CS remains one of the most devastating and challenging sequelae of musculoskeletal trauma. CS develops secondary to increased pressure within a closed osseofascial compartment, most commonly following a tibial diaphyseal fracture.[1–8] Elevated ICP compromises the microcirculation, leading to decreased oxygen and nutrient delivery, tissue anoxia and, ultimately, myonecrosis.[4,9,10]
The pathophysiology of CS-associated tissue injury is only partially understood. While ischemia-reperfusion (I/R) injury appears to be the main driving force, there is a critical distinction between CS and complete I/R: unlike complete I/R, tissue necrosis and ischemic injury in CS ensue despite a patent macrocirculatory system and palpable distal pulses. Previous studies have demonstrated that CS-induced tissue injury is more extensive than that of a complete ischemic insult of the same duration.
Normal microvascular perfusion is comprised of predominantly continuously perfused capillaries (CPC). Elevated ICP results in a shift of perfusion towards IPC and NPC,[13,14] leading to a “low flow” ischemic state. As a result, the metabolic demands of the tissue cannot be met, resulting in the production of reactive oxygen species (ROS). ROS, generated following the re-introduction of oxygen into previously ischemic tissue are known to trigger the acute inflammatory cascade and eventual cellular death via apoptosis or necroptosis.[15,16] Unlike a complete I/R (as observed following organ transplantation and re-vascularization procedures[17,18]), the defined phases of injury (ischemia, reperfusion) cannot be clearly delineated in a “low-flow” ischemia, initiating an early and ongoing reperfusion injury concurrent with ischemic period. The reperfusion injury not only persists throughout the duration of the CS, but also it is further intensified by the restoration of normal blood flow into the capillary bed after fasciotomy.
Activated leukocytes appear to play a major role in CS pathophysiology. While leukopenia does not restore or maintain perfusion, it appears to provide significant protection against muscle injury. The pro-inflammatory cytokine TNF-α has been shown to be acutely upregulated following reperfusion in multiple I/R studies.[19–21]
The purpose of this study was to examine the specific cytokines/chemokines released during CS in a rat model, as well as to evaluate the microvascular dysfunction, tissue injury, and leukocyte activation following the neutralization of pro-inflammatory cytokines TNF-α and IL-1β at the time of fasciotomy. We hypothesized that CS is associated with the initiation of the systemic inflammatory cascade, and that neutralization of ischemia-reperfusion injury-linked cytokines would diminish the microvascular dysfunction and tissue injury associated with CS.
Materials and methods
The experimental protocol was approved by the Canadian Council on Animal Care and the Animal Use Subcommittee at The University of Western Ontario. Male Wistar rats (body weight 180–250 g) were used for all experiments.
Animals were anesthetized with isoflurane (5% induction, 2% maintenance) in a 1:1 O2:N2 mixture. The left carotid artery was cannulated for the continuous monitoring of blood pressure, fluid replacement, and blood sampling; core body temperature was maintained at 37°C by the means of a heat lamp. A normotensive and normothermic model of CS was used.
CS was induced by an infusion of isotonic saline into the anterior compartment of the right-hind limb via a 24-gauge angiocatheter to raise the ICP, as previously described. Compartment pressures were measured by an electronic compartmental pressure monitoring system (Synthes USA, Paoli, Pennsylvania), inserted into the posterior compartment via a 14-gauge angiocatheter. Elevated ICP was maintained at 30 to 40 mm Hg for 2 hours, followed by fasciotomy and 45 minutes of reperfusion. The skeletal muscle was then imaged by intravital video microscopy (IVVM).
Animals were randomly assigned into 1 of 5 groups: Sham (n = 8), CS (with isotype control) (n = 8), CS with TNF-α neutralization (CS+TNF-α) (n = 4), CS with IL-1β neutralization (CS+IL-1β) (n = 4), and CS with neutralization of both TNF-α and IL-1β (CS+Combo) (n = 4). Sham animals underwent all the procedures as the CS groups, but they did not receive saline infusion into the anterior compartment of the hind limb and the ICP was maintained at the baseline level of 0 mm Hg.
All blood samples were drawn from the indwelling carotid line at 7 different time points: baseline (just prior to the elevation of ICP); at 1 hour CS; at 2 hours CS, just prior to fasciotomy; at 10 minutes reperfusion, at 20 minutes reperfusion, at 30 minutes reperfusion; at 45 minutes reperfusion, just prior to IVVM. At each time point, 0.1 mL of blood was withdrawn and replaced with 0.1 mL of normal saline.
