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. Author manuscript; available in PMC: 2018 Oct 1.
Published in final edited form as: Biochim Biophys Acta. 2017 Aug 26;1863(10 Pt B):2526–2533. doi: 10.1016/j.bbadis.2017.08.024

Mitochondrial dysfunction in rat splenocytes following hemorrhagic shock

Marie Warren 1,1, Kumar Subramani 1,1, Richard Schwartz 1, Raghavan Raju 1,*
PMCID: PMC5653436  NIHMSID: NIHMS902215  PMID: 28844961

Abstract

The regulation of mitochondrial function is critical in cellular homeostasis following hemorrhagic shock. Hemorrhagic shock leads to fluid loss and reduced availability of oxygen and nutrients to tissues. The spleen is a secondary lymphoid organ playing a key role in ‘filtering the blood’ and in the innate and adaptive immune responses. To understand the molecular basis of hemorrhagic shock, we investigated the changes in splenocyte mitochondrial respiration, and concomitant immune and metabolic alterations. The hemorrhagic injury (HI) in our rat model was induced by bleeding 60% of the total blood volume followed by resuscitation with Ringers lactate. Another group of animals was subjected to hemorrhage, but did not receive fluid resuscitation. Oxygen consumption rate of splenocytes were determined using a Seahorse analyzer. We found a significantly reduced oxygen consumption rate in splenocytes following HI compared to sham operated rats. The mitochondrial stress test revealed a decreased basal oxygen consumption rate, ATP production, maximal respiration and spare respiratory capacity. The mitochondrial membrane potential, and citrate synthase activity, were also reduced in the splenocytes following HI. Hypoxic response in the splenocyte was confirmed by increased gene expression of Hif1α. Elevated level of mitochondrial stress protein, hsp60 and induction of high mobility group box1 protein (HMGB1) were observed in splenocytes following HI. An increased inflammatory response was demonstrated by significantly increased expression of IL-6, IFN-β, Mip-1α, IL-10 and NFκbp65. In summary, we conclude that splenocyte oxidative phosphorylation and metabolism were severely compromised following HI.

Introduction

Severe hemorrhage leads to dysregulation of multiple biochemical pathways leading to cell apoptosis, organ damage and mortality. Severe hemorrhage, which often occurs with traumatic injuries, causes whole body hypoxia, metabolic perturbations and systemic inflammatory response (1–4). Patients who survive the initial traumatic insult remain susceptible to multiple organ failure and death (5, 6). Tissue damage occurs in many organs, including liver, intestine, lungs, heart and spleen depending on the severity of hemorrhagic injury (HI), and these alterations persist for a prolonged period of time despite fluid resuscitation (7–9). HI in the human as well as in animal models triggers a systemic inflammatory response and decreased splenic function (8, 10). Studies from other laboratories demonstrated a declined phagocytic function for splenic macrophage, reduced splenic blood flow and declined lymphokine release following HI (11–13). Previous studies demonstrated alterations in a number of genes related to mitochondria and glucose oxidation following HI in animal models (14, 15). Mitochondrial functional preservation is critical in energy homeostasis following HI.

Hypoxia may be implicated as one of the causes for the increased tissue and systemic inflammatory responses seen in traumatic injuries (16, 17). Furthermore, the decreased oxygen availability in tissues result in declined OXPHOS activity and ATP production. To further confirm the dysregulation of mitochondrial metabolism in HI, our laboratory and others have demonstrated that agents which potentiate mitochondrial function can improve organ function and survival following HI in experimental models of hemorrhagic shock (18–20). However, the understanding of the role of mitochondria in HI is still evolving and the effect of HI on splenocyte mitochondrial respiration remains unknown. In this study we sought to determine the alteration of mitochondrial respiration in splenocytes, and to further investigate the changes to molecular mediators of stress and inflammation following hemorrhagic shock in a rat model.

