of view of mRNA.Conclusion1. LDPI can provide qualitative and quantitative hemodynamics analysis of acute limb ischemia/reperfusion rat model, established by tourniquet and vascular clamp. Ischemia limb presented a persistent hypo-perfusion and reperfusion limb presented a characteristic time profile of hypo-perfusion within24hours. Heparin administration could not restore the hypo-perfusion either in ischemia limbs or in reperfusion limbs. The transient hemodynamics fluctuation of contralateral limbs might be caused by surgical procedure and a systemic vascular reaction in rats.2. Acute limb ischemia and post-ischemic reperfusion promoted strong inflammation reaction of artery tissue, characterized by of elevation of HMGB1protein and mRNA. Acute ischemia and post-ischemia reperfusion resulted in elevated HMGB1protein and mRNA in the arterial tissue in different profiles, helping to clarify the reaction process of artery vessel. Heparin significantly decreased HMGB1protein and mRNA level, possibly through interfering positive feedback loop of HMGB1expression.3. Attenuation of vascular inflammation restored blood perfusion significantly in reperfusion limbs, while could not improve hypo-perfusion in ischemia limbs. Acute ischemia and post-ischemia reperfusion caused inflammatory injury on ECs and SMCs, vascular remodeling and dysfunction, which were responsible for the limb hemodynamics fluctuation. Elevation of HMGB1could result in inflammatory injury on ECs and SMCs, vascular remodeling and dysfunction by promoting expression of related inflammation cytokines. |
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