Aims

Compartment syndrome results from increased intra-compartmental pressure (ICP) causing local tissue ischaemia and cell death, but the systemic effects are not well described. We hypothesised that compartment syndrome would have a profound effect not only on the affected limb, but also on remote organs.

Compartment syndrome, a devastating consequence of limb trauma, is characterised by severe tissue injury and microvascular perfusion deficits. We hypothesised that leucopenia might provide significant protection against microvascular dysfunction and preserve tissue viability. Using our clinically relevant rat model of compartment syndrome, microvascular perfusion and tissue injury were directly visualised by intravital video microscopy in leucopenic animals. We found that while the tissue perfusion was similar in both groups (38.8% (standard error of the mean (sem) 7.1), 36.4% (sem 5.7), 32.0% (sem 1.7), and 30.5% (sem 5.35) continuously-perfused capillaries at 45, 90, 120 and 180 minutes compartment syndrome, respectively versus 39.2% (sem 8.6), 43.5% (sem 8.5), 36.6% (sem 1.4) and 50.8% (sem 4.8) at 45, 90, 120 and 180 minutes compartment syndrome, respectively in leucopenia), compartment syndrome-associated muscle injury was significantly decreased in leucopenic animals (7.0% (sem 2.0), 7.0%, (sem 1.0), 9.0% (sem 1.0) and 5.0% (sem 2.0) at 45, 90, 120 and 180 minutes of compartment syndrome, respectively in leucopenia group versus 18.0% (sem 4.0), 23.0% (sem 4.0), 32.0% (sem 7.0), and 20.0% (sem 5.0) at 45, 90, 120 and 180 minutes of compartment syndrome in control, p = 0.0005). This study demonstrates that the inflammatory process should be considered central to the understanding of the pathogenesis of cellular injury in compartment syndrome.

Cite this article: Bone Joint J 2015;97-B:539–43

Purpose

Compartment syndrome is a limb threatening condition. Prior research has been limited by an inability to assess functional and histologic changes in muscle over time. This study was designed to assess and quantify functional deficits and histologic changes following acute compartment syndrome of the lower limb in a novel rat model.

Purpose: Compartment syndrome is a limb-threatening condition. Treatment is urgent decompression by fas-ciotomy. However, orthopedic surgeons are often confronted by a limb at risk for compartment syndrome, in which treatments to preserve tissue might be considered. Hypothermia has shown promise as a technique of maintaining tissue viability in transplant surgery, replant surgery and soft tissue injury. Cooling reduces microvascular dysfunction, inflammation and edema. This study was designed to determine whether tissue cooling might reduce muscle damage in the setting of elevated intracompartmental pressure. Purpose This study investigated the effect of hypothermia on tissue perfusion, viability and the inflammatory response in an animal model of elevated intracompartmental pressure. We hypothesize that hypothermia will preserve muscle tissue viability in an animal model of elevated intracom-partmental pressure.

Method: Twenty Wistar rats were randomized. Five animals had elevated intracompartmental pressure for 2 hours (CS). Five had elevated pressure and hindlimb cooling to 25oC (CS-HY). Five had hindlimb cooling to 25oC (HY) and 5 were control animals (C). All animals were anaesthesized for study. Core temperature was maintained over 30oC. Elevated ICP was maintained (30mmHg) using a saline infusion technique (groups CS and CS-HY). After 2 hours, fasciotomies were completed and intravital microscopy was used to measure tissue viability, microvascular perfusion and inflammation.

Results: The use of hypothermia reduced tissue damage by approximately 50% in the CS-HY group (8.2% injured cells) compared with the CS group (16.5% injured cells). There was no difference in capillary perfusion comparing the CS and CS-HY groups (p> 0.05). The number of adherent inflammatory cells was fewer comparing the CS-HY with the CS groups, but this did not reach statistical significance with the numbers available for study.

Conclusion: Hypothermia preserved tissue viability in an animal model of elevated intracompartmental pressure. Fasciotomy remains the gold standard treatment for established compartment syndrome. However cooling may be useful to preserve tissue viability in extremities that are at risk of developing compartment syndrome. The clinical utility of hypothermia for compartment syndrome requires further study.