Serum levels of cytokines/chemokines were assessed by xMAP Luminex assay (EMD Millipore, Mississauga, Ontario), as per manufacturer's instructions, simultaneously testing 24 different cytokines/chemokines: TNF-α, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-13, IL-17, IL-18, IFN-γ, Eotaxin, IP-10, MIP-1α, Leptin, MCP-1, GRO/KC, RANTES, GM-CSF, G-CSF, and VEGF. The samples were analyzed using Luminex 200 instrument (EMD Millipore, Mississauga, Ontario).
Neutralization of serum TNF-α and IL-1β
NA to TNF-α and/or IL-1β (eBioscience, San Diego, CA) was administered to CS animals just before fasciotomy at a dose of 3.5×104 neutralizing units/100 g body weight (0.3 mL IV). Syrian hamster IgG (eBioscience, San Diego, California) was used as an isotype control. Neutralization of serum TNF-α and IL-1β was confirmed by enzyme-linked immunosorbent assay (ELISA, Pierce Biotechnology, c/o Thermo Scientific, Rockford, Illinois) as per manufacturer's instructions. ELISAs were sensitive to <5 pg/mL.
Intravital video microscopy (IVVM)
Following fasciotomy, the extensor digitorum longus (EDL) muscle was prepared for IVVM, as previously described.[13,22–24] Briefly, following isolation of EDL, the animal was transferred onto the stage of an inverted microscope (Nikon, Nikon Instruments, Inc., Mississauga, Ontario, Canada); the EDL muscle was detached from its bony insertion and reflected into a saline bath containing 5 μg/mL each of the fluorescent vital dyes bisbenzimide (BB; excitation wavelength (Ex) 343 nm, emission wavelength (Em) 483 nm), and ethidium bromide (EB; Ex 482 nm, Em 616 nm). BB stains nuclei of all cells while EB stains the nuclei of cells with damaged cell membranes. The EB/BB ratio, therefore, was used as a measure of tissue injury.[13,22]
Microvascular perfusion and leukocyte activation within the post capillary venules were recorded by translumination with 20× and 40× objectives, respectively, in 5 adjacent fields of view, and captured into a computer for offline video analysis. Fluorescence microscopy was used to visualize the BB- and EB- labeled cells from the same fields of view. At the conclusion of the experiment, the animals were euthanized by an overdose of anesthetic agent.
Offline video analysis
Capillary perfusion was evaluated by counting the number of CPCs, IPCs, and NPCs capillaries crossing 3 parallel lines drawn perpendicular to the capillary axis on the computer monitor, and was expressed as a percentage (%) of total capillaries.[13,22]
Tissue injury was evaluated by counting the number of EB- and BB-labeled nuclei, and expressed as EB/BB ratio.[13,22]
Leukocyte activation (inflammation) was assessed by counting the number of rolling and adherent leukocytes in the postcapillary venules, and conveyed as cells per unit area (i.e., 1000 μm2). Venular area was measured using ImageJ software (NIH, Bethesda, Maryland). A leukocyte was deemed adherent if it remained immobile for at least 30 seconds and rolling if it remained in contact with the wall of the vessel but remained in motion.[13,22]
All data were expressed as mean ± SEM (standard error of the mean). Serum levels of cytokines/chemokines were analyzed by repeated measures analysis of variance (ANOVA) (GraphPad Prism v. 5.0c for Mac, San Diego, California); each sample within the time course was compared to its baseline levels. One-way ANOVA was used to compare the degree of perfusion, tissue injury, and leukocyte activation (adhesion, rolling). Newman—Keuls multiple comparison post-hoc test was used where appropriate. A sample size calculation was performed (StatMate, GraphPad Inc, San Diego, California), with power set at 85%. Statistical significance was defined as P < .05.
Sixteen of the 24 cytokines/chemokines sampled were detectable using xMAP Luminex technology. These included TNF-α, IL-1α, IL-1β, IL-10, IL-12p70, IL-13, IL-18, IFN-γ, Eotaxin, MIP-1α, Leptin, MCP-1, GRO/KC, IP-10, RANTES, and IL-6. Six of the detectable cytokines/chemokines were significantly elevated from their baseline levels at 2-hour CS (Table 1). These included pro-inflammatory cytokines TNF-α and IL-1β, anti-inflammatory cytokine IL-10, and chemokines GRO/KC, MCP-1, and MIP-1α.
Pro-inflammatory cytokine neutralization
Systemic TNF-α increased from 18.1 ± 3.4 pg/mL at baseline to 707.1 ± 181.9, 1495.0 ± 152.7, 1573.0 ± 188.0, 1460.4 ± 283.3, 1624.3 ± 546.9, and 2303.7 ± 394.9 pg/mL at 1-hour CS, 2-hour CS, and 10 minutes, 20 minutes, 30 minutes, and 45 minutes post-fasciotomy, respectively (P < .05). Administration of TNF-α NA at fasciotomy resulted in a complete inhibition of TNF-α to a nondetectable level, where it remained for the rest of the experiment (P < .05) (Fig. 1A).
