Time-related morphological changes in cold-stored rat livers. A comparison of Euro-Collins solution with UW solution

Rat livers were stored in cold UW solution and Euro-Collins solution for various periods. Morphological investigations were performed using light microscopy, as well as scanning and transmission electron microscopy. In the UW-stored livers, the appearance of blebs derived from hepatocytes and the destruction of sinusoidal endothelial cells (SEC) occurred more slowly than in the EC-stored livers. Almost no ultrastructural damage in the hepatocytes was observed even after 48 hr of storage in UW solution, while extremely swollen and degenerated hepatocytes were observed in the 48-hr EC-stored livers. After 48-hr of storage, livers stored in UW solution lost 7.9% of their weight though EC-stored livers gained 29.7% of weight. Light microscopic morphometry revealed that there was a significant increase of 24.3% in the mean hepatocyte area of 24-hr EC-stored livers, whereas the UW-stored hepatocytes did not show any significant increase even after 48 hr of storage. After perfusion fixation, livers stored for more than 8 hr in EC solution showed a mosaic pattern of uneven fixation indicating a microcirculatory disturbance, whereas the UW-stored livers showed a rather uniform fixation after 12 hr of storage. It is suggested that the microcirculatory disturbance occurred more slowly in the UW-stored livers than in the EC-stored livers, which might be due to the protection of SEC and the suppression of bleb formation and the swelling of hepatocytes by UW solution.     

Quantitative analysis of the microcirculation of xenogeneic haemoperfused rat livers by intravital microscopy

Abstract  Livers from male Spra-gue-Dawley rats were perfused with heparinised, unmodified isogeneic rat blood ( n = 6) or xenogeneic human blood. The microcirculation of these livers, as the primary manifestation of hyperacute xenogeneic rejection, was directly observed and quantified by using fluorescence videomicroscopy. Bile flow and enzyme release of the isogeneic perfused livers were in the physiological range, whereas bile flow was significantly reduced and enzyme release increased during xenogeneic perfu-sion. In contrast to an almost physiological acinar (90.4 %) and sinusoi dal (93.6 %) perfusion rate in the isogeneic group, a rapid breakdown of microcirculation with an acinar perfusion index of 47.5 % and a sinusoidal perfusion rate of 67.1 % were found in the xenogeneic group. This direct quantification of micro-circulatory parameters is a step forward towards sensitive and early characterisation of the severity of the xenogeneic rejection of the liver.     

Study of the microcirculation in hDAF transgenic rat livers xenoperfused with human blood

Abstract: Background: The microcirculation was assessed in the livers of human decay accelerating factors (hDAF) and wild‐type transgenic rats by fluorescent intravital microscopy, histology and histomorphology to determine the benefits of hDAF expression for the microcirculation of a rat liver xenograft perfused with human blood. Methods: Male hDAF transgenic rats (group A; n = 20) and wild‐type Sprague–Dawley rats (group B; n = 20) were xenoperfused with human blood, while other male wild‐type Sprague–Dawley rats (group C; n = 10) were perfused with allogeneic blood. Following plasma and leukocyte staining with fluorescein sodium, and platelet staining with rhodamine, the right lobe of the liver was assessed by intravital microscopy, counting the numbers of perfused sinusoids and leukocytes adhering to the endothelium per mm², and calculating the acinar perfusion index (Pi). The liver underwent histological assessment at the end of each experiment. Mean ± SEM values were calculated and the Mann–Whitney U‐test was used for statistical analysis. Results: The number of perfused sinusoids was higher in the group of hDAF rat livers (group A) and controls (group C) than in the group of non‐transgenic rat livers perfused with human blood (group B) (P < 0.05), although only group C still had a significantly more perfused sinusoids than the other groups after 90 min of perfusion (P < 0.05). The acinar perfusion index was higher in groups A and C than in group B (P < 0.05); here again, only group C still had a significantly higher Pi than group B after 90 min of perfusion (P < 0.05). There was a massive accumulation of leukocytes that peaked after 5 min and persisted throughout the perfusion in all three groups. Histology showed portal and subendothelial hepatic vein hemorrhage, necrosis and inflammatory reaction, which were particularly evident in group B. Conclusion: In our study, rat livers transgenic for hDAF were better protected against early tissue damage by perfusion with human blood, but this did not result in a longer xenograft survival.     

