The purpose of this study is to determine gender-specific differences in the development of necrosis in persistent ischemic tissue and to analyze whether differences are due to gender-specific loss of vascular reactivity or change in ischemic tolerance.Hairless mice (skh-1) of both genders were assigned to three groups of adolescent, adult, and senescent age. Critical ischemia was induced by transection of the two distal pedicles of the animal's ear. Microcirculation was assessed over a 5-day period using intravital epifluorescence microscopy. Tissue necrosis, blood flow, functional capillary density (FCD), red blood cell (RBC) velocity, and capillary diameter were analyzed.Induction of persistent ischemia caused an age-dependent demarcation of nonperfused flap tissue. Adult and senescent females developed markedly more necrosis than age-matched males (49 +/-1% vs. 37 +/-3% and 53 +/- 3% vs. 44 +/- 2%, respectively; p<0.05), whereas no gender-specific difference in flap necrosis was observed in adolescent animals (31 +/- 2% vs. 33 +/- 3%). Gender did not affect the amount of microcirculatory dysfunction in the flap. Thus, age-matched females and males exhibited a comparable decrease of FCD, RBC velocity, and capillary dilatory response.Both age and female gender may predispose for an increased susceptibility to develop ischemic tissue necrosis. The increased necrosis in female animals does not apply to an aggravated microvascular dysfunction, but rather to a reduced ischemic tissue tolerance.

BACKGROUND:Erythropoietin (Epo), the primary regulator of erythropoiesis, has recently been shown to exert antiinflammatory and antiapoptotic properties in neuronal and myocardial tissue. We herein studied whether Epo pretreatment can reduce cell death and ischemic necrosis in a chronic in vivo model. METHODS:C57BL/6 mice were treated daily for 3 consecutive days with either 500 IU EPO/kg body weight (bw) (group Epo 500, n = 8) or 5000 IU EPO/kg bw (group Epo 5000, n = 8) administered intraperitoneally 24 hours before surgery. Thereafter, a random pattern myocutaneous flap subjected to acute persistent ischemia was elevated and fixed into a dorsal skinfold chamber. Flap elevation in animals receiving the water-soluble vitamin E analog Trolox (6-hydroxy-2, 5, 7, 8-tetramethylchroman-2-carboxylic acid) served as a nonspecific antiinflammatory agent control group (Tro); untreated control animals (Con) received saline only. Capillary perfusion, leukocyte-endothelial cell interaction, apoptotic cell death, and tissue necrosis were determined over a 10-day observation period using intravital multifluorescence microscopy. RESULTS:Epo 5000 (44 +/- 26 cm/cm(2)) but, more noticeably, Epo 500 (116 +/- 32 cm/cm(2)) improved capillary perfusion compared with the two control groups, particularly the Con group (9 +/- 7 cm/cm(2); P < .05). The ischemia-associated leukocytic inflammation was found drastically attenuated in both Epo-pretreatment groups. Epo 500 further decreased apoptotic cell death and was effective in significantly reducing tissue necrosis (16% +/- 4% vs Tro: 48% +/- 7% and Con: 52% +/- 4%; P < .001). No angiogenic blood vessel formation could be observed in either of the Epo groups. Of interest, Epo 5000-but not Epo 500-increased systemic hematocrit. CONCLUSION:Despite the lack of neovascularization, Epo pretreatment was capable of reducing ischemic tissue necrosis by protecting capillary perfusion, ie, nutrition of the tissue. Low-dose pretreatment was more effective, a result that was most likely due to the better perfusion conditions without an increase of the hematocrit values. Thus, low-dose Epo pretreatment might represent a promising strategy to protect critically perfused ischemic tissue.