Purpose: Severe compartment syndrome is associated with renal failure, end organ damage, and systemic inflammatory response syndrome (SIRS). Intravital videomicroscopy (IVVM) is a useful tool to study capillary perfusion and inflammation in end organs such as the liver and lungs. In this study, the systemic effect of hindlimb compartment syndrome was studied using hepatic IVVM. The purpose was to measure the effect of increased hindlimb intracompartmental pressure on hepatocyte viability, inflammation, and blood flow in a rodent model.

Method: Ten Wistar rats were randomised into control (C) and Compartment Syndrome (CS) groups. Animals were anaesthetized with 5 % isoflurane. Mean arterial pressure was monitored using a carotid artery catheter. Elevated intracompartmental pressure (EICP) was induced by saline infusion into the anterior compartment of the hind limb and maintained for 2 hours between 30–40mmHg in the CS group. Two hours following fasciotomy, the liver was analyzed using IVVM to quantify capillary perfusion as a measure of microvascular dysfunction. The numbers of adherent and rolling leukocytes in venules and sinusoids were quantified to measure the inflammatory response. Irreversible hepatocyte injury was measured using a fluorescent vital dye which labels the nuclei of severely injured cells.

Results: Hepatocellular injury was significantly higher in the CS group (325±103 PI labeled cells/10-1 mm2) compared to controls (30±12 PI labeled cells/10-1 mm2)(p=0.0087). The number of adherent venular white blood cells (WBC) was significantly higher for the CS group (5±2/hpf) than controls (0.2±0.2)(p=0.0099). Volumetric blood flow was not significantly different between CS and controls.

Conclusion: After only 2 hours of compartment syndrome in this animal model, the number of activated white blood cells increased 25-fold and liver cellular injury increased 10-fold compared to controls. Marked systemic inflammation and hepatocellular damage was detected in response to isolated limb compartment syndrome. Compartment syndrome is a low-flow ischemia/reperfusion injury with a profound inflammatory response. Further research into the severe end-organ damage associated with compartment syndrome is required.

Purpose: Compartment syndrome is a limb-threatening complication of skeletal trauma. Both ischemia and inflammation may be responsible for tissue necrosis in compartment syndrome (CS). In this study, normal rodents were compared with neutropenic animals to determine the importance of inflammation as a mechanism of cellular damage using techniques of intravital videomicroscopy (IVVM) and histochemical staining.

Method: Forty Wistar rats were randomised. Twenty animals served as a control (group C). Twenty rats were rendered neutropenic using cyclophosphamide (250mg/kg) (group N). Animals were anaesthetised with 5 % isoflurane. Elevated intracompartmental pressure was induced by saline infusion into the anterior hindlimb compartment and maintained at 30–40 mmHg for 0, 15, 45 or 90 minute time intervals. Following fasciotomy, the EDL muscle was analyzed using IVVM to quantify tissue injury, capillary perfusion, and inflammatory response.

Results: The proportion of injured cells decreased in group N compared to group C at all time intervals of EICP (p< 0.05). The proportion of injured cells in group N was 8 % after 0 minutes EICP, and 12, 15, and 10 % at 15, 45, and 90 min of EICP. In group C injured cells increased from 8 % to 20, 22, and 21 % at 15, 45, and 90 minutes EICP respectively. Groups N and C both demonstrated a time-dependent reduction in capillary perfusion. In group N continuously-perfused capillaries decreased from 79±4/mm with 0 min of EICP, to 48±11/mm (15min), 36±7/mm (45min), and 24±10/mm (90min) (p < 0.05). Overall, There was no difference between groups N and C with regards to perfusion (p> 0.05).

Conclusion: This study demonstrates the importance of inflammation as a cause of injury in compartment syndrome. There was a 50% decrease in injury in neutropenic animals compared to controls after 90 minutes of elevated intracompartmental pressure. Microvascular perfusion analysis demonstrated a time-dependent decrease in capillary perfusion in both neutropenic and control animals. Blocking of the inflammatory response via neutropenia was protective against tissue injury. These results provide evidence toward a potential therapeutic benefit for anti-inflammatory treatment of elevated intra-compartmental pressure.

