Tourniquet Model Research Articles

ProBDNF-p75NTR signaling in sterile inflammation

Trauma, injury, and surgical procedures cause tissue damage, sterile inflammatory responses, and significant immune system stress leading to immune dysregulation and increased susceptibility to secondary infections. The spleen is a reservoir of myeloid immune cells that are involved in inflammation following various tissue injuries. One potential signaling pathway that may be involved in this process is proBDNF-p75NTR signaling in myeloid cells. Brain derived neurotrophic factor (BDNF) is a previously reported myokine, however there is little known about its role in skeletal muscle injury. We recently found that p75NTR, the receptor for proBDNF, was significantly upregulated (p=0.004) in myeloid cells isolated from the spleen of animals with hindlimb IR injury compared to sham controls, potentially in a sexually dimorphic manner. Our previous studies have shown that proBDNF, the precursor protein to BDNF, is the predominant form of BDNF expressed in skeletal muscle tissue and is significantly upregulated following tourniquet induced hind limb IR injury in mice (p=0.013). We hypothesize that proBDNF released from injured skeletal muscle could act through p75NTR in myeloid cells to regulate inflammatory responses following IR injury. The hind limb IR injury was induced using a tourniquet model in which an orthodontic rubber band placed on the hind limb at the level of the hip and left in place for 1.5 hours followed by tourniquet removal and reperfusion. A series of in vitro experiments were conducted on myeloid cells isolated from the spleen of sham mice, or mice with skeletal muscle IR injury. As a model of secondary infection, myeloid cells were stimulated with lipopolysaccharide (LPS) and cytokine release into culture media was measured using ELISA assays. We found that in myeloid cells isolated from IR injured animals, LPS stimulated cytokine release was increased. Further, treating isolated myeloid cells from IR injured animals with recombinant cleavage resistant proBDNF increased cytokine release compared to controls. To confirm that p75NTR on myeloid cells was at least partially responsible for the induction of cytokine expression following LPS or proBDNF stimulation, myeloid cells were treated with LM11A-31, a modulator of p75NTR in combination with LPS or proBDNF. Treatment with C31 (100ng) blunted cytokine release following LPS and proBDNF stimulation. These results suggest that the p75NTR pathway is involved in the innate immune function of splenic myeloid cells in injury induced inflammation and therefore is potentially a novel therapeutic target. This work was funded by NIH grant R01HL147105, NIH Training Grant T32-GM136503, and NIH F31 Fellowship F31HL167480. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A low-cost, DIY tourniquet simulator with built-in self-assessment for prehospital providers in Guatemala city.

Hemorrhage is the leading cause of preventable death after trauma. In high-income countries first responders are trained in hemorrhage control techniques but this is not the case for developing countries like Guatemala. We present a low-cost training model for tourniquet application using a combination of virtual and physical components. The training program includes a mobile application with didactic materials, videos and a gamified virtual reality environment for learning. Additionally, a physical training model of a bleeding lower extremity is developed allowing learners to practice tourniquet application using inexpensive and accessible materials. Validation of the simulator occurred through content and construct validation. Content validation involved subjective assessments by novices and experts, construct validation compared pre-training novices with experts. Training validation compared pre and post training novices for improvement. Our findings indicate that users found the simulator useful, realistic, and satisfactory. We found significant differences in tourniquet application skills between pre-training novices and experts. When comparing pre- and post-training novices, we found a significantly lower bleeding control time between the groups. This study suggests that this training approach can enhance access to life-saving skills for prehospital personnel. The inclusion of self-assessment components enables self-regulated learning and reduces the need for continuous instructor presence. Future improvements involve refining the tourniquet model, validating it with first-responder end users, and expanding the training program to include other skills.

Prospective Analysis of Simulated Pneumatic Tourniquet Use and Oedema Following Axillary Lymph Node Dissection.

Background: Tourniquet use during upper extremity surgery in patients with a history of axillary lymph node dissection (ALND) remains controversial due to the perceived but unproven risk of lymphoedema. We prospectively evaluated upper extremity swelling in patients with a history of unilateral ALND using a standardised tourniquet model. Methods: A tourniquet was applied to the upper arm bilaterally, with the unaffected side serving as an internal control. Each arm was subsequently held in an elevated position to reduce swelling. Hand volume was measured using an aqueous volumeter. Results: The patients' ALND arms experienced slightly greater increases in volume following tourniquet application compared to their healthy control arms. However, this amount of oedema was temporary and reversible, as both arms experienced spontaneous resolution of swelling with no significant difference in residual hand volume at the conclusion of the study. Conclusions: Tourniquet use may be safe in patients with a history of ALND. Further investigation is needed to verify this in a surgical setting. Level of Evidence: Level II (Therapeutic).

