Heat Shock Preconditioning Reduces the Formation of 8-hydroxy-2′-deoxyguanosine and 4-hydroxy-2-nonenal Modified Proteins in Ischemia-reperfused Liver of Rats

Heat shock preconditioning (HSPC) is a promising strategy for providing ischemic tolerance. The objective of this study is to investigate the effectiveness of HSPC in preventing oxidative damage of cellular proteins and DNA during ischemia-reperfusion of the liver. Male Wistar rats were divided into a heat shock group (group HS) and control (group C). Forty-eight hours prior to ischemia, rats in group HS received HSPC at 42°C for 15 &#117 min. All rats received hepatic warm ischemia for 30 &#117 min and subsequent reperfusion. The formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG), 4-hydroxy-2-nonenal (HNE) modified proteins in liver tissue, survival rate of the animals, and changes in biochemical and histological parameters were compared between groups. Heat shock protein 72 was produced only in group HS. The 7-day survival of rats was significantly better in group HS (10/10) than in group C (5/10) ( p <0.01). The serum release of alanine aminotransferase ( n =10, p <0.01) and the concentration of adenosine triphosphate in liver tissue ( n =10, p <0.01) 40 &#117 min after reperfusion was significantly better in group HS than in group C. The formation of 8-OHdG in liver tissue measured by high-performance liquid chromatography was suppressed in group HS ( p <0.01). The production of HNE-modified proteins as determined by Western-blot analysis was also decreased in group HS. These results were also confirmed by immunohistochemical analysis. As determined by levels of 8-OHdG and HNE-modified proteins produced during ischemia-reperfusion of the liver, HSPC reduced the oxidative injury of cellular proteins and DNA in the liver tissue.     

Heat shock preconditioning reduces the formation of 8-hydroxy-2'-deoxyguanosine and 4-hydroxy-2-nonenal modified proteins in ischemia-reperfused liver of rats

Heat shock preconditioning (HSPC) is a promising strategy for providing ischemic tolerance. The objective of this study is to investigate the effectiveness of HSPC in preventing oxidative damage of cellular proteins and DNA during ischemia-reperfusion of the liver. Male Wistar rats were divided into a heat shock group (group HS) and control (group C). Forty-eight hours prior to ischemia, rats in group HS received HSPC at 42 degrees C for 15 min. All rats received hepatic warm ischemia for 30min and subsequent reperfusion. The formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG), 4-hydroxy-2-nonenal (HNE) modified proteins in liver tissue, survival rate of the animals, and changes in biochemical and histological parameters were compared between groups. Heat shock protein 72 was produced only in group HS. The 7-day survival of rats was significantly better in group HS (10/10) than in group C (5/10) (p < 0.01). The serum release of alanine aminotransferase (n = 10, p < 0.01) and the concentration of adenosine triphosphate in liver tissue (n = 10, p < 0.01) 40min after reperfusion was significantly better in group HS than in group C. The formation of 8-OHdG in liver tissue measured by high-performance liquid chromatography was suppressed in group HS (p < 0.01). The production of HNE-modified proteins as determined by Western-blot analysis was also decreased in group HS. These results were also confirmed by immunohistochemical analysis. As determined by levels of 8-OHdG and HNE-modified proteins produced during ischemia-reperfusion of the liver, HSPC reduced the oxidative injury of cellular proteins and DNA in the liver tissue.     

Local hypothermia during early reperfusion protects skeletal muscle from ischemia-reperfusion injury