Purpose: Compartment syndrome is a severe complication of skeletal trauma. Intravital microscopy (IVVM) has demonstrated an inflammatory response to compartment syndrome (CS). The molecular mechanisms underlying this inflammatory response are unknown. The purpose of this study was threefold. First, a broad inflammatory cytokine profile was examined to determine the molecules responsible for white cell recruitment. As well, skeletal muscle expression of white cell adhesion molecules including P-Selectin, E-Selectin, Mac-1 and ICAM-1 were examined to assess the extent of white cell activation in target tissues. Finally, skeletal muscle apoptosis was measured to determine the magnitude of cell death.

Method: Normal and neutropenic rats were randomised to either compartment syndrome or control groups. CS Animals were treated with 45 minutes of elevated intra-compartmental pressure (EICP) of the hindlimb. Fasciotomy was then performed, followed by 60 minutes of reperfusion. Control animals experienced no EICP. Blood was collected from carotid arterial lines used for pressure monitoring. Skeletal muscle tissue samples were collected from the EDL following reperfusion. Blood samples were obtained from carotid arterial lines and skeletal muscle was collected following reperfusion. A Multiplex assay was used to examine serum levels of 24 proinflammatory cytokines/chemokines. Skeletal muscle mRNA levels of P-Selectin, E-Selectin, Mac-1 and ICAM-1 were evaluated using real-time PCR. Finally, skeletal muscle apoptosis was measured by DNA laddering and a caspase-3 assay.

Results: Neutropenic CS animals demonstrated a continuous increase in TNF-alpha levels, peaking at 700+/−350pg/ml by 60 minutes of reperfusion. TNF-alpha values for other groups did not increase. A 104-fold increase in ICAM-1 mRNA levels was observed in neutropenic CS rats while other groups showed no significant increase. There was no significant increase in any group for P-Selectin, E-Selectin, or Mac-1.

Conclusion: This study is the first to attempt to describe the molecular inflammatory response in CS. Neutropenic CS animals demonstrated an upregulation in TNF-alpha and ICAM-1 mRNA levels. This likely represents an attempt to generate an inflammatory response in the neutropenic animals. Additional data at incremental timepoints is necessary to further characterize the molecular mechanisms. However, both TNF-alpha and ICAM-1 appear to be important in the mechanism of inflammatory activation in compartment syndrome.

Purpose: Indomethacin may preserve tissue viability in compartment syndrome. The mechanism of improved tissue viability is unclear, but the anti-inflammatory effects may alter the relative contribution of tissue necrosis versus apoptosis to cellular injury. Existing studies have only considered indomethacin administration prior to induction of compartment syndrome. The purpose of this study was to determine the effect of timing of indomethacin administration on muscle damage in compartment syndrome, and to assess apoptosis as a cause of tissue demise.

Method: Twenty-four Wistar rats were randomized to elevated intracompartmental pressure (EICP) for either 45 or 90 minutes (30mm Hg). In the 45 min group, indomethacin was withheld (group 1), given prior to induction of EICP (group 2) or given 15 min prior to fasciotomy (group 3). In the 90 min group, indomethacin was withheld (group 4) or provided 30 or 60 minutes prior to fasciotomy (groups 5 and 6). Intravital microscopy and histochemical staining assessed capillary perfusion, cell damage and inflammatory activation within EDL muscle. Apoptosis was assessed using ELISA staining for caspase-3. Groups were compared with one-way ANOVA (p< 0.05).

Results: Perfusion improved in indomethacin-treated groups. Nonperfused capillaries decreased from group 1 (50.1±2.5), to groups 2 (38.4±1.8) and 3 (14.13±1.73)(p< 0.0001). Similarly, groups 5 and 6 had 25% fewer non-perfused capillaries compared to group 4 (p< 0.0001). Tissue viability improved in indo-methacin-treated groups. Groups 2 and 3 showed fewer damaged cells (1±0.5% and 8.7±2%) compared to group 1 (20±14%)(p< 0.0001). Groups 5 and 6 showed decreased cell damage (13±1% and 11±1%) compared to group 4 (18±1%) (p< 0.01). Apoptotic activity was present in compartment syndrome. At 30 minutes there were elevated caspase levels in EICP groups (0.47±0.08) compared to controls (0.19±0.02). However, indomethacin treated groups did not differ from controls with regards to caspase levels (p> 0.05).