Background: Critical perfusion conditions may impair the function of surgical flaps resulting in partial or even total necrosis. Statins have shown to exert protection in myocardial ischemia through antioxidative, antiapoptotic, anti-inflammatory and angiogenic effects. Herein we studied, whether simvastatin, an inhibitor of the HMG-CoA-reductase, may be effective to confine necrosis in critically perfused myocutaneous flaps by reducing microvascular dysfunction and ischemia-induced inflammation. Methods: A randomly perfused musculocutaneous flap was elevated and fixed into a dorsal skinfold chamber of C57BL/6 mice. Microcirculation in arterioles and capillaries, ischemia-induced inflammatory response, i.e. leukocyte-endothelial cell interaction and apoptotic cell death, neovascularization and tissue necrosis were determined over a 10-day period by intravital fluorescence microscopy. Animals were pretreated with simvastatin (5 mg/kg body weight per injection) 24h and 30 min before flap elevation (n=7; simvastatin). Additional simvastatin was administered daily for 3 consecutive days after flap elevation. Saline-treated animals served as controls (n=7; control). Results: Persistent ischemia in controls induced a considerable inflammatory response characterized by leukocyte-endothelial cell interaction whereas simvastatin was able to significantly reduce this response (control: day 1: 408 ± 47 vs simvastatin: 175 ± 64 adherent leukocytes/mm2 endothelial surface; p < 0.05 vs control). Also, simvastatin resulted in a marked decrease of apoptotic cell death (control: day 3: 218 ± 22 vs simvastatin: 92 ± 12 cells/mm2; p < 0.05). Despite a comparable dilatory response observed in all animals, we noted significant decrease of capillary perfusion within the critically perfused flap tissue only in untreated mice (control: day 1: 84 ± 27 cm/cm2; day 10: 48 ± 20 cm/cm2; simvastatin: day 1: 220 ± 12 cm/cm2; day 10: 244 ± 12 cm/cm2; p < 0.001). The new formation of microvascular structures was observed only in simvastatin pretreated animals. Taken together, these effects resulted in a significant reduction of tissue necrosis (control; day 10: 50 ± 4% vs simvastatin; 23 ± 5% of common flap surface; p < 0.05). Conclusions: These finding indicate, that pretreatment with simvastatin of critically perfused flap tissue is able to reduce ischemia-induced inflammatory response and apoptotic cell death, which is associated with the maintenance of nutritive capillary perfusion and an angiogenic response. Taken together, this interplay of simvastat in-mediated cellular and microvascular protection significantly reduces ischemia-induced tissue necrosis and may thus represent an alternative to pharmacologically manipulate the survival of ischemically challenged flaps.

Background: Erythropoietin (EPO), the main regulator of erythropoiesis, is used to treat anemias of different causes. Recently, EPO has additionally been attributed tissue-protective effects, particularly after severe ischemia in the brain, the heart and the liver, irrespective of its erythropoietic properties. The aim of this study was therefore to elaborate the effect of EPO on critically perfused flap tissue and to study underlying cellular mechanisms. Material & Methods: Chronic ischemia was induced by elevating a randomly perfused flap in the back of C57BL/6-mice subsequently fixed into a dorsal skinfold chamber (control group (CON): n = 8). In the second group EPO (500 IU/kg bodyweight; n = 8) was administered intraperitoneally 30 minutes before as well as 30 minutes and 24 hours after flap elevation (EPO). Repetitive epi-illumination fluorescence microscopy was performed over ten days, assessing arteriolar diameter, flap necrosis, functional capillary density (FCD) and angiogenesis (mean vessel density; MVD). Ischemia-induced inflammatory response was determined by apoptotic cell death and leukocyte-endothelial cell interaction. Immunohistochemistry and Western blot analyses were performed to quantify the expression of inducible nitric oxide synthase (iNOS), endothelial NO-synthase (eNOS) and vascular endothelial growth factor (VEGF). Rheology was repetitively measured in separate animals (n = 8). Results: Increased iNOS (day 1; 2-fold vs basal expression) and eNOS (day 5; 2-fold; p < 0.05) expression of EPO-pretreated mice correlated with significant arteriolar dilation (CON: day1: 49 ± 3 µm, day 10: 52 ± 5 µm; EPO: day 1: 61 ± 5 µm, day 10: 75 ± 5 µm; p < 0.05 vs CON) and hyperperfusion, which resulted in maintained FCD at day 10 (CON: 0 ± 0cm/cm2; EPO: 72 ± 26 cm/cm2; p < 0.05). We further observed a significantly decreased apoptotic cell death (CON: 208 ± 24 cells/mm2; EPO: 90 ± 5 cells/mm2; p < 0.05; day 3) and a reduction of leukocytes adhering to the endothelium of venules (CON: 691 ± 50 stickers/mm2; EPO: 111 ± 14 stickers/mm2; p < 0.05; day 3) in all animals receiving EPO. Moreover, administration of EPO induced an early VEGF-upregulation (2-fold), resulting in newly formed capillaries first visible at day 5 (MVD: day 10; CON: 0 ± 0cm/cm2; EPO: 33 ± 3cm/cm2; p < 0.05). Consequently, EPO-pretreatment resulted in a significantly reduced flap necrosis (CON: 48 ± 2 %; EPO: 20 ± 3 %; p < 0.05; day 10). Hematocrit did not change following EPO-administration (CON: 41.3 ± 0.7 %; EPO: 41.9 ± 1.0 %; day 6). Conclusion: Systemic EPO-administration before induction of ischemia initiates a NO-dependent arteriolar dilation and hence hyperperfusion resulting in maintained capillary perfusion. Furthermore, upregulation of VEGF triggers the formation of newly formed functional capillaries which results in significantly improved tissue survival. Thus, pretreatment with EPO seems to be a promising non-invasive method to reduce ischemia-related complications in flap surgery without stimulating objectionable side effects of erythropoiesis.