Autologous mitochondrial transplantation in male mice as a strategy to prevent deleterious effects of peripheral ischemia-reperfusion.

Ischemia-reperfusion (IR) is known to induce severe tissue damage, notably through mitochondrial dysfunction. Mitochondrial transplantation has emerged as a promising therapeutic strategy in cardiac IR; however, few studies have previously assessed its efficacy in the context of peripheral IR. Therefore, the objective of this study was to assess the effect of mitochondrial transplantation in a hindlimb model of IR injury. Thirty-six SWISS mice were divided into three groups: control (CTL, n = 12), ischemia-reperfusion (IR, n = 12), and IR with mitochondrial transplantation (MT, n = 12). Ischemia (2 h) was induced using the tourniquet model around the right hind limb in the IR and MT groups. In MT group, mitochondria isolated from the right rectus muscle, a nonischemic region, were injected shortly before reperfusion. Mitochondrial respiration, calcium retention capacity, and Western blotting analysis were performed 2 h after reperfusion. Compared with the CTL group, IR led to a decrease in the mitochondrial respiratory capacity, particularly for the basal state (-30%; P = 0.015), oxidative phosphorylation (-36%; P = 0.024), and calcium retention capacity (-45%; P = 0.007). Interestingly, mitochondrial transplantation partially restored these functions since no differences between MT and CTL groups were found. In addition, the administration of healthy mitochondria resulted in a positive regulation of redox balance and mitochondrial dynamics within the skeletal muscle. Although further investigations are needed to better characterize underlying mechanisms, mitochondrial transplantation represents a promising strategy in the setting of IR-induced muscular damage.NEW & NOTEWORTHY Ischemia-reperfusion injury leads to severe muscular damage. Even if prompt revascularization is the treatment of choice, muscular alterations can lead to severe sequalae as mitochondrial dysfunction. Accordingly, adjunctive strategies are needed to overcome the muscular damage. Mitochondrial transplantation has shown beneficial effects in cardiac ischemia-reperfusion, but its role in peripheral muscle is not well established. In this study, we found that mitochondrial transplantation partially restored muscular function when submitted to ischemia reperfusion.

An optimized low-pressure tourniquet murine hind limb ischemia reperfusion model: Inducing acute ischemia reperfusion injury in C57BL/6 wild type mice.

Acute ischemia reperfusion injury in skeletal muscle remains an important issue in several fields of regenerative medicine. Thus, a valid model is essential to gain deeper insights into pathophysiological relations and evaluate possible treatment options. While the vascular anatomy of mice regularly prevents sufficient vessel occlusion by invasive methods, there is a multitude of existing models to induce ischemia reperfusion injury without surgical procedures. Since there is no consensus on which model to prefer, this study aims to develop and evaluate a novel, optimized low-pressure tourniquet model. C57BL/6 mice underwent an ischemic procedure by either tourniquet or invasive artery clamping. A sham group served as control. With exception of the sham group, mice underwent 2 hours of ischemia followed by 4 hours of reperfusion. Groups were compared using microcirculatory and spectroscopic measurements, distinctions in tissue edema, histological and immunohistochemical analyses. Both procedures led to a significant decrease in tissue blood flow (- 97% vs. - 86%) and oxygenation (- 87% vs. - 75%) with a superiority of the low-pressure tourniquet. Tissue edema in the tourniquet cohort was significantly increased (+ 59%), while the increase in the clamping cohort was non-significant (+ 7%). Haematoxylin Eosin staining showed significantly more impaired muscle fibers in the tourniquet group (+ 77 p.p. vs. + 11 p.p.) and increased neutrophil infiltration/ROI (+ 51 vs. + 8). Immunofluorescence demonstrated an equal increase of p38 in both groups (7-fold vs. 8-fold), while the increase in apoptotic markers (Caspase-3, 3-Nitrotyrosine, 4-Hydroxynonenal) was significantly higher in the tourniquet group. The low-pressure tourniquet has been proven to produce reproducible and thus reliable ischemia reperfusion injury. In addition, significantly less force was needed than previously stated. It is therefore an important instrument for studying the pathophysiology of ischemia reperfusion injury and for the development of prophylactic as well as therapeutic interventions.