Serum IL-1β was elevated from the baseline of 40.0 ± 26.7 pg/mL to 97.6 ± 32.1, 141.3 ± 13.1, 102.2 ± 22.1, 119.9 ± 17.6, 46.0 ± 46.0, and 61.2 ± 31.7 pg/mL at 1-hour CS, 2-hour CS, and 10 minutes, 20 minutes, 30 minutes, and 45 minutes post-fasciotomy, respectively (P < .05). Administration of IL-1β NA at fasciotomy resulted in a complete inhibition of IL-1β, to a nondetectable level, for the rest of the experiment (P < .05) (Fig. 1B).
Serum TNF-α and IL-1β levels in the sham group showed no significant changes from the baseline levels of 12.1 ± 6.9 pg/mL and 12.3 ± 7.1 pg/mL, respectively. Levels of TNF-α at 2 hours and 45 minutes reperfusion time-matched points were 14.8 ± 3.8 pg/mL, and 12.6 ± 5.9 pg/mL, respectively, while those of IL-1β were 11.0 ± 6.0 pg/mL and 14.0 ± 8.0 pg/mL, respectively (Fig. 1).
Elevation of ICP resulted in significant changes to microvascular perfusion, as shown in Figure 2. The number of CPC decreased from 75 ± 2% in the sham to 31 ± 3%, 36 ± 4%, 31 ± 3%, and 30 ± 2% in the CS, TNF-α, IL-1β and Combo neutralizing groups, respectively (P < .001). There were no significant differences in CPC between the CS and any of the neutralizing groups.
The number of IPC increased from 11 ± 2% in sham to 19 ± 1%, 25 ± 3%, 36 ± 4% and 38 ± 2% in the CS and TNF-α, IL-1β and Combo neutralizing groups, respectively (P < .001). The number of IPC in all neutralizing groups was significantly higher than that in CS group (P < .05).
The number of NPC increased from 14 ± 1% in sham to 50 ± 3%, 39 ± 3% 33 ± 2% and 32 ± 2% in the CS and TNF-α, IL-1β and Combo neutralizing groups, respectively (P < .001). The number of NPC in the CS+TNF-α neutralizing group was significantly lower as compared to the CS group (P < .05).
Elevation of ICP resulted in a significant increase in tissue injury, from 0.04 ± 0.02 in sham to 0.33 ± 0.04 in CS group (P < .001) (Fig. 3). Administration of TNF-α NA resulted in a decrease to 0.19 ± 0.04 (P < .001) and 0.13 ± 0.02 (P < .001) in CS+TNF+α and CS + Combo groups, respectively, while neutralization of IL-1β had no effect (0.29 ± 0.02 in CS+IL-1β group, not significant).
Elevation of ICP resulted in an increase in leukocyte rolling, from 2 ± 1 leukocytes/30 s/1000 μm2 in sham to 10 ± 3 leukocytes/30 s/1000 μm2 in CS group (P < .001). Administration of TNF-α NA resulted in a significant decrease to 2 ± 1 and 3 ± 1 rolling leukocytes/30 s/1000 μm2 in CS+TNF+α and CS+Combo groups, respectively (P < .01) (Fig. 4A). Administration of IL-1β following CS had no effect (15 ± 3 rolling leukocytes/30 s/1000 μm2, not significant).
There was a significant increase in leukocyte adherence following elevation of ICP, from 2 ± 1 to 14 ± 2 leukocytes/30 s/1000 μm2 in CS group (P < .001) (Fig. 4B). Administration of TNF-α NA produced a significant decrease to 1 ± 0 adherent leukocytes/30 s/1000 μm2 in both CS+TNF+α and CS+Combo groups (P < .001). Administration of IL-1β also significantly decreased leukocyte adherence (6 ± 1 adherent leukocytes/30 s/1000 μm2, P < .05).
In spite of the fact that CS has been a recognized complication of musculoskeletal trauma since Richard von Volkmann's description of ischemic contracture in 1881, the pathophysiology of CS remains largely unknown. The use of IVVM imaging has been instrumental in illustrating the derangement of microvascular perfusion following a CS insult. Under live conditions, we were able to observe the presence of CPC, IPC, and NPC within the same capillary bed, validating CS as a “low-flow” ischemic environment.
Inflammation has been shown to be a major contributor to the microvascular dysfunction associated with I/R (TNF-α has been shown to be elevated following a complete I/R injury[19,20]), and hence CS. The purpose of this study was to identify the mediators (i.e., cytokines/chemokines) involved, and to observe the effect of neutralizing 2 major pro-inflammatory cytokines, TNF-α, and IL-1β, on microvascular perfusion, tissue injury, and leukocyte activation in an animal model of CS.