Acute effects of periarterial sympathectomy on the cutaneous microcirculation

The clinical effects of peripheral sympathectomy on patients with vaso-occlusive disease are often dramatic and include relief of pain, improved quality of life, and healing of ulcers. Peripheral periarterial sympathectomy is known to increase skin temperature and to maximize the nutritional component of peripheral blood flow, but the pathophysiology of vaso-occlusive disease and the physiologic mechanisms of this treatment are unknown. In this study, the acute effects of periarterial sympathectomy were directly observed in a rabbit ear model of digital microcirculation (arterioles, arteriovenous anastomoses, and venules). The effects of periarterial sympathectomy on cutaneous perfusion and total flow were also examined using laser Doppler. Perfusion imaging and digital temperature measurements. The central auricular artery became dilated (50–100%) immediately after sympathectomy; the arterioles, arteriovenous anastomoses, and venules dilated to 165,156, and.223%, respectively, at 30 minutes and to 187,174, and 204%, respectively, at 60 minutes, relative to their baseline diameters prior to sympathectomy. Laser Doppler perfusion imaging values and ear temperatures were noted to increase after sympathectomy (8.9%, 3°C), although the core temperature of the rabbit did not change. Thus, acute periarterial sympathectomy can (a) effectively reduce the vascular tone of the distal microvasculature and (b) increase total microcirculatory perfusion—cutaneous and thermoregulatory—by both venular and arteriolar dilation. Periarterial sympathectomy has the clinical potential to increase nutritional blood flow, thereby ameliorating the signs and symptoms of ischemia associated with thermoregulatory abnormalities. Dilation of the arteriovenous anastomoses, with a subsequent reduction in vascular resistance, may contribute to the increased cutaneous temperature noted after sympathectomy.     

缺血预处理对大鼠移植肝脏微循环的保护作用

目的：探讨早期再灌注损伤中缺血预处理（IP）对大鼠移植肝脏微循环的保护作用。方法：采用SD大鼠原位肝移植动物模型，供肝冷保存时间100min，无肝期25min。32只SD大鼠随机平均分成两组，每组16只。对照组：获取供肝前仅以肝素生理盐水经门静脉灌注；IP组；获取供肝前阻断肝门血供10min，再灌注10min，然后再以肝素生理盐水经门静脉灌注。移植肝脏再灌注2h后取血及肝脏检测。结果：IP组的肝脏抗氧化酶活力明显高于对照组（P＜0．01），血清丙氨酸转氨酶（ALT）、门冬氨酸转氨酶（AST）、乳酸脱氢酶（LDH）及肝组织中的过氧化产物丙二醛（MDA）含量均明显低于对照组（P＜0．001）；肝组织损伤以窦状内皮细胞为主，并且是以凋亡的方式发生死亡，IP组窦状内皮细胞损伤明显轻于对照组（P＜0．001）。结论：IP对大鼠移植肝脏微循环的早期再灌注损伤有保护作用。     

Acute effects of cigarette smoking on microcirculation of the thumb.

The acute effect of smoking on the microcirculation of the skin of the thumb was investigated in healthy volunteers. Twenty-two were smokers and 10 were non-smokers. The flow was assessed by means of laser Doppler flowmetry. The smokers inhaled 2 cigarettes. During smoking of their first and second cigarette respectively, a mean decrease in laser Doppler flow of 23.8% and 29.0% was seen (p = 0.03; p = 0.01). Ten minutes after smoking this decrease was recovered by half. This experiment confirms that one should prohibit smoking of cigarettes pre- and postoperatively for optimal wound healing conditions.     