Materials and Methods

Animals and hemorrhagic injury procedures

Male Sprague Dawley (Charles River Laboratory Wilmington, MA, USA) rats were used. The animal use protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at Augusta University. The animals were subjected to sham or hemorrhagic injury (HI) as described before (18). The animals were anesthetized with 2.5% isoflurane, a midline laparotomy was performed, and the incision closed to induce soft tissue trauma. Both femoral arteries and one femoral vein were cannulated (PE-50 tubing) and one artery was connected to a blood pressure analyzer (Digi-Med; Micro-Med Inc., Louisville, KY, USA) while hemorrhage was performed through the other artery. The resuscitation fluid was administered through the femoral vein. All surgical sites were bathed with bupivacaine. Sham animals were not subjected to bleeding or resuscitation. The animals in the HI groups were bled for 45 min, maintaining the low MAP of 40±5, until 60% of circulatory blood volume was withdrawn. The animals were maintained at this low pressure for another 45 min. The HI animals were divided into two groups; one group was resuscitated with Ringer’s lactate for one hour while the other group was not resuscitated. The animals that were resuscitated were observed for 2 hours, euthanized and tissues collected. The animals in the group that were not resuscitated were euthanized when their mean arterial pressure (MAP) dropped below 30 mmHg.

Isolation of Splenocytes

A part of each animal’s spleen was harvested and submerged in RPMI 1640 (Thermo Scientific, Chicago, IL). Red blood cells were lysed using lysis buffer (Thermo Scientific, Chicago, IL) and vigorous trituration. Remaining splenocytes were centrifuged and resuspended in RPMI 1640 supplemented with 10% FBS.

Mitochondrial respiration

The Seahorse XFp Analyzer (Seahorse Biosciences, North Billerica, MA) was used according to the manufacturer’s protocol to measure oxygen consumption rate (OCR) of the isolated rat splenocytes. Splenocytes were plated in XFp base medium, minimal DMEM (Seahorse Biosciences, North Billerica, MA) supplemented with 1mM pyruvate, 2mM glutamine, 10mM glucose (Sigma) at a density of 3 × 105 cells per well in specialized XFp miniplates pretreated with Cell Tak (Fisher). Plates were spun and then incubated for 30 minutes at 37 °C prior to loading into the Seahorse analyzer. Three baseline OCR measurements were taken for each well in the first 35 minutes, and then the following mitochondrial inhibitors were sequentially injected: oligomycin (1 μM), carbonycyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) (0.3 μM), antimycin A and rotenone (1 μM). Three OCR values were automatically calculated after each injection by the Seahorse XFp software. Data were obtained as the mean value ±SEM for each time point in pmol per minute (n = 3 replicates per treatment group). For each experimental Sham and HI animal pair, OCR values of animals in HI group were normalized to the sham.

TMRE Staining

Both sham and HI splenocytes were stained with 1μM TMRE for 30 minutes alongside a baseline control first treated with 20μM FCCP for 10 minutes. Fluorescence was read at Ex/Em = 549/575, and TMRE fluorescence was normalized to the sham values for each experiment.

Western blot analysis

The spleen tissue was homogenized in Pierce RIPA lysis buffer (Thermo Scientific, Chicago, IL) containing 25 mmol/L Tris-HCl pH 7.6, 150 mmol/L NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS and protease inhibitor cocktail (Sigma Aldrich, St. Louis, MO, USA). Protein samples were resolved on SDS polyacrylamide gel, transferred to PVDF membrane, blocked using 5% (w/v) nonfat dried milk or 5% BSA in Tris-buffered saline containing 25 mmol/L Tris-HCl (pH 7. 4), 0.13 mol/L NaCl, 0.0027 mol/L KCl and 0.1% Tween 20 for 1 h at room temperature (RT) and then incubated with respective antibodies overnight at 4°C or for 1 h at RT. The membranes were probed with antibodies to GAPDH, VDAC, HMGB1, heat shock protein 60 (hsp60) (Thermo Scientific). The membranes were washed and incubated with horseradish peroxidase conjugated secondary antibody to mouse IgG or rabbit IgG (Cell Signalling) followed by enhanced western lightning plus-ECL (PerkinElmer). The protein bands were detected by autoradiography and quantitated by densitometry using the ImageJ software (NIH, Rockville, MD).