Conclusion: Indomethacin decreased cell damage and improved perfusion in compartment syndrome. The benefits of indomethacin were partially time dependent; some improvement in tissue viability occurred regardless of timing of administration. Although apoptosis was common in compartment syndrome, the protective effect of indomethacin does not appear to be related to apoptosis.

Purpose: This study determined the relative role of inflammation and ischemia in cell damage using an animal model of compartment syndrome.

Method: Forty adult Wistar rats were studied according to a protocol approved by the animal care committee at our institution. Twenty rats were used as control animals, while an additional 20 rats were pretreated with cyclophosphamide to create a leucocyte-deplete state. Animals were anesthetized using 5% isoflurane. Mean arterial pressure was maintained at 80 mm Hg and core temperature was maintained at 36 degrees. Animals were then randomly assigned to one of 4 groups, in which hindlimb compartment pressure was maintained at 30 mm Hg for 0, 15, 45, or 90 minutes. Intravital microscopy was then utilized to study capillary perfusion, white blood cell activation, and cellular damage in the hindlimb EDL muscle.

Results: Inflammation: White blood cell activation was dampened in the neutropenic animals by approximately 85 % at all time periods. Capillary Perfusion: Perfusion was similar between the neutropenic and control animals. Both groups demonstrated a gradual decrease in the number of continuously perfused capillaries, from 80 % at 0 min of elevated intracompartmental pressure (EICP) to 30 % after 90 minutes of EICP. Cellular damage: Cellular damage, measured using a differential staining technique, decreased by 55 % in the neutropenic group after 90 minutes of EICP (p< 0.005).

Conclusion: Compartment syndrome is an important clinical problem resulting in severe muscle damage. In this study, inflammation was confirmed as an important causative element of cell damage. Based upon the results of this study, adjuvant treatment to fasciotomy designed to reduce inflammation and cellular damage may have important clinical benefit.

Purpose: This study was designed to determine whether indomethacin, a potent anti-inflammatory agent, reduces muscle damage secondary to elevated ICP.

Method: 16 adult Wistar rats were randomized to 4 groups. In group 1 (control), no intervention occurred. Group 2 (indo) rats were administered indomethacin (12mg/kg) with no elevation of ICP. Group 3 (CS) rats had elevated ICP (30–40mmHg × 45 minutes) using saline injection. Group 4 rats (CS/indo) had elevated ICP and indomethacin administration. After 45 minutes, hindlimb fasciotomy was performed. The extensor digitorum longus muscle was reflected onto an intravital microscope. Capillary perfusion was measured by comparing the number of continuously perfused capillaries to intermittent and non perfused capillaries. Inflammation was determined using the number of activated (rolling and adherent) white blood cells. Muscle cell damage was measured using differential fluorescent staining. Perfusion, inflammation, and muscle damage were compared in all 4 groups using a one-way ANOVA (p< 0.05).

Results: Perfusion: Indomethacin treatment (CS/indo) increased the proportion of intermittently perfused capillaries (39.1 ± 2.2 vs 30.3 ± 2.7) and decreased nonperfused capillaries (38.4 ± 1.8 vs 50.1 ±2.5) compared to CS (p=0.0002). Control and indo groups demonstrated more continuously perfused capillaries compared to CS or CS/indo groups (p0.05).

Conclusion: Treatment of elevated ICP with indomethacin improved microvascular perfusion and reduced cell damage. The protective mechanism of indomethacin is unknown, but may be related to an anti-oxidative and vasodilatory effect. Treatment of elevated intracompartmental pressure with indomethacin dramatically reduces muscle damage and may have important future clinical benefit. Further research is required to determine the mechanism of action.

Elevated intracompartmental pressure (ICP) results in muscle damage. Previous studies identified severe inflammation associated with elevated ICP. This study was designed to determine whether indomethacin, a potent anti-inflammatory agent, reduces muscle damage secondary to elevated ICP. We hypothesised that administration of indomethacin reduces muscle damage from elevated ICP.