Amputated tissue maintained in a hypothermic environment can endure prolonged ischemia and improve replantation success. The authors hypothesized that local tissue hypothermia during the early reperfusion period may provide a protective effect against ischemia-reperfusion injury similar to that seen when hypothermia is provided during the ischemic period. A rat gracilis muscle flap model was used to assess the protective effects of exposing skeletal muscle to local hypothermia during ischemia only (p = 18), reperfusion only (p = 18), and both ischemia and reperfusion (p = 18). Gracilis muscles were isolated and exposed to hypothermia of 10 degrees C during 4 hours of ischemia, the initial 3 hours of reperfusion, or both periods. Ischemia-reperfusion outcome measures used to evaluate muscle flap injury included muscle viability (percent nitroblue tetrazolium staining), local edema (wet-to-dry weight ratio), neutrophil infiltration (intramuscular neutrophil density per high-power field), neutrophil integrin expression (CD11b mean fluorescence intensity), and neutrophil oxidative potential (dihydro-rhodamine oxidation mean fluorescence intensity) after 24 hours of reperfusion. Nitroblue tetrazolium staining demonstrated improved muscle viability in the experimental groups (ischemia-only: 78.8 +/- 3.5 percent, p < 0.001; reperfusion-only: 80.2 +/- 5.2 percent, p < 0.001; and ischemia-reperfusion: 79.6 +/- 7.6 percent, p < 0.001) when compared with the nonhypothermic control group (50.7 +/- 9.3 percent). The experimental groups demonstrated decreased local muscle edema (4.09 +/- 0.30, 4.10 +/- 0.19, and 4.04 +/- 0.31 wet-to-dry weight ratios, respectively) when compared with the nonhypothermic control group (5.24 +/- 0.31 wet-to-dry weight ratio; p < 0.001, p < 0.001, and p < 0.001, respectively). CD11b expression was significantly decreased in the reperfusion-only (32.65 +/- 8.75 mean fluorescence intensity, p < 0.001) and ischemia-reperfusion groups (25.26 +/- 5.32, p < 0.001) compared with the nonhypothermic control group (62.69 +/- 16.93). There was not a significant decrease in neutrophil CD11b expression in the ischemia-only group (50.72 +/- 11.7 mean fluorescence intensity, p = 0.281). Neutrophil infiltration was significantly decreased in the reperfusion-only (20 +/- 11 counts per high-power field, p = 0.025) and ischemia-reperfusion groups (23 +/- 3 counts, p = 0.041) compared with the nonhypothermic control group (51 +/- 28 counts). No decrease in neutrophil density was observed in the ischemia-only group (40 +/- 15 counts per high-power field, p = 0.672) when compared with the nonhypothermic control group (51 +/- 28 counts). Finally, dihydrorhodamine oxidation was significantly decreased in the reperfusion-only group (45.83 +/- 11.89 mean fluorescence intensity, p = 0.021) and ischemia-reperfusion group (44.30 +/- 11.80, p = 0.018) when compared with the nonhypothermic control group (71.74 +/- 20.83), whereas no decrease in dihydrorhodamine oxidation was observed in the ischemia-only group (65.93 +/- 10.3, p = 0.982). The findings suggest a protective effect of local hypothermia during early reperfusion to skeletal muscle after an ischemic insult. Inhibition of CD11b expression and subsequent neutrophil infiltration and depression of neutrophil oxidative potential may represent independent protective mechanisms isolated to local tissue hypothermia during the early reperfusion period (reperfusion-only and ischemia-reperfusion groups). This study provides evidence for the potential clinical utility of administering local hypothermia to ischemic muscle tissue during the early reperfusion period.     

Heat shock does not attenuate low-frequency fatigue.

Whole-body hyperthermia or heat shock confers protection to myocardial contractility against reperfusion-induced injury. The purpose of this study was to determine whether heat shock could provide similar protection to skeletal muscle contractility against low-frequency fatigue. Male Sprague-Dawley rats (6 rats/group) were heat shocked at 41.5 degrees C for 15 min either 24 h or 4 days prior to fatiguing stimulation to compare the contractile responses of the plantaris muscle with those of a nonheated group. Both 24 h and 4 days after heat shock, the 72-kDa heat shock protein (HSP72) was elevated above control levels. There were no differences between the heat-shocked and non-heat-shocked animals in measures of contractility prior to fatiguing contractions or in resistance to fatigue. Heat-shock preconditioning did not lead to improved postfatigue force recovery above control responses and, in fact, delayed the recovery of force. This study does not support the use of heat-shock therapy to improve skeletal muscle contractile performance under fatiguing conditions.     