Real-time Polymerase Chain Reaction

Total RNA was isolated from spleen tissue using the TRIzol method as previously described (21). 300 ng of total RNA isolated was reverse transcribed to cDNA using reverse transcription kit (Promega). Quantitative real-time PCR was performed using Agilent Technologies Stratagene Mx3000P real-time PCR machine. The primer sequences are provided in Supplementary Table 1. The thermal cycling conditions were 95°C for 5 min followed by 40 cycles of 95°C for 15 seconds and 60°C for 50 seconds. Results are expressed as a ratio of expression to beta-actin and normalized to the values obtained for samples in sham group.

Citrate synthase activity measurement

Citrate synthase activity was determined using a Citrate Synthase Activity Assay Kit (Sigma, St Louis, MO) according to the manufacturer’s instructions. Briefly, 20mg of spleen tissue was homogenized in 200uL citrate synthase assay buffer. The citrate synthase activity level was then determined using a coupled enzyme reaction, which results in a colorimetric (412nm) product proportional to the enzymatic activity present. The enzyme activity values were then normalized to the total protein content of the spleen homogenate and was further normalized to sham values.

Statistics

Multi-group comparisons were carried out and significance determined by one-way ANOVA followed by Tukey’s test using GraphPad software (Graphpad Prism, WA). Two-group comparisons for significance were done by Mann–Whitney nonparametric test using GraphPad software. A p value less than 0.05 is considered significant.

Results

Hemorrhagic Injury and fluid resuscitation

A sub-group of rats were subjected to HI and following a hypotensive period, Ringer’s lactate was administered for fluid resuscitation. The fluid resuscitation resulted in a significant increase in MAP (Fig 1A). Consistent with the blood loss (Fig 1B) and contributed to by the consequential hypoxia, plasma lactate levels were significantly elevated at 2 hours following the end of resuscitation (Fig 1C). However, in another sub-group, when animals were not resuscitated by the fluid, none of them survived more than 1 hour after the shock period (Fig 1D).

Figure 1. Hemodynamics and hemorrhagic injury.

Figure 1

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(A) Mean arterial pressure (MAP) at different time points during hemorrhagic injury, resuscitation and recovery. MBO=maximum bleed out time. (n=4). * indicates p<0.05 compared to MAP at the start of resuscitation. (B) Hematocrit (HCt) values at the start of hemorrhage procedure and 2 hours post resuscitation. * indicates p<0.05 compared to sham. (C) Plasma lactate values for sham and HI + res 2 hours post resuscitation; * indicates p<0.05 compared to sham. (D) Kaplan-Meier survival curves for HI without resuscitation. Time 0 is time at the end of shock period (n = 4),

Mitochondrial oxygen consumption and ATP production following HI

In order to determine mitochondrial functional integrity following HI, we tested the OCR of splenocytes isolated from rats subjected to HI and fluid resuscitation. The animals were sacrificed two hours after the end of fluid resuscitation. There was a decrease of approximately 30% in basal OCR in splenocytes of animals subjected to HI and resuscitation compared to that in sham animals (Fig. 2). The splenocytes were sequentially treated with oligomycin (ATP synthase inhibitor), FCCP (membrane depolarizer) and rotenone/antimycin A (complex I and complex III inhibitors). As shown in Fig 2, reduced ATP production, decreased maximal respiration and declined spare respiratory capacity were observed in the splenocytes from animals subjected to HI and resuscitation, as compared to the splenocytes harvested from the sham animals (Fig 2A–F). We further repeated this experiment in animals that were not subjected to fluid resuscitation, the splenocytes were harvested upon euthanasia when the MAP dropped below 30 mm Hg and found that basal OCR and ATP production did not significantly differ from the resuscitated animals (Fig 2A,C,D). However there was a decrease in maximal respiration and spare respiratory capacity in the group of animals that did not receive fluid resuscitation, compared to the fluid resuscitated animals, though only maximal respiration showed statistical significance (Fig E, F).