Sixteen adult Wistar rats were randomised to four groups. In group One (control), no intervention occurred. Group Two (indo) rats were administered indomethacin (12mg/kg) with no elevation of ICP. Group Three (CS) rats had elevated ICP (30–40mmHg X 45 minutes) using saline injection. Group Four rats (CS/indo) had elevated ICP and indomethacin administration. After forty-five minutes, hindlimb fasciotomy was performed. The extensor digitorum longus muscle was reflected onto an intravital microscope. Capillary perfusion was measured by comparing the number of continuously perfused capillaries to intermittent and non perfused capillaries. Inflammation was determined using the number of activated (rolling and adherent) white blood cells. Muscle cell damage was measured using differential fluorescent staining. Perfusion, inflammation, and muscle damage were compared in all four groups using a one-way ANOVA (p< 0.05).

Perfusion: Indomethacin treatment (CS/indo) increased the proportion of intermittently perfused capillaries (39.1 ± 2.2 vs 30.3 ± 2.7) and decreased nonperfused capillaries (38.4 ± 1.8 vs 50.1 ±2.5) compared to CS (p=0.0002). Control and indo groups demonstrated more continuously perfused capillaries compared to CS or CS/indo groups (p< 0.0001). Muscle damage: Indomethacin treatment of elevated ICP reduced the proportion of damaged cells from 0.20 ± 0.14 (CS) to 0.01 ± 0.0.005 (CS/indo, p< 0.0001). There were no differences between CS/indo, control, or indo groups. Inflammation: CS and CS/indo groups demonstrated greater inflammatory activation compared to control and indo groups (p< 0.001). There were no differences in inflammatory activation between CS and CS/indo (p> 0.05).

Treatment of elevated ICP with indomethacin improved microvascular perfusion and reduced cell damage. The protective mechanism of indomethacin is unknown, but may be related to an anti-oxidative and vasodilatory effect. Treatment of elevated intracompartmental pressure with indomethacin dramatically reduces muscle damage and may have important future clinical benefit.

Elevated intracompartmental pressure (ICP) results in tissue damage due to impaired microcirculatory function. The nature of microcirculatory impairment in elevated ICP is not well understood. This study was designed to measure the effects of increased ICP on skeletal muscle microcirculation, inflammation and cell viability using intravital videomicroscopy.

Twenty adult male Wistar rats were randomised to four groups: the control group (control) had no intervention; while three experimental groups had elevated ICP maintained for fifteen (15m), 45 (45m), or ninety (90m) minutes. Compartment pressure was continuously monitored and controlled between 30¡V40mmHg in the posterior hindlimb using saline infusion into the anterior hindlimb. Mean arterial pressure was maintained between 80 and 120mmHg. Fasciotomy was then performed and the Extensor Digitorum Longus muscle studied using intravital videomicroscopy. Perfusion was measured by comparing the numbers of continuous, intermittent, and nonperfused capillaries. Inflammation was measured by counting the number of activated (rolling and adherent) leukocytes in post-capillary venules. Muscle cellular Injury was measured using fluorescent vital staining of injured cell nuclei.

Perfusion: The number of continuously perfused capillaries decreased from 77 ± 3/mm (control) to 46 ± 10/mm (15m),40±10/mm(45m)and27±8/mm(90m)(p< 0.05). Non-perfused capillaries increased from 13 ± 1 (control) to 16 ± 4 (15m), 30 ± 7 (45m), and 39 ± 5 (90m) (p< 0.05). Inflammation: Activated leukocytes increased from 3.6 ± 0.7/(100ƒÝ)2 (control) to 5.9 ± 1.3 (15m), 8.6 ± 1.8 (45m), and 10.9 ± 3.0/(100ƒÝ)2 (90m) (p< 0.01). Injury: The proportion of injured cells increased from 5 ± 2 % in the control group to 12 ± 3 (15m), 16 ± 7 (45m) and 20 ± 3 % (90m) (p< 0.05).

As little as fifteen minutes of 30mmHg ICP caused irreversible muscle damage and microvascular dysfunction. With increased duration, further decreases in capillary perfusion and increases in injury are noted. A severe inflammatory response accompanies elevated ICP. The role of inflammation in compartment syndrome is unknown, but may contribute to cell injury and reduced capillary perfusion.