Effects of L-NAME and L-arginine on ischemia-reperfusion injury in rat skeletal muscle

The involvement of nitric oxide in ischemia-reperfusion injury remains controversial and has been reported to be both beneficial and deleterious, depending on the tissue and model used. This study evaluated the effects of the nitric oxide synthase inhibitor N(G)-nitro-L-arginine-methyl ester (L-NAME) and the substrate for nitric oxide synthase, L-arginine on skeletal muscle necrosis in a rat model of ischemia-reperfusion injury. The rectus femoris muscle in male Wistar rats (250 to 500 g) was isolated on its vascular pedicle and subjected to 4 hours of complete arteriovenous occlusion. The animals were divided into five groups: (1) sham-raised control, no ischemia, no treatment (n = 6); (2) 4 hours of ischemia (n = 6); (3) vehicle control, 4 hours of ischemia + saline (n = 6); (4) 4 hours of ischemia + L-arginine infusion (n = 6); and (5) 4 hours of ischemia + L-NAME infusion (n = 6). The infusions (10 mg/kg) were administered into the contralateral femoral vein beginning 5 minutes before reperfusion and during the following 30 to 45 minutes. Upon reperfusion, the muscle was sutured in its anatomic position and all wounds were closed. The percentage of muscle necrosis was assessed after 24 hours of reperfusion by serial transections, nitroblue tetrazolium staining, digital photography, and computerized planimetry. Sham (group 1) animals sustained baseline necrosis of 11.9 +/- 3.0 (percentage necrosis +/- SEM). Four hours of ischemia (group 2) significantly increased necrosis to 79.2 +/- 1.4 (p < 0.01). Vehicle control (group 3) had no significant difference in necrosis (81.17 +/- 5.0) versus untreated animals subjected to 4 hours of ischemia (group 2). Animals treated with L-arginine (group 4) had significantly reduced necrosis to 34.6 +/- 7.5 versus untreated (group 2) animals (p < 0.01). Animals infused with L-NAME (group 5) had no significant difference in necrosis (68.2 +/- 6.7) versus untreated (group 2) animals. L-Arginine (nitric oxide donor) significantly decreased the severity of muscle necrosis in this rat model of ischemia-reperfusion injury. L-arginine is known to increase the amount of nitric oxide through the action of nitric oxide synthase, whereas L-NAME, known to inhibit nitric oxide synthase and decrease nitric oxide production, had comparable results to the untreated 4-hour ischemia group. These results suggest that L-arginine, presumably through nitric oxide mediation, appears beneficial to rat skeletal muscle subjected to ischemia-reperfusion injury.     

Preconditioning of latissimus dorsi muscle flaps with monophosphoryl lipid a

The use of dynamic myoplasty to restore function to failing organs is an exciting new application of skeletal muscle flaps. A complication of large flap elevation that can compromise flap function is ischemia-induced necrosis; one approach to minimizing this is to pretreat tissues with ischemic preconditioning. The purpose of this study was to determine whether systemic administration of monophosphoryl lipid A, a drug known to mimic late-phase ischemic preconditioning in the heart, could reduce ischemia-induced necrosis in latissimus dorsi muscle flaps. Forty latissimus dorsi muscle flaps from 20 Sprague-Dawley rats were allocated into four groups. In group I (n = 10), flaps were not preconditioned and served as controls. In group II (n = 10), flaps received ischemic preconditioning with two 30-minute periods of ischemia interspersed by 10 minutes of reperfusion. In group III (n = 10), rats received an intravenous bolus of approximately 0.3 ml of monophosphoryl lipid A vehicle only. In group IV (n = 10), rats received an intravenous bolus of 450 microg/kg of monophosphoryl lipid A and vehicle. Twenty-four hours after treatment, all latissimus dorsi muscle flaps were elevated on a single neurovascular pedicle and subjected to 4 hours of ischemia. After 72 hours of reperfusion, latissimus dorsi muscles were harvested, weighed, stained with nitroblue tetrazolium, and assessed for percent necrosis using digitized images of muscle sections and computerized planimetry. The percent necrosis in ischemic preconditioning-treated flaps (group II) was significantly reduced by 57 percent (p < 0.05) compared with control flaps (group I). The percent necrosis in flaps treated with monophosphoryl lipid A (group IV) was significantly reduced by 58 percent (p < 0.05) compared with vehicle-control flaps (group III). There was no difference in mean percent necrosis between ischemic preconditioning (group II) and monophosphoryl lipid A-treated (group IV) flaps or between ischemic preconditioning-control (group I) and monophosphoryl lipid A vehicle-control (group III) flaps. Intravenous administration of systemic monophosphoryl lipid A mimics the late-phase protective effect of ischemic preconditioning in the authors' rat latissimus dorsi muscle flap model.     