Interoperable Readiness to Use Tourniquets by One's Familiarity With Different Models.

We investigated interoperability for a first aid provider to perform simulated use of three tourniquet models of maximal, moderate, and minimal familiarity. The experiment was focused on the tourniquets used by an expert who rendered aid on a manikin by using three models of tourniquet with different extents of familiarity: The familiarity with Combat Application Tourniquet (C-A-T) was maximal; that for Special Operations Forces Tactical Tourniquet (SOFTT) was moderate, and that for Military Emergency Tourniquet (MET) was minimal. Each model had a band-and-rod design. Interoperability changes as intermodel differences were beneficial or costly in that performances were improved or impaired in units of time, ease, blood, and pressure. Each model had 10 tests, and test order was randomized by model. The HapMed Leg Tourniquet Trainer simulated a limb amputation. In comparison of interoperability burdens, sums of 10 test durations by model for C-A-T, SOFTT, and MET were 38, 77, and 64 minutes, respectively; C-A-T was fastest (p ≤ .002, both). The sums of times to stop bleeding for C-A-T, SOFTT, and MET were 334, 953, and 826 seconds, respectively; C-A-T was fastest (p ≤ .0013, both). The sums of blood losses for C-A-T, SOFTT, and MET were 2105, 3287, and 4256mL, respectively; that for C-A-T was least (p ≤ .0005, both). The mean ease of use differed, with C-A-T being easiest (p ≤ .0046, both). The mean pressure differed, with C-A-T being higher than SOFTT (p = .0073). Timesaving strongly favored the model with which the user had maximal familiarity. In theory and simulation, interoperability bears costs in successfully attaining it, in maintaining it, and in failing either. The user's familiarity with tourniquet model was associated with improved interoperability as seen by improved performances. If multiple models are fielded, then organizations may plan on extra spending, supplying, training, and managing.

New and Established Models of Limb Tourniquet Compared in Simulated First Aid.

The performance of a new tourniquet model was compared with that of an established model in simulated first aid. Four users applied the Combat Application Tourniquet (C-A-T), an established model that served as the control tourniquet, and the new SAM Extremity Tourniquet (SXT) model, which was the study tourniquet. The performance of the C-A-T was better than that of the SXT for seven measured parameters versus two, respectively; metrics were statistically tied 12 times. The degree of difference, when present, was often small. For pretime, a period of uncontrolled bleeding from the start to a time point when the tourniquet first contacts the manikin, the bleeding rate was uncontrolled at approximately 10.4mL/s, and for an overall average of 39 seconds of pretime, 406mL of blood loss was calculated. The mean time to determination of bleeding control (± standard deviation [SD]) was 66 seconds (SXT, 70 ± 30 seconds; C-A-T, 62 ± 18 seconds; p = .0075). The mean ease-of-use score was 4 (indicating easy) on a scale of 1 to 5, with 5 indicating very easy (mean ± SD: SXT, 4 ± 1; C-A-T, 5 ± 0; p < .0001). C-A-T also performed better for total trial time, manikin damage, blood loss rate, pressure, and composite score. SXT was better for pretime and unwrap time. All users intuitively self-selected the speed at which they applied the tourniquets and that speed was similar in all of the required steps. However, by time segments, one user went slowest in each segment while the other three generally went faster. In simulated first aid with tourniquets, better results generally were seen with the C-A-T than with the SXT in terms of performance metrics. However, the degree of difference, when present, was often small.

Assessment of Groin Application of Junctional Tourniquets in a Manikin Model.