Objective: The objective of this study was to analyze whether erythropoietin (EPO) protects from necrosis of critically perfused musculocutaneous tissue and the mechanisms by which this protection is achieved.Background: EPO is the regulator of erythropoiesis and is used to treat patients with anemia of different causes. Recent studies suggest that EPO has also other tissue-protective effects, irrespective of its erythropoietic properties.Material and Methods: C57BL/6-mice were treated with 3 doses of EPO at 500 IU/kg intraperitoneally. EPO was given either before (preconditioning, n = 7), before and after (overlapping treatment, n = 7), or after (treatment, n = 7) surgery. Animals receiving only saline served as controls (CON). Acute persistent ischemia was induced by elevating a randomly perfused flap in the back of the animals. This critically perfused tissue demonstrates all initial microvascular failure of similar to 40%, resulting in similar to 50% tissue necrosis if kept untreated. Repetitive fluorescence microscopy was performed over 10 days, assessing angiogenesis, functional capillary density, inflammatory leukocyte-endothelial cell interaction, apoptotic cell death, and tissue necrosis. Additional molecular tissue analyses included the determination of inducible nitric oxide synthase, erythropoietin receptor (EPO-R), and vascular endothelial growth factor (VEGF).Results: EPO preconditioning did not affect hematocrit and EPO-R expression, but increased inducible nitric oxide synthase in the critically perfused tissue. This correlated with a significant arteriolar dilation, which resulted in a maintained functional capillary density (CON: 0 +/- 0 cm/cm(2); preconditioning: 37 +/- 21 cm/cm(2); overlapping treatment: 72 +/- 26 cm/cm(2); P < 0.05). EPO pretreatment further significantly reduced microvascular leukocyte adhesion and apoptotic cell death. Moreover, EPO pretreatment induced an early VEGF upregulation, which resulted in new capillary network formation (CON: 0 +/- 0 cm/cm(2); preconditioning: 40 +/- 3 cm/cm(2); overlapping treatment: 33 +/- 3 cm/cm(2); p < 0.05). Accordingly, EPO pretreatment significantly reduced tissue necrosis (CON: 48% 2%; preconditioning: 26% +/- 3%; overlapping treatment: 20% +/- 3%; P < 0.05). of interest, EPO treatment was only able to alleviate ischemia-induced inflammation but could not improve microvascular perfusion and tissue survival.Conclusions: EPO pretreatment improves survival of critically perfused tissue by nitric oxide -mediated arteriolar dilation, protection of capillary perfusion, and VEGF-initiated new blood vessel formation.