Heat stress attenuates air bubble-induced acute lung injury: a novel mechanism of diving acclimatization.

Diving acclimatization refers to a reduced susceptibility to acute decompression sickness (DCS) in individuals undergoing repeated compression-decompression cycles. We postulated that mechanisms responsible for the acclimatization are similar to that of a stress preconditioning. In this study, we investigated the protective effect of prior heat shock treatment on air embolism-induced lung injury and on the incidence of DCS in rats. We exposed rats (n = 31) to a pressure cycle that induced signs of severe DCS in 48% of the rats, greater wet-to-dry ratio (W/D) of lung weight compared with the control group (5.48 +/- 0.69 vs. 4.70 +/- 0.17), and higher protein concentration in bronchoalveolar lavage (BAL) fluid (362 +/- 184 vs. 209 +/- 78 mg/l) compared with the control group. Rats with DCS expressed more heat shock protein 70 (HSP70) in the lungs than those without signs of disease. Prior heat shock (n = 12) increased the expression of HSP70 in the lung and attenuated the elevation of W/D of lung weight (5.03 +/- 0.17) after the identical decompression protocol. Prior heat shock reduced the incidence of severe DCS by 23%, but this failed to reach statistical significant (chi(2) = 1.94, P = 0.163). Venous air infusion (1.0 ml/40 min) caused profound hypoxemia (54.5 +/- 3.8 vs. 83.8 +/- 3.2 Torr at baseline; n = 6), greater W/D of lung weight (5.98 +/- 0.45), and high protein concentration in BAL fluid (595 +/- 129 mg/l). Prior heat shock (n = 6) did not alter the level of hypoxemia caused by air embolism, but it accelerated the recovery to normoxemia after air infusion was stopped. Prior heat shock also attenuated the elevation of W/D of lung weight (5.19 +/- 0.40) and the increase in BAL protein (371 +/- 69 mg/l) in air embolism group. Our results showed that the occurrence of DCS after rapid decompression is associated with increased expression of a stress protein (HSP70) and that prior heat shock exposure attenuates the air bubble-induced lung injury. These results suggest that bubble formation in tissues activates a stress response and that stress preconditioning attenuates lung injury on subsequent stress, which may be the mechanism responsible for diving acclimatization.     

Improved postischemic function following acute exercise is not mediated by nitric oxide synthase in the rat heart