Introduction To aid in preparation of military medic trainers for a possible new curriculum in teaching junctional tourniquet use, the investigators studied the time to control hemorrhage and blood volume lost in order to provide evidence for ease of use. Hypothesis Models of junctional tourniquet could perform differentially by blood loss, time to hemostasis, and user preference. In a laboratory experiment, 30 users controlled simulated hemorrhage from a manikin (Combat Ready Clamp [CRoC] Trainer) with three iterations each of three junctional tourniquets. There were 270 tests which included hemorrhage control (yes/no), time to hemostasis, and blood volume lost. Users also subjectively ranked tourniquet performance. Models included CRoC, Junctional Emergency Treatment Tool (JETT), and SAM Junctional Tourniquet (SJT). Time to hemostasis and total blood loss were log-transformed and analyzed using a mixed model analysis of variance (ANOVA) with the users represented as random effects and the tourniquet model used as the treatment effect. Preference scores were analyzed with ANOVA, and Tukey's honest significant difference test was used for all post-hoc pairwise comparisons. All tourniquet uses were 100% effective for hemorrhage control. For blood loss, CRoC and SJT performed best with least blood loss and were significantly better than JETT; in pairwise comparison, CRoC-JETT (P .5, all models). The CRoC and SJT performed best in having least blood loss, CRoC performed best in having least time to hemostasis, and users did not differ in preference of model. Models of junctional tourniquet performed differentially by blood loss and time to hemostasis. Kragh JF Jr , Lunati MP , Kharod CU , Cunningham CW , Bailey JA , Stockinger ZT , Cap AP , Chen J , Aden JK 3d , Cancio LC . Assessment of groin application of junctional tourniquets in a manikin model. Prehosp Disaster Med. 2016;31(4):358-363.

Postischemic conditioning does not reduce muscle injury after tourniquet-induced ischemia-reperfusion injury in rats

BackgroundThe widespread application of tourniquets has reduced battlefield mortality related to extremity exsanguinations. Tourniquet-induced ischemia-reperfusion injury (I/R) can contribute to muscle loss. Postischemic conditioning (PostC) confers protection against I/R in cardiac muscle and skeletal muscle flaps. The objective of this study was to determine the effect of PostC on extremity muscle viability in an established rat hindlimb tourniquet model. MethodsRats were randomly assigned to PostC-1, PostC-2, or no conditioning ischemic groups (n = 10 per group). Postischemic conditioning, performed immediately after tourniquet release, consisted of four 15-second cycles (PostC-1) or eight 15-second cycles (PostC-2) of alternating occlusion and perfusion of hindlimbs. Twenty-four hours later, muscles were excised. The primary end points were muscle edema and viability; secondary end points were histologic and markers of oxidative stress. ResultsIschemia-reperfusion injury decreased viability in all tourniquet limbs, but viability was not improved in either PostC group. Likewise, I/R resulted in substantial muscle edema that was not reduced by PostC. The predominant histologic feature was necrosis, but no significant differences were found among groups. Markers of oxidative stress were increased similarly among groups after I/R, although myeloperoxidase activity was significantly increased only in the no conditioning ischemic group. A protective effect from PostC was not observed in our model suggesting that PostC was not effective in reducing I/R skeletal muscle injury or any benefits of PostC were not sustained for 24 hours when tissues were assessed. ConclusionThese negative findings are pertinent as the military investigates different strategies to extend the safe time for tourniquet application.

Contribution of RhoA/Rho-kinase/MEK1/ERK1/2/iNOS pathway to ischemia/reperfusion-induced oxidative/nitrosative stress and inflammation leading to distant and target organ injury in rats

The small G protein RhoA and its downstream effector Rho-kinase play an important role in various physiopathological processes including ischemia/reperfusion (I/R) injury. Reactive oxygen and nitrogen species produced by iNOS and NADPH oxidase are important mediators of inflammation and organ injury following an initial localized I/R event. The aim of this study was to determine whether RhoA/Rho-kinase signaling pathway increases the expression and activity of MEK1, ERK1/2, iNOS, gp91phox, and p47phox, and peroxynitrite formation which result in oxidative/nitrosative stress and inflammation leading to hindlimb I/R-induced injury in kidney as a distant organ and gastrocnemius muscle as a target organ. I/R-induced distant and target organ injury was performed by using the rat hindlimb tourniquet model. I/R caused an increase in the expression and/or activity of RhoA, MEK1, ERK1/2, iNOS, gp91phox, p47phox, and 3-nitrotyrosine and nitrotyrosine levels in the tissues. Although Rho-kinase activity was increased by I/R in the kidney, its activity was decreased in the muscle. Serum and tissue MDA levels and MPO activity were increased following I/R. I/R also caused an increase in SOD and catalase activities associated with decreased GSH levels in the tissues. Y-27632, a selective Rho-kinase inhibitor, (100µg/kg, i.p.; 1h before reperfusion) prevented the I/R-induced changes except Rho-kinase activity in the muscle. These results suggest that activation of RhoA/Rho-kinase/MEK1/ERK1/2/iNOS pathway associated with oxidative/nitrosative stress and inflammation contributes to hindlimb I/R-induced distant organ injury in rats. It also seems that hindlimb I/R induces target organ injury via upregulation of RhoA/MEK1/ERK1/2/iNOS pathway associated with decreased Rho-kinase activity.