Background. Tissues are endowed with protective mechanisms to counteract chronic ischemia. Previous studies have demonstrated that endogenous heme oxygenase (HO)-1 may protect parenchymal tissue from inflammation- and reoxygenation-induced injury. Nothing is known, however, on whether endogenous HO-1 also plays a role in chronic ischemia to protect from development of tissue necrosis. The aim of this study is, therefore, to evaluate in vivo whether endogenous HO-1 exerts protection on chronically ischemic musculocutaneous tissue, and whether this protection is mediated by an attenuation of the microcirculatory dysfunction.Materials and methods. In C57BL/6-mice, a chronically ischemic flap was elevated and fixed into a dorsal skinfold chamber. In a second group, tin-protoporphyrin-IX was administrated to competitively block the action of HO-1. Animals without flap elevation served as controls. With the use of intravital fluorescence microscopy, microcirculation, apoptotic cell death, and tissue necrosis were analyzed over a 10-day observation period. The time course of HO-1 expression was determined by Western blotting.Results. Chronic ischemia induced an increase of HO-1 expression, particularly at day 1 and 3. This was associated with arteriolar dilation and hyperperfusion, which was capable of maintaining an adequate capillary perfusion density in the critically perfused central part of the flap, demarcating the distal necrosis. Inhibition of endogenous HO-1 by tin-protoporphyrin-IX completely abrogated arteriolar dilation (44.6 +/- 6.2 mu m versus untreated flaps: 71.3 +/- 7.3 mu m; P < 0.05) and hyperperfusion (3.13 +/- 1.29 nL/s versus 8.55 +/- 3.56 nL/s; P < 0.05). This resulted in a dramatic decrease of functional capillary density (16 +/- 16 cm/ cm(2) versus 84 +/- 31 cm/cm(2); P < 0.05) and a significant increase of apoptotic cell death (585 +/- 51 cells/mm(2) versus 365 +/- 53 cells/mm(2); P < 0.05), and tissue necrosis (73% +/- 5% versus 51% +/- 5%; P < 0.001).Conclusion. Thus, our results suggest that chronic ischemia-induced endogenous HO-1 protects ischemically endangered tissue, probably by the vasodilatory action of the HO-1-associated carbon monoxide. (C) 2008 Elsevier Inc. All rights reserved.

BACKGROUND:The purpose of the present study was to develop of a rodent model of abdominal compartment syndrome (ACS), which allows detailed analysis of intra-abdominal hypertension (IAH)- and decompression-associated reperfusion injury. METHODS:In 20 anesthetized and ventilated Sprague-Dawley rats an IAH of 20 mmHg was induced for 3 h by intraperitoneal infusion of gelatin polysuccinate. After decompression, an additional 3-h period of reperfusion was studied. Sham-operated animals, undergoing identical procedures without IAH induction, served as controls. Controlled hyperventilation and intravenous fluid substitution were adapted to keep PCO(2) <60 mmHg and mean arterial pressure (MAP) >100 mmHg. RESULTS:IAH of 20 mmHg could successfully be maintained for the entire 3-h period. MAP was not affected during IAH, however, decreased upon decompression despite forced fluid resuscitation. CVP was markedly elevated during IAH, but returned to baseline after decompression. Of interest, the IAH-induced reduction of PaO(2) did not recover to baseline after decompression, indicating a persistent deterioration of gas exchange. In contrast, IAH-associated elevation of PaCO(2) normalized during reperfusion. IAH was further accompanied by metabolic acidosis, which persisted after decompression, indicating reperfusion injury. IAH was further associated with a significant increase of serum potassium, lactate, AST, LDH, bilirubin, urea, and creatinine as well as creatine kinase (CK) and CK-MB. Histomorphological analysis revealed parenchymal injury in liver, lung, intestine, and myocardium. CONCLUSION:We established an easily reproducible ACS model in the rat, demonstrating hemodynamic deteriorations and organ dysfunctions similar as known from patients with IAH. Decompression did not restore functional deteriorations, indicating persistent post-ACS reperfusion injury. The model may be suitable to study mechanisms and novel treatment strategies in ACS.

Objective: To investigate whether microdialysis is capable of assessing metabolic derangements during intra-abdominal hypertension (IAH), and whether monitoring of the rectus abdominis muscle (RAM) by microdialysis represents a reliable approach in the early detection of organ dysfunctions in abdominal compartment syndrome (ACS). Design: Prospective, randomized, controlled animal study. Setting: University animal research facility. Subjects: Fifteen isoflurane-anesthetized and mechanically ventilated Sprague -Dawley rats. Interventions: IAH of 20 mmHg was induced for 3 h and followed by decompression and reperfusion for another 3-h period (n = 10). Five sham-operated animals served as controls. Microdialysis was performed in the anterior gastric wall, liver, kidney, and RAM. The anterior cervical muscles served as distant reference. Glucose, lactate, pyruvate, and glycerol was analyzed throughout the 6-h experiment. Measurements and main results: Prolonged IAH induced significant cardiopulmonary dysfunction and persistent abdominal organ injury. Microdialysis revealed a significant increase of lactate/pyruvate and glycerol in kidney, intestine and liver, indicating ischemia, energy failure, and cell membrane damage. In addition, at 3 h IAH glucose was significantly decreased in all organs studied. The distant reference did not show any alteration of lactate/pyruvate, glycerol, and glucose over the entire 6-h observation period. In contrast to the other organs, microdialysis of the RAM showed an early and more pronounced increase of lactate, lactate/pyruvate and glycerol already at 1 h IAH. It is noteworthy that lactate, glycerol, and glucose did not completely recover upon decompression of IAH. Conclusions: Our data suggest that continuous microdialysis in the RAM may represent a promising tool for early detecting IAH-induced metabolic derangements.