The mediators of acute exercise-induced preconditioning against ischemia-reperfusion injury are not understood. This study assesses the role of nitric oxide synthase (NOS), a reported mediator of other forms of preconditioning. Male Fischer 344 rats were divided into five groups (n = 6-7): sedentary (Sed); exercised 2 days on a treadmill at 20 m/min, 6 degrees grade, for 60 min (Run); sedentary, perfused with 100 microM N(omega)-nitro-l-arginine methyl ester hydrochloride (l-NAME) to inhibit NOS (Sed/L-N); exercised, perfused with l-NAME (Run/L-N); and exercised in a 4 degrees C environment, perfused with l-NAME (CRun/L-N). Twenty-four hours following exercise, isolated, perfused working hearts were subjected to 22.5 min of global ischemia plus 30 min of normoxic reperfusion. Left ventricle contents of several putative preconditioning mediators were determined. Postischemic recovery of cardiac output times systolic pressure was better in Run than Sed (78.4 vs. 50.2% of preischemia, P < 0.05). Inhibition of NOS did not abrogate the improved recovery in the exercise groups or alter recovery in Sed. All exercise groups also displayed improved myocardial efficiency (cardiac output times systolic pressure/oxygen consumption) postischemia and less lactate dehydrogenase release (P < 0.05). l-NAME appeared to lower lactate dehydrogenase release independent of exercise. The only change in antioxidant enzyme activity was a decrease in manganese superoxide dismutase in CRun/L-N (P < 0.05). Heat shock protein 72 expression increased only in Run and Run/L-N and endothelial NOS only in CRun/L-N (P < 0.05). Acute exercise-induced preconditioning of the Fischer 344 rat heart is not mediated by NOS and does not require increases in heat shock protein 72 or antioxidant enzymes.     

Heat stress attenuates skeletal muscle atrophy in hindlimb-unweighted rats.

This study tested the hypothesis that elevation of heat stress proteins by whole body hyperthermia is associated with a decrease in skeletal muscle atrophy induced by reduced contractile activity (i.e. , hindlimb unweighting). Female adult rats (6 mo old) were assigned to one of four experimental groups (n = 10/group): 1) sedentary control (Con), 2) heat stress (Heat), 3) hindlimb unweighting (HLU), or 4) heat stress before hindlimb unweighting (Heat+HLU). Animals in the Heat and Heat+HLU groups were exposed to 60 min of hyperthermia (colonic temperature approximately 41.6 degrees C). Six hours after heat stress, both the HLU and Heat+HLU groups were subjected to hindlimb unweighting for 8 days. After hindlimb unweighting, the animals were anesthetized, and the soleus muscles were removed, weighed, and analyzed for protein content and the relative levels of heat shock protein 72 (HSP72). Compared with control and HLU animals, the relative content of HSP72 in the soleus muscle was significantly elevated (P < 0.05) in both the Heat and Heat+HLU animals. Although hindlimb unweighting resulted in muscle atrophy in both the HLU and Heat+HLU animals, the loss of muscle weight and protein content was significantly less (P < 0.05) in the Heat+HLU animals. These data demonstrate that heat stress before hindlimb unweighting can reduce the rate of disuse muscle atrophy. We postulate that HSP70 and/or other stress proteins play a role in the control of muscle atrophy induced by reduced contractile activity.     

Role of priming stresses and Hsp70 in protection from ischemia-reperfusion injury in cardiac and skeletal muscle

Ischemia-reperfusion injury limits the survival of muscle involved in tissue trauma or transfers during microsurgical reconstruction. Priming stresses such as ischemic preconditioning or mild hyperthermia have frequently been associated with improved survival of ischemic-reperfused cardiac muscle, such protection coinciding with induction of the stress-related heat shock protein 70 (Hsp70). Little is known about the response of skeletal muscle to priming stresses. This review summarizes the current knowledge on the use of priming stresses as protective strategies against the consequences of ischemia-reperfusion in cardiac and skeletal muscle and the potential role of Hsp70.     

The effects of the nitric oxide donor SIN-1 on ischemia-reperfused cutaneous and myocutaneous flaps