Glutamine preconditioning protects against tour-niquet-induced local and distant organ injury in a rodent ischemia-reperfusion model

Introduction Ischemia-reperfusion (IR) injury is a common surgical event, with tourniquet use being a recognized cause in orthopedic surgery. Preconditioning is a highly evolutionarily conserved endogenous protective mechanism, but finding a clinically safe, acceptable method of induction has proven difficult. Glutamine, a known inducer of the heat shock protein response, offers pharmacological modulation of injury through clinically acceptable preconditioning. Our aim was to test the hypothesis that glutamine preconditioning protects against tourniquet-induced regional and remote IR injury in a rodent model.Animals and methods 40 adult male Sprague-Dawley rats were randomized into 4 groups: control, IR injury, normal saline-pretreated and IR injury, and glutamine-pretreated and IR injury. Pretreated groups received either normal saline or glutamine by intravenous bolus 24 h before injury. A bilateral hindlimb tourniquet model was used. Blood samples were analyzed, bronchioalveolar lavage (BAL) performed, and skeletal muscle and lung harvested for evaluation.Results The glutamine-pretreated group showed significantly lower muscle myeloperoxidase (MPO) content and creatine kinase levels than the untreated or salinepretreated injury groups. Lung tissue showed reduced MPO content and a significantly reduced neutrophil count by BAL fluid microscopy.Interpretation These data suggest that preconditioning with glutamine offers local and distant organ protection in the setting of tourniquet-induced IR injury.

Evaluation of limb compartments with increased interstitial pressure. An improved noninvasive method for determining quantitative hardness

The purpose of this project was to study a noninvasive method of evaluating limbs at risk of compartment syndrome. Untreated limb compartment syndrome may lead to irreversible dysfunction, chronic pain and contracture. An improved means of obtaining quantitative hardness measurements is reported. The handheld noninvasive compartment syndrome evaluator (NCSE) device formulates a quantitative hardness curve of force verse depth of indentation by applying a 5.0 mm diameter indenter to a limb muscle compartment. Eighteen volunteers (9 men, 9 women) had elevated intracompartmental interstitial pressures by application of custom-made tourniquets. Comparison of interstitial pressure to quantitative hardness measurements in 71 compartments tested showed a statistically significant strong linear relationship with an average Pearson correlation coefficient of .84 (range .78–.9 P < . 0001 ). In addition, for all compartments tested, the mean hardness value for the group with interstitial pressure greater than 50 mm Hg (considered compartment syndrome) was close to 2 x greater and was statistically different than the mean hardness for the group with interstitial pressure less than 30 mm Hg (no compartment syndrome, t-test: P < . 0001 ). This study utilizes a device that has improved upon previous quantitative hardness measurement techniques. In addition, by utilizing a tourniquet model to create elevated interstitial intracompartmental pressure in volunteers, data collection was easier and faster. In the future, quantitative hardness measurements may accurately predict interstitial intracompartmental pressure for most patients. This technique may greatly enhance the medical community's ability to diagnose compartment syndrome with a noninvasive means.

Changes in mRNA expression patterns for cytokines in blood leukocytes of a rat tourniquet model

We examined changes in mRNA expression patterns for proinflammatory cytokines and growth factors in blood samples after application of a tourniquet to the rat hind limb. Slight upregulations of interferon (IFN)-γ, macrophage colony-stimulating factor (M-CSF) and transforming growth factor (TGF)-β1 mRNA began at 2 h after tourniquet application and were short-lived. The levels of activating transcription factor (ATF)-3, a stress-inducible gene, had increased at 1 h after tourniquet application. No significant expression of interleukin (IL)-6 mRNA was observed in most samples. There were no significant temporal changes in the levels of IL-1β, cardiotrophin (CT)-1 mRNA compared to the control levels, but, downregulation of gp130, a receptor of the IL-6 family, began at 1 h after tourniquet application. Nerve growth factor (NGF) mRNA gradually increased and reached a significantly high level at 4 h after application of the tourniquet. Gene expression induction in blood leukocytes occurred soon after application of the tourniquet and was short-lived. The transient mRNA expressions probably trigger secondary events that may be beneficial to wound repair and regeneration.