Background and aims Insufficient perfusion of distal flap areas, which may lead to partial necrosis, still represents a challenge in reconstructive surgery. In the process of microvascular and endothelial dysfunction, endothelins (ETs) and their receptors may play an important role. Therefore, the aim of the study was to investigate in a chronic in vivo model the effect of various ET-receptor antagonists in critically perfused flap tissue.Materials and methods A random pattern musculocutaneous flap was elevated in the back of 25 C57BL/6 mice and fixed into a dorsal skinfold chamber. Repetitive intravital fluorescence microscopy was performed over a 10-day observation period, assessing arteriolar diameter, arteriolar blood flow (aBF), functional capillary density (FCD), the area of tissue necrosis, and the development of newly formed blood vessels. ET-receptor blockers were administrated intraperitoneally 30 min before induction of ischemia, as well as daily during the subsequent 4-day period, including (1) BQ-123, a specific ET-A-receptor antagonist (ET-A=1 mg/kg), (2) BQ-788, a selective ET-B-receptor antagonist (ET-B=1 mg/kg), and (3) PD-142893, a nonse-lective ET-AB-receptor antagonist (ET-AB=0.5 mg/kg). Animals receiving saline only served as controls (n=7).Results Despite an increase in aBF during the 10-day observation period (day 1=1.92 +/- 0.29 nl/s; day 10=4.70 +/- 1.64 nl/s), the flaps of saline-treated controls showed a distinct decrease in FCD (94 +/- 12 cm/cm(2)). This perfusion failure resulted in flap necrosis of 52 +/- 3%. Selective blockade of the ET-B receptor caused a further increase in aBF already at day 1 (2.97 +/- 0.42 nl/s), which persisted during the following 10-day observation period (day 10=5.74 +/- 0.69 nl/s). Accordingly, adequate FCD could be maintained (day 10=215 +/- 8 cm/cm(2); p < 0.05 vs control), resulting in a significant reduction in flap necrosis (day 10=25 +/- 4%; p < 0,05). In contrast, neither selective blockade of the ET-A receptor nor nonselective ET-A- and ET-B-receptor blockade were able to significantly affect aBF when compared to controls (day 1=ET-A=1.39 +/- 0.10 nl/s; ET-AB=1.53 +/- 0.80 nl/s; n.s.). Accordingly, flap necrosis after ET-A- and ET-AB-receptor inhibition did not differ from that of controls (day 10=ET-A: 46 +/- 10%; ET-AB=51 +/- 7%).Conclusion Our data show that only selective ET-B-receptor inhibition is capable of maintaining nutritive perfusion and, hence, reducing necrosis in critically perfused flap tissue. Accordingly, administration of ET-B-receptor antagonists may be considered in the treatment of critically perfused flaps.