Ischemia-reperfusion injury causes tissue damage that leads to a decrease in bioavailability of nitric oxide. The authors hypothesized that an exogenous supply of nitric oxide will have beneficial effects on survival of skin and skeletal muscle subjected to ischemia-reperfusion injury. By using the nitric oxide donor SIN-1 (3-morpholino-sydnonimine) the effects of direct intraarterial infusion of an exogenous source of nitric oxide in reperfused flaps was studied. Bilateral island buttock skin flaps and latissimus dorsi myocutaneous flaps were elevated in eight pigs, for a total of 32 flaps. Flaps were subjected to 6 hours of ischemia followed by 18 hours of reperfusion. Flaps on one side of each animal were randomized to be treated with the nitric oxide donor (treatment group). The contralateral side was treated with an equivalent volume of saline vehicle (infusion control) SIN-1, or saline was administered as a continuous direct intraarterial infusion at the onset of reperfusion and continued during the observation period. Outcomes measured were tissue neutrophil accumulation by using myeloperoxidase assay and tissue survival (intravenous fluorescein and nitroblue tetrazolium for skin and muscle, respectively). In both skin and myocutaneous flaps, SIN-1 treatment caused a significant improvement in survival and a decrease in neutrophil accumulation. Nitric oxide may play an important role in the pathophysiologic process of ischemia-induced reperfusion injury in skin and skeletal muscle. Nitric oxide donors may be a promising family of therapeutic agents for the prevention of ischemia-induced reperfusion injury in cutaneous and myocutaneous flaps.     

Heat shock prevents simulated ischemia-induced apoptosis in renal tubular cells via a PKC-dependent mechanism

Heat shock produces cellular tolerance to a variety of adverse conditions; however, the protective effect of heat shock on renal cell ischemic injury remains unclear. Protein kinase C (PKC) has been implicated in the signaling mechanisms of acute preconditioning, yet it remains unknown whether PKC mediates heat shock-induced delayed preconditioning in renal cells. To study this, renal tubular cells (LLC-PK1) were exposed to thermal stress (43 degrees C) for 1 h and heat shock protein (HSP) 72 induction was confirmed by Western blot analysis. Cells were subjected to simulated ischemia 24 h after thermal stress, and the effect of heat shock (delayed preconditioning) on ischemia-induced apoptosis (terminal deoxynucleotidyl transferase dUTP nick-end labeling) and B cell lymphoma 2 (Bcl(2)) expression (Western) was determined. Subsequently, the effect of PKC inhibition on HSP72 induction and heat stress-induced ischemic tolerance was evaluated. Thermal stress induced HSP72 production, increased Bcl(2) expression, and prevented simulated ischemia-induced renal tubular cell apoptosis. PKC inhibition abolished thermal induction of HSP72 and prevented heat stress-induced ischemic tolerance. These data demonstrate that thermal stress protects renal tubular cells from simulated ischemia-induced apoptosis through a PKC-dependent mechanism.     

Early and late effects of ischemic preconditioning on microcirculation of skeletal muscle flaps

The present study was designed to investigate the early and late effects of ischemic preconditioning on muscle flap perfusion and reperfusion-induced skeletal muscle damage. Thirty-six Sprague-Dawley rats were divided into six experimental groups of six animals each. The cremaster muscle flap model and the intravital microscopy system were used to observe microcirculatory changes associated with ischemia-reperfusion injury and ischemic preconditioning. In groups 1, 2, and 3, microcirculatory measurements were taken on the same day; however, in groups 4, 5, and 6, measurements were taken a day after surgery. Group 1 served as a control. The cremaster muscle was prepared as a tube flap, subjected to an hour of perfusion without ischemia. In group 2 (ischemic preconditioning + ischemia group), the cremaster muscle tube flap was subjected to 30 minutes of ischemia and 30 minutes of reperfusion, followed by 4 hours of total ischemia. In group 3 (ischemia alone), the flap was submitted to 4 hours of ischemia alone. In group 4 (control), the cremaster muscle flaps were dissected out, preserved in the subcutaneous tunnel, and submitted to 24 hours of perfusion only. In group 5 (ischemic preconditioning + 24 hours of perfusion + 4 hours of ischemia), the ischemic preconditioning protocol was followed by 24 hours of perfusion and 4 hours of ischemia. In group 6 (24 hours of perfusion + ischemia), the same protocol was used as in group 5 without ischemic preconditioning. Functional capillary perfusion, and the diameters of the arterioles of the first, second, and third order were significantly increased in the ischemic preconditioning group during the early period, but not after 24 hours of perfusion. No differences in the red blood cell velocities of arterioles of the first, second, or third order were found in either the early-effect or late-effect groups. The numbers of rolling, adhering, and transmigrating leukocytes, however, were significantly lower in the ischemic preconditioning group at both early and late follow-up. Ischemic preconditioning of the skeletal muscle flap has both an early and a late protective effect against reperfusion injury. Ischemic preconditioning at the early interval significantly improves muscle flow hemodynamics of the flap and attenuates leukocyte-mediated reperfusion injury. After 24 hours of reperfusion, however, ischemic preconditioning failed to improve the flow hemodynamics of the flap, yet it still protected the skeletal muscle flap from leukocyte-mediated reperfusion injury.     