Expression of cytokines, neurotrophins, neurotrophin receptors and NOS mRNA in dorsal root ganglion of a rat tourniquet model

We studied temporal changes in mRNA expression patterns for nitric oxide synthase (NOS), cytokines, neurotrophins and neurotrophin receptors in the dorsal root ganglion (DRG) of the rat, after application of a tourniquet to the hind limb. Collapsed myelin and degenerated axons were observed in the tourniquet segment of the sciatic nerve. Gene expression level of inducible nitric oxide synthase (iNOS) and neuronal nitric oxide synthase (nNOS) was significantly increased in ipsilateral DRG samples at 4 h after application of the tourniquet but not in the contralateral or control DRG samples. Upregulation of tumor necrosis factor (TNF)-α, activating transcription factor (ATF)-3 and neurotrophin-3 (NT3) expressions began at 1 h after application of the tourniquet in ipsilateral DRGs. It is likely that transient expression of these molecules triggers secondary events that may be beneficial to wound repair and regeneration.

Prior heat stress improves survival of ischemic-reperfused skeletal muscle in vivo

The ability of heat stress to improve the survival of ischemic-reperfused skeletal muscle in vivo was investigated. Ischemia-reperfusion was applied using the rat hindlimb tourniquet model. The viability of ischemic-reperfused muscle (11 +/- 1%) was increased by prior mild heat stress (86 +/- 2%). To investigate whether heat shock protein 70 (Hsp 70) expression in the muscle of the heated limb was responsible for this protection, the survival of Hsp 70-expressing transduced myoblasts and myocytes was measured after exposure to mediators of ischemia-reperfusion injury. Survival was improved in Hsp 70-positive myoblasts but not in myocytes, suggesting that the mechanism of protection conferred by heat stress in vivo cannot be explained by the expression of Hsp 70 in myocytes and may involve a more complex mechanism. In conclusion, prior heat stress is effective in protecting mature skeletal muscle in vivo against necrosis after ischemia-reperfusion and has potential for use in microsurgical procedures requiring tourniquet applications.

Ischaemia-reperfusion injury in mouse skeletal muscle is reduced by N ω-nitro- l-arginine methyl ester and dexamethasone

We have developed a model of ischaemia-reperfusion injury in C57BL/6 mice involving ischaemia for 0.5 to 2.5 h with an elastic tourniquet on one hind limb and reperfusion for 24 h, analogous to a well-established model of ischaemia-reperfusion injury in the rat. Viability was assessed in tissue homogenates of the gastrocnemius muscles from the affected and contralateral control limb by a triphenyl tetrazolium chloride dye reaction, measuring the activity of the oxidative mitochondrial enzymes. After 1.5 h ischaemia and 24 h reperfusion, viability in the ischaemic-reperfused limb was 13%, with the control muscle regarded as 100% viable. Significant improvements in viability to 86% ( P<0.05) and 56% ( P<0.05) were achieved, with administration 30 min prior to tourniquet release, of the nitric oxide (NO) synthase inhibitor nitro- l-arginine methyl ester ( l-NAME, 30 mg/kg) and the anti-inflammatory glucocorticoid dexamethasone (2.5 mg/kg) respectively, with similar findings in the rat tourniquet model.

Hyperbaric oxygen treatment attenuates glutathione depletion and improves metabolic restitution in postischemic skeletal muscle.

Glutathione serves as an important intracellular defence against reactive oxygen metabolites and has been shown to be depleted from a number of tissues upon oxidative stress. In the present study we have investigated the levels of total glutathione (reduced + oxidized) in skeletal muscle of the rat after prolonged ischemia and reperfusion with and without treatment with hyperbaric oxygen (HBO) for the initial 45 minutes immediately following reperfusion. A tourniquet model for temporary, total ischemia was used, in which one hind leg was made ischemic for 3 or 4 hours. Muscle biopsies were taken after 5 hours of reperfusion. In postischemic muscle there was a significant decrease of total glutathione compared to control muscle, but in the 3-hour-ischemia-groups the loss of total glutathione was less in HBO treated animals than in untreated. HBO treatment also preserved ATP and PCr and decreased edema formation in the postischemic muscle following 3 hours of ischemia and reperfusion when compared to untreated animals. However, after 4 hours of ischemia, HBO treatment failed to improve any of these parameters in the postischemic muscle. Thus, our results demonstrate that HBO treatment lessens the metabolic, ischemic derangements and improves recovery in postischemic muscle after 3 hours of ischemia followed by reperfusion.