This study confirms that CS induces a systemic response, as evidenced by the upregulation of pro-inflammatory cytokines/chemokines in circulation. Of the 14 cytokines/chemokines detected in the serum of rats challenged with CS, 6 were significantly elevated from their baseline levels after 2 hours of ICP elevation (P < .05): TNF-α, IL-1β, GRO/KC, MCP-1, MIP-1α, and IL-10. With the exception of IL-10, (which is an anti-inflammatory cytokine), all detected cytokines/chemokines are pro-inflammatory.[27–35]
TNF-α is a powerful pro-inflammatory cytokine released following trauma, inflammation, or infection. TNF-α acts as a chemoattractant for neutrophils, causes up-regulation of cytokine/chemokine production, and activates cell death via apoptosis and necroptosis.[27–30,37] Similarly, IL-1β is another pro-inflammatory cytokine that plays an important role in sterile inflammation. IL-1β is produced by activated macrophages, monocytes, endothelial cells, fibroblasts, and appears to be involved in cell proliferation, differentiation and apoptosis, mediating an inflammatory sequence similar to that of TNF-α. As both TNF-α and IL-1β have been demonstrated to play a significant role in I/R,[29,38] our current study focused on assessing the contribution of these 2 cytokines to the pathophysiology of CS.
CS resulted in a significant rise in systemic TNF-α and IL-1β, in conjunction with a marked increase in activated leukocytes. Modulating the inflammatory response significantly decreased leukocyte activation, illustrated by a decrease in both rolling and adherent leukocytes within the post-capillary venules (P < .05 and P < .0001, respectively).
Tempering the inflammatory response through neutralization of TNF-α (alone or in combination with IL-1β) also led to a significant relative reduction of approximately 40% in tissue injury following 2 hours of elevated ICP. Neutralization of IL-1β by itself had no appreciable effect; however, when given in combination with TNF-α, it augmented the reduction in parenchymal injury to levels previously found in a leukopenic model of CS. One possible explanation as to why neutralization of IL-1β alone failed to offer any protection may be due to the existence of temporal relationship between TNF-α and IL-1β release. In I/R injury, TNF-α release appears to precede that of IL-1β; this would indicate that IL-1β probably plays a more significant role at later stages of ischemia. Thus, our findings suggest that, although TNF-α appears to play a significant role in the inflammatory response associated with CS, the contribution of other cytokines/chemokines (e.g., IL-1β) to the pathophysiology is not inconsequential.
Neutralizing TNF-α and/or IL-1β at the time of fasciotomy did not provide any protection to the microvascular perfusion following a CS insult. These results corroborate previous work by our group, whereby leukopenia was not protective in preventing the microvascular dysfunction associated with CS. Again, a distinction from complete I/R must be highlighted: following complete ischemia, microvascular dysfunction, and tissue injury were significantly decreased in a leukopenic I/R animal model. Our current study, therefore, provides further evidence that the pathologic changes in microvascular dysfunction following CS have a distinct pathophysiology compared to complete I/R injury.
This study demonstrates that CS is associated with a significant activation of the systemic inflammatory response, and its initiation should, therefore, be considered in the pathophysiology of CS. In addition to its role as an acute inflammatory mediator, TNF-α has also been implicated in initiating cell death via apoptosis and necroptosis. The necroptotic pathway is also associated with the tissue injury following I/R.[40,41] In contrast to apoptosis, necroptosis generates a systemic inflammatory response. It can be inferred from our findings that this pathway of programmed cell death may contribute to the tissue injury associated with CS. It is plausible that the early and sustained reperfusion injury associated with ‘low-flow’ ischemia induces an early and sustained stimulus for the necroptotic pathway. This could in part, explain the greater tissue injury associated with CS versus complete I/R.
Surprisingly, neutralization of TNF-α (alone or in combination with IL-1β) did not confer the same type of protection to the microvasculature as that seen following administration of carbon monoxide (CO) donor, CORM-3, in a rat model of CS. CO has been shown to exhibit potent vasodilatory, anti-inflammatory and anti-apoptotic properties in various models of disease.[43,44] As such, the results of our current study confirm that inflammatory response is not the only component of CS pathophysiology, although it plays a major role. Hence, neutralization of pro-inflammatory cytokines does not address other factors that contribute to CS-associated damage (e.g., vasodilation, ROS production, apoptosis) and thus is insufficient in restoring the microcirculation back to normal.
To our knowledge, this is the first study that directly confirms CS as an inflammatory process. The systemic inflammatory cascade is complex and redundant, and these results are merely the beginning steps in an attempt to extrapolate the pathophysiologic mechanism by which CS injury occurs.
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