Figure 2. Mitochondrial respiration in sham and HI splenocytes.

Figure 2

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(A) HI splenocytes demonstrate decreased oxygen consumption. Mitostress test was performed by sequential addition of oligomycin (complex V inhibitor), FCCP (mitochondrial membrane depolarization), rotenone (complex I inhibitor) and antimycin (Ant A- complex III inhibtior). (n=3–6). (B) A diagrammatic representation of mitostress test and functional significance of area under the curve (C–F) Basal respiration, ATP production, maximal respiration and spare capacity was calculated from the data in panel A by the method shown in panel B.; * indicates p<0.05 compared to sham; # indicates p<0.05 compared to HI+res.

Mitochondrial oxidation and membrane permeability

Citrate synthase functions at the entry gate of acetyl CoA in the Krebs cycle within the mitochondrial matrix by condensing the 2-carbon acetyl CoA with 4-carbon oxaloacetate to produce 6-carbon citrate. As shown in Fig 3A, there was a significant decrease in the activity of citrate synthase in splenocytes following HI. When compared to sham control the mean activity was decreased by 10%. Further, the mitochondrial permeability was assessed by TMRE uptake assay and the splenocytes from HI animals treated with TMRE showed significantly decreased fluorescence intensity compared to the control splenocytes (Fig 3B). The cell permeant cationic dye TMRE accumulates in negatively charged mitochondria whereas mitochondrial depolarization reduces membrane potential and sequestration of TMRE, resulting in reduced fluorescence intensity. There was also a concomitant increase in mitochondrial stress as demonstrated by the elevated level of HSP60, a mitochondrial chaperone involved in protein folding in the mitochondria (Figure 4).

Figure 3. Citrate synthases activity and TMRE fluorescence.

Figure 3

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(A) Citrate synthase activity is significantly reduced following HI compared to sham; n=4 each * indicates p<0.05 compared to sham, repeated 3 times (B) TMRE fluorescence is reduced in HI + res splenocytes compared to sham; * indicates p<0.05 compared to sham, repeated twice.

Figure 4. Stress protein changes in HI.

Figure 4

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Hsp 60 protein expression in sham and HI splenocytes. GAPDH (shared with Fig 5A) served as loading control. (n=6–7);* indicates p<0.05 compared to sham.

Alterations in hypoxic and immune response genes following HI

In order to understand the hypoxic response in splenocytes we tested the gene expression changes of HIF1α, a master regulator of hypoxia responsive genes and hexokinase 2 (HK2), the first enzyme in glycolytic pathway. As shown in Figure 5A and B, by real time PCR, we demonstrate a significant increase in the expression of both HIF1α and HK-2 in the splenocytes of animals subjected to HI.

Figure 5. Glycolytic shift following HI.

Figure 5

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HIF1α and HK2 mRNA expression in HI+res splenocytes relative to sham was determined by real time PCR (n=6);* indicates p<0.05 compared to sham.

The proinflammatory protein high-mobility group box 1 (HMGB1) is a well-characterized damage associated molecular pattern (DAMP) or alarmin that is ubiquitously expressed. Its expression has been found to be significantly increased following injury or infection and it is considered a promoter of inflammatory response (22). The protein expression of HMGB1 was tested by Western blot and HI significantly increased the level of this protein in splenocytes (Fig 6).

Figure 6. HMGB1 protein level increases in splenocytes after HI.

Figure 6

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HMGB1 protein expression in splenocytes was assessed by Western blot. GAPDH (shared with Fig 5B) served as loading control. (n=6–7); * indicates p<0.05 compared to sham.