高压氧预处理对大鼠局灶性脑缺血再灌注损伤的保护作用

目的:研究高压氧预处理对大鼠脑缺血再灌注损伤的保护作用。方法:36只SD大鼠随机分为假手术组、模型组及高压氧预处理组,每组12只。高压氧预处理组大鼠在造模前5天给予高压氧预处理。采用线栓法建立大鼠脑缺血再灌注模型,观察高压氧预处理对脑缺血再灌注损伤大鼠神经功能缺损评分、脑梗死面积的影响,检测大鼠缺血脑组织COX-2 mRNA和蛋白的表达以及IL-1β、TNF-α、MDA的含量。结果:高压氧预处理可明显改善脑缺血再灌注大鼠神经功能缺损评分,减少脑梗死面积,降低COX-2m RNA和蛋白表达量,抑制IL-1β、TNF-α的表达,降低MDA水平。结论:高压氧预处理对大鼠脑缺血再灌注损伤具有明显的保护作用,其机制可能与抑制IL-1β、TNF-α、COX-2的表达以及减弱脂质过氧化反应有关。     

Differing flow patterns between ischemically challenged flap skin and flap skeletal muscle: implications for salvage regimens.

In this study, the authors tested the hypothesis that there is a significant difference in spatial patterns of reflow in skin as opposed to skeletal muscle after an ischemic insult. The authors believe that this pathophysiologic difference between the two flap types has significant implications for flap salvage strategies. Bilateral buttock skin flaps (10 x 18 cm) and latissimus dorsi myocutaneous flaps (10 x 20 cm) were elevated in Landrace pigs (n = 7). Flaps on one side of the animal were randomly assigned to 6 hours of arterial occlusion, with the contralateral side acting as control. At 15 minutes, 1 hour, and 4 hours after reflow, radioactive microspheres (15 microm) were injected into the left ventricle. After 18 hours of reperfusion, skin and muscle viability were estimated by intravenous fluorescein and soaking in nitroblue tetrazolium, respectively. Flow rates in the skin with an ischemia-reperfusion injury were significantly reduced (30 to 53 percent), at all time intervals, compared with controls. The flow rate in the fluorescent skin with ischemia-reperfusion injury of the latissimus dorsi flaps (0.037 ml/min/g at 15 min) was greater than in that of the buttock flaps (0.018 ml/min/g). The muscle flaps with ischemia-reperfusion injury had significantly higher flow rates than control muscle flaps at all time intervals studied (at 1 hour, 0.32 ml/min/g compared with 0.16 ml/min/g, respectively). In flap skeletal muscle, an early hyperemic phase during reperfusion maintains a significant blood flow to all regions, including the area of the flap that is destined for necrosis. In flap skin, however, there is a marked decrease in flow rates. These differences have important implications for the intravascular delivery of therapeutic agents to the damaged portions of the flap. Transdermal drug delivery systems should be explored as an alternative to intravascular regimens for the salvage of flap skin with ischemia-reperfusion injury.     