Local hypothermia during ischemia or reperfusion in skeletal muscles.

A modified rat hindlimb tourniquet model was used to measure postischemic muscle necrosis. The effect of moderate local hypothermia to 20 degrees C during ischemia and reperfusion was investigated. Eighteen animals were kept in an incubator at 27 degrees C, and complete circulatory arrest was maintained for 3.5 h before release of the vascular occlusion. After survival for 72 h the degree of necrosis in the anterior tibial muscles was measured morphometrically on histological slides. Areas of necrosis with intact capillary structure and resorption of muscle fibers, and areas without resorption and capillary disintegration were measured separately. Three experimental groups (six animals in each) were included in the series. In the first group local cooling to 20 degrees C was performed during the initial 1.5 h of ischemia. The second group was cooled for 1.5 h during the initial phase of reperfusion. The animals in the third group served as controls without cooling. The total areas of necrosis in the three groups were 0, 90 and 90%, and the areas of no-resorption 0, 23 and 39%, respectively. Cooling during ischemia thus had a marked effect, while no significant differences were found between the control group and the group cooled during reperfusion. The study shows that moderate cooling during initial ischemia protects effectively against postischemic muscle necrosis, while cooling during reperfusion has no such effect.

Histological Studies on Postischemic Rat Skeletal Muscles

Several studies have suggested that the damage after ischemic injuries increases during reperfusion and that this reperfusion damage is mediated through granulocytes which invade the damaged area. The present study was undertaken to test these hypotheses by histological investigations of the anterior tibial muscles in a rat hind limb tourniquet model after 4.5 h of complete ischemia and graded periods of reperfusion. Uptake of albumin into damaged muscle fibers was demonstrated immunohistochemically and used to determine the extent of the ischemic lesions. The size of the lesions was measured morphometrically on immunostained sections from paraffin-embedded material. Granulocytes in and outside the capillaries were counted on slides from historesin-embedded material. Generally, there was a compact central area in the muscles which showed necrosis of all fibers and many capillaries, little interstitial edema, and little or no invasion of granulocytes or macrophages. The central core did not increase in size during the postoperative period. Outside this are there was a zone of partial tissue destruction with quite marked interstitial edema. This zone also remained unchanged in size but it is uncertain whether the number of necrotic fibers increased with time. There was an increasing invasion of granulocytes in this outer zone from 5 to 24 h after release of the occlusion and monocyte/macrophage invasion was seen from 48 h. Outside this zone there was a subfacial zone with normal muscle fibers in all cases. It is concluded that the central area of complete tissue destruction was present at the time of release of the occlusion and did not increase in size during the further postoperative course. There was no indication that granulocytes participated in this damage. The extent of the partially damaged area also remained unchanged during the postoperative course. It is conceivable that granulocytes aggravated the lesions by increasing the number of necrotic fibers in this peripheral area but not before 5 h after release of the occlusion. This is later than described in previous studies.

Protective effect of low-grade hypothermia in experimental skeletal muscle ischemia.

In the present study, a rat hindlimb tourniquet model was used to investigate the effect of moderate hypothermia on ischemic muscle necrosis. Complete circulatory arrest was maintained for 4.5 h. During the ischemic period the animals were kept in an infant incubator at different temperatures. After 72 h survival the percentage of necrosis in the anterior tibial muscle was measured morphometrically on histological slides. At an ambient temperature of 24 degrees C there was 80% necrosis in the anterior tibial muscle. At 22 degrees C the necrosis was reduced to 29%. This reduction corresponds to more than 30 min shortening of the ischemia time. Differences in tissue temperature may explain some of the discrepancies reported in tolerance limits for muscle ischemia. To achieve consistent results in experimental muscle ischemia, it is necessary to control the ambient temperature.