Normothermic ischemia effects in renal microcirculation

We report the results obtained in a experimental work designed to evaluate the consequences of warm ischemia in hypothermic isolated renal perfusion. We perfused a number of kidneys after a period of 45 min of vascular occlusion. An alternative group of kidneys were perfused without previous warm ischemia. Ureter was canulated in all the procedures and output collected during the HP. Creatinine was added to the perfusion solution initially in order to determine creatinine clearance. HP hydrodynamics was recorded on real time through a computerised system. According to the results, renal vascular resistance as well as CrCl were higher in ischemic kidneys. Both facts along with minimal differences in pathologic study suggest an increase in vascular tone of efferent-postglomerular arteriole during HP. HP was an adequate technique to minimize histologic consequences of ischemia. Mycrovascular an biochemical changes produced during HP may be produced, essentially, by dynamic causes.     

In vivo effects of hypothermia on the microcirculation during extracorporeal circulation.

OBJECTIVE: Induced hypothermia has been shown to be protective during cardiac surgery, but also in traumatic, ischemic, burn, and neurological injury. In previous in vivo animal experiments, we documented increased leukocyte/endothelial (L/E) cell interaction following normothermic extracorporeal blood circulation (ECC). This study was carried out to investigate whether reduced core temperature during ECC affects the damage to the microcirculation as evidenced by leukocyte adherence and edema formation. METHODS: Intravital fluorescence microscopy was used on the dorsal skinfold chamber preparation in Syrian golden hamsters. ECC was introduced via a micro-rollerpump (1 ml/min) and a 60 cm silicon tube (1mm inner diameter) shunted between the carotid artery and the jugular vein after application of 300IE Heparin/kg per body weight. Experiments were performed in chronically instrumented, awake animals (age 10-14 weeks, weight 65-75 g). Animals of the experimental group were cooled to 18 degrees C body temperature while ECC, followed by a rewarming period (n=7), controls experienced ECC under normothermia (37 degrees C, n=7). RESULTS: 30 min ECC at 18 degrees C resulted in a decrease of rolling and adherent leucocytes (stickers) in postcapillary venules after 1, 4 and 8h compared with the control group (119+/-46 vs. 274+/-113 n/mm2, P<0.05, mean+/-SD; n=7 in each group). Functional capillary density was significantly reduced during hypothermia (80+/-16 vs. 148+/-16 cm/cm2, P<0.05), but restored after rewarming. In contrast, edema formation was markedly increased during hypothermia. CONCLUSIONS: Hypothermia during ECC significantly reduced L/E cell interaction in the early post-ECC period. Hypothermia markedly reduced microvascular perfusion, but was completely restored upon rewarming. Despite a reduced number of adherent leukocytes, no protection of endothelial barrier function was seen as a consequence of induced hypothermia.     

Synergistic effects of brain death and liver steatosis on the hepatic microcirculation

BACKGROUND: The routine transplantation of steatotic livers could potentially mitigate the donor shortage, but so far is associated with a high rate of graft dysfunction. Steatosis and brain death have been perceived as independent risk factors, but they may synergistically target the hepatic microcirculation. This study compares the effects of brain death on the microcirculation of steatotic and normal livers. METHODS: Brain death was induced in obese and lean Zucker rats. Lean and obese sham-operated animals served as controls. Liver microcirculation was investigated using intravital fluorescence microscopy. Serum liver enzyme and reduced glutathione, expression of P-selectin, ICAM-1 and VCAM-1 mRNA in the liver were determined. The ultrastructural alterations were compared by electron microscopy. RESULTS: In nonbrain-dead animals, liver steatosis was associated with smaller sinusoidal diameters, but did not impair sinusoidal perfusion. During brain death, sinusoidal diameter and perfusion were reduced in normal and, to a greater extent, in steatotic livers. Also, more leukocytes were recruited to the microvasculature of steatotic livers than to normal livers in brain-dead state. The highest liver enzyme activities and the lowest hepatic GSH concentrations were measured in brain-dead animals with steatotic livers; only in these organs was endothelial cell swelling regularly observed. In brain-dead state, only the P-selectin mRNA expression was increased in steatotic livers as compared to normal livers. CONCLUSIONS: Brain death amplifies the adverse effects of steatosis on the hepatic microcirculation. Our results underline the need for therapeutic intervention in brain-dead state when steatotic livers are to be used for transplantation.     