In order to further confirm the immune response regulation in splenocytes following HI, we tested the gene expression changes of a battery of inflammation-related genes; IL-6, IL-10, IL-1 β, IL-2, MIP-1α and IFN-β by real time PCR. The expression level of IL-6, IL-10, IFN-β and Mip-1α following HI (Fig 7A – F) was significantly higher compared to that in sham operated animals. IL-6 and IL-10 demonstrated the largest increase following HI, 60 and 20-fold respectively. Though IL-2 showed an increase in expression following HI, the change was not statistically significant. Furthermore we also tested the expression of the p65 subunit of NF-kβ and found a significant increase in its expression in the animals subjected to HI (Fig 7G).

Figure 7. Inflammatory gene expression in splenocyte after HI.

Figure 7

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The expression of IL-6, IL-10, IFNβ, MIP-1α, IL-1β, IL-2 and Nfκb p65 mRNA expression was determined by real time PCR. The expression values were initially normalized to beta-actin and further normalized to sham. (n=6); * indicates p<0.05 Sham vs HI + res.

Discussion

Severe hemorrhage leads to tissue hypoxia and nutrient deprivation, cellular apoptosis and organ dysfunction (4, 15, 20, 23–25). The decreased availability of oxygen and nutrients coupled with fluid loss present physiological and biochemical challenges in cellular homeostasis (26–28). Mitochondria is a major subcellular target of ischemia/reperfusion injury and HI has been known to result in declined mitochondrial function in various tissues as demonstrated by decreased ATP production and decrease in the expression of mitochondrial respiratory complexes (29, 30). The hypoxia due to blood loss as well as the release of damage associated molecular patterns (DAMPs) are also known to cause profound inflammatory response following HI (31, 32). A systemic inflammatory response as evidenced by an increase in the levels of pro- and anti-inflammatory cytokines is a hallmark of HI (33). Our recent studies demonstrated a significant increase in the plasma levels of cytokines such as IL-6, TNF-α and IL-10 and activation of NF-κb (34). The hypoxic response of the host is complex as reduced oxygen availability can lead to altered mitochondrial energy metabolism as well as an inflammatory trigger (35, 36). The spleen is an organ that is important in both adaptive and innate immunity (37) and the hypoxic effect of HI on mitochondrial respiration in splenocytes has not been investigated.

We used a well-standardized HI model (18, 38, 39) and the hemodynamic (Fig 1A and B) and metabolic (Fig 1C) impact of the severe blood loss in this model is evident from the data presented. The study clearly demonstrates that at two hours following the end of fluid resuscitation, there was a significant decrease in basal oxygen consumption in splenocytes when compared to splenocytes harvested from sham operated animals. However, the basal respiration did not significantly change between splenocytes from fluid resuscitated and non-resuscitated animals. In splenocytes from animals subjected to HI followed by fluid resuscitation or no resuscitation, there was a decrease in ATP production, maximal respiration and spare respiratory capacity as compared to the splenocytes from sham controls.

It is pertinent to note that during preparation of splenocytes and subsequent oxygen consumption measurement, the cells were exposed to atmospheric oxygen levels. This might have affected any reversible hypoxic response in splenocytes prevailed in the in vivo environment following severe blood loss. Nevertheless, there was a measurable and significant decrease in oxygen consumption in the splenocytes following HI suggesting the impact of blood loss on mitochondrial function. Furthermore, the lack of any significant difference between fluid resuscitated and non-resuscitated splenocytes with respect to OCR demonstrates the severity of the effect of blood loss on mitochondrial metabolism, irrespective of fluid resuscitation. This severe impact on mitochondrial function is consistent with the observation that despite fluid resuscitation almost half of the animals die within 24 hours (18). The reversibility of the apparent metabolic and respiratory derangement following treatment with adjuncts to fluid resuscitation also suggest that any defect to the respiratory complexes may be reversed by early intervention, if mitochondrial dysfunction is the causative factor.