In the present study in a murine model of chronic ischaemia, we analysed: (i) whether aging was associated with an increased susceptibility to ischaemic necrosis, and (ii) whether this was based on microvascular dysfunction or reduced ischaemic tolerance. An ischaemic pedicled skin flap was created in the ear of homozygous hairless mice. The animals were assigned to three age groups, including adolescent (2+/-1 months), adult (10+/-2 months) and senescent (19+/-3 months). Microvascular perfusion of the ischaemic flap was assessed over 5 days by intravital microscopy, evaluating FCD (functional capillary density), capillary dilation response and the area of tissue necrosis. Expression of the stress-protein HO (haem oxygenase)-1 was determined by immunohistochemistry and Western blotting. Induction of chronic ischaemia stimulated a significant expression of HO-1 without a significant difference between the three age groups. This was associated with capillary dilation, which, however, was more pronounced in adolescent (10.5+/-2.8 microm compared with 3.95+/-0.79 microm at baseline) and adult (12.1+/-3.1 microm compared with 3.36+/-0.45 microm at baseline) animals compared with senescent animals (8.5+/-1.7 microm compared with 3.28+/-0.69 microm at baseline; P value not significant). In senescent animals, flap creation further resulted in complete cessation of capillary flow in the distal area of the flap (FCD, 0+/-0 cm/cm(2)), whereas adult (11.9+/-13.5 cm/cm(2)) and, in particular, adolescent animals (58.4+/-33.6 cm/cm(2); P<0.05) were capable of maintaining residual capillary perfusion. The age-associated microcirculatory dysfunction resulted in a significantly increased flap necrosis of 49+/-8% (P<0.05) and 42+/-8% (P<0.05) in senescent and adult animals respectively, compared with 31+/-6% in adolescent mice. Of interest, functional inhibition of HO-1 by SnPP-IX (tin protoporphyrin-IX) in adolescent mice abrogated capillary dilation, decreased functional capillary density and aggravated tissue necrosis comparably with that observed in senescent mice. Thus aging is associated with an increased susceptibility to tissue necrosis, which is due to a loss of vascular reactivity to endogenous HO-1 expression, rather than a reduction in ischaemic tolerance.

Long-term function of three-dimensional (3D) tissue constructs depends on adequate vascularization after implantation. Accordingly, research in tissue engineering has focused on the analysis of angiogenesis. For this purpose, 2 sophisticated in vivo models (the chorioallantoic membrane and the dorsal skinfold chamber) have recently been introduced in tissue engineering research, allowing a more detailed analysis of angiogenic dysfunction and engraftment failure. To achieve vascularization of tissue constructs, several approaches are currently under investigation. These include the modification of biomaterial properties of scaffolds and the stimulation of blood vessel development and maturation by different growth factors using slow-release devices through pre-encapsulated microspheres. Moreover, new microvascular networks in tissue substitutes can be engineered by using endothelial cells and stem cells or by creating arteriovenous shunt loops. Nonetheless, the currently used techniques are not sufficient to induce the rapid vascularization necessary for an adequate cellular oxygen supply. Thus, future directions of research should focus on the creation of microvascular networks within 3D tissue constructs in vitro before implantation or by co-stimulation of angiogenesis and parenchymal cell proliferation to engineer the vascularized tissue substitute in situ.

BACKGROUND:The present study aimed at quantitatively evaluating the impact of severity of local trauma on manifestation of soft-tissue injury-associated microcirculatory and microlymphatic dysfunctions in a chronic model that allowed repeated analyses by intravital fluorescence microscopy.METHODS:C57BL/6 mice were chronically instrumented with dorsal skinfold chambers and subjected to mild (180 J/m2, n = 6), moderate (270 J/m2, n = 6), or severe trauma (450 J/m2, n = 6; 540 J/m2, n = 6). Nontraumatized animals served as controls (sham; n = 8). Intravital microscopy was performed before and at 5 minutes, 1 hour, 8 hours, 24 hours, 3 days, and 5 days after trauma, and included the analysis of (1) blood and lymph microvessel rupture, (2) hematoma formation and lymph leakage, (3) arteriolar and venular constriction, (4) capillary perfusion failure, (5) arteriolar and venular leukocyte adhesion, and (6) interstitial edema formation.RESULTS:Mild trauma did not induce any changes of microcirculatory and microlymphatic functions. Moderate trauma did not affect lymphatics but provoked arteriolar constriction, capillary perfusion failure, leukocyte-endothelial cell interactions, and minor blood vessel ruptures with hematoma formation. These alterations, however, recovered within the first 24 hours after trauma. Severe trauma also did not affect the lymphatic microvasculature, but resulted in massive hematoma formation, arteriolar constriction, and capillary perfusion failure, which was associated with marked arteriolar and venular leukocyte recruitment and edema formation, and which did not recover to normal over a 5-day observation period.CONCLUSION:Only severe trauma of > 450 J/m2 provokes irreversible microcirculatory dysfunction in soft tissue, however, without affecting the integrity of lymphatic microvessels. Of interest, trauma-induced microcirculatory alterations are neither dominated solely by microcirculatory dysfunction nor by leukocytic inflammation. Instead, both pathologies develop in parallel, generating a vicious circle, which may be responsible for the compromised healing of severely traumatized soft tissue frequently observed in clinical practice.