In vivo effects of diadenosine polyphosphates on rat renal microcirculation

BACKGROUND: Diadenosine polyphosphates (APXA) are vasoactive nucleotides that elicit effects via purinoceptors. Recent data suggest differential effects of APXA on kidney vasculature. METHODS: The in vivo effects of AP3A, AP5A, and adenosine on renal microvessels and the role of purinoceptors were investigated by the application of agonists to the hydronephrotic rat kidney and preincubation with respective antagonists. RESULTS: The addition of the agonists (10-7 mol/L up to 10-4 mol/L) resulted in a concentration-dependent transient vasoconstriction [interlobular artery (ILOB): adenosine 30 +/- 7%, N = 7, AP3A 35 +/- 10%, N = 5; AP5A 66 +/- 19%, N = 5; 10-5 mol/L each] lasting up to one minute, followed by a concentration-dependent vasodilation (ILOB: adenosine 10 +/- 3%, N = 6; AP3A 19 +/- 4%, N = 5; AP5A 12 +/- 5%, N = 6; 10-5 mol/L each). In ILOB and in the afferent arteriole (AFF), the constrictory effects of AP5A were more pronounced than those of AP3A and adenosine. In the efferent arteriole (EFF), vascular tone was only slightly affected by all agonists. The dilatory potency was comparable for all agonists in ILOB and EFF. No significant vasodilation occurred in AFF. The application of the selective A1 receptor antagonist DPCPX (10-5 mol/L) completely abolished the adenosine-induced vasoconstriction, whereas the A2 receptor antagonist DMPX and the P2 purinoceptor antagonists PPADS and A3P5P (all 10-5 mol/L) did not affect adenosine-induced constriction. The AP3A-induced constriction was abolished by DPCPX and was partially inhibited by PPADS. The constriction induced by AP5A was less sensitive to DPCPX but more sensitive to PPADS. In ILOB and EFF, DMPX or A3P5P abolished dilation after the addition of the agonists. The dilation after AP5A was not significantly reduced. In AFF, no significant dilation was observed with these agonists alone, but it was clearly visible in the presence of DPCPX or PPADS. CONCLUSIONS: APXA evoke transient constrictions in vessels of the hydronephrotic rat kidney, which are mediated by A1 and P2 purinoceptors. The length of the phosphate chain determines the degree of vasoconstriction and the extent to which the substances exert effects on the P2 purinoceptor subtypes. ILOB and AFF are more potently affected by APXA than EFF. Afferent vasodilation is partially overridden by sustained vasoconstriction.     

中药复方“清下1号”对急性水肿性胰腺炎局部微循环损伤的作用

目的探讨大鼠急性水肿性胰腺炎(AEP)动物模型的早期微循环改变及中药复方清下1号（MCP-1）对AEP胰腺微循环的作用。方法用异硫氰酸荧光素-荧光标记红细胞(FITC-RBC)胰腺活体微循环技术、微血管树脂/墨汁灌注光镜和扫描电镜、透射电镜技术,用蛙皮缩胆囊肽诱发大鼠急性胰腺炎（AP）动物模型,观察36只Wistar大鼠的早期微循环改变、MCP-1应用后胰腺局部微循环的反应。结果与AEP自然病程组比较,MCP-1治疗组血清淀粉酶由（2997.7±801.4）?IU/L降至（1909.7±295.5）?IU/L（P<0.01）;胰腺间质炎性细胞浸润减少;腺泡细胞胞浆内空泡减少;毛细血管密度由（52.8±6.1）%增至（63.2±5.5）%（P<0.01);微血管管径由（4.5±0.4）?μm增至（5.9±0.6）?μm（P<0.05）;FITC-RBC显示胰腺微循环流速、流量增加,灌流稳定(0.87±0.06)nl/min（P<0.01)。结论MCP-1具有显著改善AP胰腺微循环的作用,抗AP胰腺局部微循环损伤是实现MCP-1疗效的重要机制。