Effect of heat shock preconditioning on NF-kappaB/I-kappaB pathway during I/R injury of the rat liver

Hepatic ischemia-reperfusion (I/R) injury continues to be a fatal complication after liver surgery. Heat shock (HS) preconditioning is an effective strategy for protecting the liver from I/R injury, but its exact mechanism is still unclear. Because the activation of nuclear factor-kappaB (NF-kappaB) is an important event in the hepatic I/R-induced inflammatory response, the effect of HS preconditioning on the pathway for NF-kappaB activation was investigated. In the control group, NF-kappaB was activated 60 min after reperfusion, but this activation was suppressed in the HS group. Messenger RNA expressions of proinflammatory mediators during reperfusion were also reduced with HS preconditioning. Concomitant with NF-kappaB activation, NF-kappaB inhibitor I-kappaB proteins were degraded in the control group, but this degradation was suppressed in the HS group. This study shows that HS preconditioning protected the liver from I/R injury by suppressing the activation of NF-kappaB and the subsequent expression of proinflammatory mediators through the stabilization of I-kappaB proteins.     

Heat stress inhibits skeletal muscle hypertrophy

Heat shock proteins (Hsps) are molecular chaperones that aid in protein synthesis and trafficking and have been shown to protect cells/tissues from various protein damaging stressors. To determine the extent to which a single heat stress and the concurrent accumulation of Hsps influences the early events of skeletal muscle hypertrophy, Sprague-Dawley rats were heat stressed (42 degrees C, 15 minutes) 24 hours prior to overloading 1 plantaris muscle by surgical removal of the gastrocnemius muscle. The contralateral plantaris muscles served as controls. Heat-stressed and/or overloaded plantaris muscles were assessed for muscle mass, total muscle protein, muscle protein concentration, Type I myosin heavy chain (Type I MHC) content, as well as Hsp72 and Hsp25 content over the course of 7 days following removal of the gastrocnemius muscle. As expected, in non-heat-stressed animals, muscle mass, total muscle protein and MHC I content were significantly increased (P < 0.05) following overload. In addition, Hsp25 and Hsp72 increased significantly after 2 and 3 days of overload, respectively. A prior heat stress-elevated Hsp25 content to levels similar to those measured following overload alone, but heat stress-induced Hsp72 content was increased significantly greater than was elicited by overload alone. Moreover, overloaded muscles from animals that experienced a prior heat stress showed a lower muscle mass increase at 5 and 7 days; a reduced total muscle protein elevation at 3, 5, and 7 days; reduced protein concentration; and a diminished Type I MHC content accumulation at 3, 5, and 7 days relative to nonheat-stressed animals. These data suggest that a prior heat stress and/or the consequent accumulation of Hsps may inhibit increases in muscle mass, total muscle protein content, and Type I MHC in muscles undergoing hypertrophy.     

Whole-body hyperthermia induces up-regulation of vascular endothelial growth factor accompanied by neovascularization in cardiac tissue

Whole-body hyperthermia (WBH) promotes cardiac protection against ischemia/reperfusion injury, in part by up-regulation of heat shock proteins (HSP). Whether heat stress also promotes up-regulation of angiogenic factors or induces endothelial cell proliferation is unknown. We studied the effects of heat stress on up-regulation of vascular endothelial growth factor (VEGF) and growth of new blood vessels following WBH. Anesthetized rats were subjected to WBH at 42 degrees C for 15 min. The control (n=23) and heated (n=55) groups were allowed to recover for 4, 12, 24, 48, or 72 h prior to harvesting the heart for Western Blot and immunohistochemical assessment of VEGF, HSP70, and platelet endothelial cell adhesion molecular-1 (PECAM-1). A significant increase in VEGF and HSP70 expression was observed as early as 4 h post-heating. The Western Blot analysis revealed a close temporal correlation between up-regulation of HSP70 and VEGF. Maximum VEGF and HSP70 expression occurred at 12 and 24 h post-heating in the left and right ventricles, respectively. The right ventricle showed the greatest expression of both VEGF and HSP70. Immunostaining revealed that VEGF was focally increased in the endothelial cells of capillaries, small arteries, and in interstitium. At 48 and 72 h post-heating, multiple areas of extensive capillary proliferation occurred in the epicardial region of the right ventricle. These observations were verified by quantitative analysis of the density of blood vessels as determined by PECAM-1 staining. Our experiments show that sublethal heat stress can lead to upregulation of both VEGF and HSP70 in cardiac tissue and promote focal endothelial proliferation in the heart.