Severe soft tissue trauma still remains a problem in surgical practice, resulting in disturbed wound healing and infection of the traumatized tissue. Delayed wound healing may be triggered by the dysbalance of reactive oxygen species and endogenous antioxidative mediators. In the present study we evaluated the effects of supplementation with vitamin E before and after isolated severe soft tissue trauma on microcirculatory, inflammatory and cellular tissue damage. Following implantation of a dorsal skinfold chamber, we induced standardized soft tissue trauma in the chamber tissue in C57BL/6J-mice. Therapy with the water-soluble vitamin E-analogue Trolox (6 mg/kg bw ip every 12 hours until day 3) was started 30min before (n=9) or 60min after trauma (n=9). Seven animals received the vehicle PBS only; animals subjected to trauma without further treatment served as controls (n=7). Arteriolar diameters and blood flow (aBF), functional capillary density (FCD), venular leukocyte adherence, endothelial permeability and the number of apoptotic cells were assessed by intravital fluorescence microscopy before and directly after trauma and at 8 h, 24 h, 3 d and 5 d.

Pancreatic cancer is associated with the worst 5-year survival rate of any human cancer. This high mortality is due, in part, to difficulties in establishing early and accurate diagnosis. Because most tumours share the ability to accumulate amino acids more effectively than normal tissues and any other pathology, assessment of amino acid transport in tumour cells using radiolabelled amino acids has become one of the most promising tools for tumour imaging. This study investigated the potential of p-[123I]iodo-l-phenylalanine (IPA) for detection of pancreatic cancer by single-photon emission tomography. IPA affinity for pancreatic tumour was investigated in human pancreatic adenocarcinoma PaCa44 and PanC1 cells, followed by analysis of the underlying mechanisms of tracer accumulation in neoplastic cells. Thereafter, IPA was evaluated for targeting of pancreatic tumours using SCID mice engrafted with primary human pancreatic adenocarcinoma cells, as well as in acute inflammation models in immunocompetent mice and rats. IPA accumulated intensively in human pancreatic tumour cells. Radioactivity accumulation in tumour cells following a 30-min incubation at 37°C/pH 7.4 varied from 41% to 58% of the total loaded activity per 106 cells. The cellular uptake was temperature and pH dependent and predominantly mediated by specific carriers for neutral amino acids, namely the sodium-independent and l-leucine-preferring (L-system) transporter and the alanine-, serine- and cysteine-preferring (ASC-system) transporter. Protein incorporation was less than 8%. Biodistribution studies showed rapid localization of the tracer to tumours, reaching 10%±2.5% to 15%±3% of the injected dose per gram (I.D./g) in heterotopic tumours compared with 17%±3.5% to 22%±4.3% I.D./g in the orthotopic tumours, at 60 and 240 min post injection of IPA, respectively. In contrast, IPA uptake in the gastrointestinal tract and areas of inflammation remained moderate and decreased with time. Excellent tumour detection was obtained by gamma camera imaging. The specific and high-level targeting of IPA to tumour and the negligible uptake in the gastrointestinal tract and areas of inflammation indicate that p-[123I]iodo-l-phenylalanine is a promising tracer for differential diagnosis of pancreatic cancer.

Die Entzündungsreaktion des traumatisierten Gewebes stellt nach wie vor eine Herausforderung in der Chirurgie dar, welche neben der Belastung für den Patienten auch hohe Kosten verursacht. Sowohl die Störung der Mikrozirkulation als auch der Anstieg des apoptoti-schen Gewebeschadens sind hier von wesentlicher Bedeutung. Ziel der vorliegenden Studie war, die Beteiligung von p53 an der TNF-α-induzierten entzündlichen Gewebereaktion durch temporäre Inhibition mit Pifithrin-α (PFT-α) zu klären.

Background. During the past decade, experimental studies have provided convincing evidence that microcirculatory dysfunction plays a pivotal role in the manifestation of tissue injury in ischemia-reperfusion and osteomyocutaneous flap transfer. The study of the mechanisms of injury, however, requires sophisticated experimental in vivo models. With the use of microsurgical techniques, osteomyocutaneous flap transfer can successfully be performed in rat hind limbs, allowing in vivo fluorescent microscopic analysis of post-ischemic microcirculatory dysfunction in all tissues involved, including periosteum, striated muscle, subcutis and skin. The drawback of this "acute" model is that the period of analysis is restricted to a few hours only. Method. To overcome this limitation, the "chronic" dorsal skinfold chamber preparation, containing striated muscle and subcutis, can be used. This model allows one to study microcirculatory dysfunction after both tourniquet-induced and pressure-induced ischemia-reperfusion-induced tissue injury over a period of up to 3 weeks. Results. With the use of these models, recent investigations have demonstrated that ischemia-reperfusion and osteomyocutaneous flap transfer are associated with capillary perfusion failure (no-reflow), mediated by intravascular hemoconcentration, endothelial swelling and endothelin (ET)-1-mediated microvascular constriction. In addition, post-ischemic reperfusion provokes an inflammatory response (reflow paradox) in post-capillary venules, which is characterized by beta(2)-integrin-mediated and intercellular adhesion molecule (ICAM)-1-mediated leukocyte adhesion and vascular hyperpermeability, which results in interstitial edema formation. Treatment studies have produced evidence that isovolemic hemodilution and heat shock protein induction are successful in ameliorating capillary no-reflow, while blockade of adhesion molecules, inactivation of oxygen radicals and, also, induction of heat shock proteins, are capable of reducing the post-ischemic inflammatory response. Conclusion. These experimental results not only demonstrate the importance of the use of advanced in vivo methods to delineate pathophysiological mechanisms in complex disease models, but may also provide a basis for potential prospective randomized trials to test the benefit for the patient in the daily clinical routine.

The protective effect of cooling on traumatized and inflamed tissue has been recognized long time ago, yet the underlying mechanisms are not fully understood. In our study we used the competitive inhibitors of constitutive NO synthase and heme oxygenase (HO-2), N G -nitro-L-arginine-methyl-ester (L-NAME) and tin-protoporphyrine IX (SnPP-IX) to evaluate the impact of NO and CO on microvascular and cellular changes of inflamed tissue after cooling. Dorsal skinfold chambers of hairless mice were exposed to 2000 U of TNF-α and subsequently cooled for 30 min by immersion with ice-cold saline to a surface temperatur of 8–10°C. Arteriolar diameters, arteriolar Volumetric blood flow (VBF), functional capillary density (FCD) and the number of permanent adherent leukocytes in venules were evaluated by intravital fluorescence microscopy at baseline and 30,60,90,120 and 180 min after exposure to TNF-α. Animals were either exposed to TNF-α and cooling (n=6) or were additonally pre-treated with L-NAME and SnPP-IX (n = 6). Animals receiving TNF-α without subsequent cooling served as controls (n=6). Data are given as % of baseline values ± SEM. ANOVA and Students’ Newman-Keuls test. In controls, arteriolar diameters remained constant after TNF-α exposure, while VBF and FCD decreased to about 60% of baseline after 180 min. Further, TNF-α induced a significant increase of the number of adherent leukocytes, reflecting the inflammatory response. Cooling of TNF-α-exposed tissue significantly attenuated all microcirculatory disorders. After pharmacological blockade of both NO and CO, hypothermia-induced vasoconstriction, hypoperfusion and reduction of FCD persisted during passive rewarming of tissue. Furthermore, the number of adherent leukocytes increased significantly despite initial cooling. To summarize, our results show that endogenously produced NO and CO are involved in the hypothermia-induced protection of inflamed tissue as blocking of both mediators led to an inhibition of the cooling-associated reconstitution of microvascular perfusion. Moreover, NO and CO seem to mediate the anti-adhesive effects of cooling and thus the reduetion of TNF-α-induced inflammatory response.

Die ischämische Präkonditionierung stellt ein interessantes Verfahren zur Gewebeprotektion bei plastisch-chirurgischen Eingriffen dar [1]. Der Mechanismus der protektiven Wirkung ist jedoch weiter ungeklärt. Neuere Untersuchungen zeigen, daß Hitzeschockprotein(HSP)-32 (Hemeoxygenase-1) einen Gewebeschaden vermindern kann [2]. Wir untersuchten daher, ob ischämische Präkonditionierung über Induktion von HSP-32-Expression gewebeprotektiv wirkt, und welche der beiden für HSP-32 beschriebenen Wirkungsmechanismen (vasoaktiv, antioxidativ) beteiligt sind.