The mitochondrial functional decline as measured by OCR was further confirmed by the observation of decreased TMRE uptake in splenocytes from animals subjected to HI as compared to the sham animals. TMRE is a cationic dye that specifically accumulates in the negative environment within the matrix of mitochondria that are not depolarized. Therefore the results indicate a significant compromise of mitochondrial membrane potential in the splenocytes following HI. Furthermore a concomitant slowdown of Krebs cycle was indicated by the decreased citrate synthase activity. Citrate synthase catalyzes the condensation of oxaloacetate with the incoming acetyl CoA in the generation of citrate, a 6-carbon intermediate, in the citric acid cycle (40).

Hypoxia is a determining factor in the maintenance of cellular homeostasis. Therefore we tested the expression of the hypoxia responsive gene HIF1α in the splenocytes from sham and HI animals and as expected its expression was significantly increased in the splenocytes of animals subjected to HI. HIF1α controls the expression of more than one hundred genes involved in host response to hypoxia (41). HIF1α promotes glycolysis and inhibits genes important in mitochondrial oxidative phosphorylation (42). We also found an increase in the expression of HK2, the first enzyme in the glycolytic process, following HI. This is consistent with our previous observation of a situation similar to Warburg effect following tissue hypoxia in HI (43). The difference being that whereas aerobic glycolysis is postulated in cancer cells (Warburg effect), in conditions such as observed in HI, the decrease in OXPHOS and increase in glycolysis may be attributed to deficiency of oxygen (44). However despite causative distinction, the metabolic outcome of mitochondrial-glycolytic shift, remains the same in both situations. The decrease in OCR and mitochondrial stress observed may be a direct effect of the hypoxia and nutrient deprivation following HI.

The exacerbated inflammatory response is also a reckoning factor in the hypoxic response leading to organ dysfunction in hemorrhagic shock. As shown in Fig 7, there is a marked increase in the level of HMGB1 protein, a master regulator of inflammatory process, following HI in splenocytes. HMGB1 is a damage associated molecular pattern that provokes inflammatory response and it is released from the nucleus in response to conditions such as cellular stress, I/R injury, infection and sepsis (22, 45–48). The increased expression of HMGB1 in splenocytes is consistent with the systemic inflammatory response observed following HI. Our lab and others have reported the increased level of plasma cytokines immediately after HI (34, 49). Some of these cytokines begin to appear as early as one hour after HI (34, 38, 50, 51). The initial inflammatory response is likely to be a repair process to balance cellular homeostasis, but persistent exacerbated inflammation following the initial insult contributes to organ dysfunction (5, 52, 53). Studies have also shown a depressed mitochondrial function in peripheral blood mononuclear cells following hemorrhagic shock (29). However, when spleen derived macrophages, dendritic cells or T cells were cultured in vitro and stimulated with LPS or anti-CD3, as the case may be, a suppressed inflammatory response was observed in cells derived from animals subjected to HI (54, 55). An important factor that is not considered in such experiments is that during these experiments the cells are exposed for several hours in normoxic environment and this prolonged effect of the environment on the cells is unknown. Our experiments using snap frozen spleens show a significantly increased expression of several cytokines including IL-6. Consistent with these gene expression changes, the expression of the p65 subunit of inflammation promoting transcription factor NF-kb was also significantly increased (34, 56–58). The observation of an active immune response in the spleen needs to be further characterized by phenotyping splenocyte subsets and correlating with intracellular cytokine expression.

Our data demonstrate a significant decrease in mitochondrial respiration following HI and an inflammatory gene expression milieu in the splenocytes following HI. The declined mitochondrial function in the splenocytes as a hypoxic response is consistent with the observation of decreased ATP production and increased expression of HIF1α and inflammatory genes. However the cause-effect relationship or the exact sequence of events involving factors such as reduced mitochondrial oxygen consumption, increased mitochondrial stress and inflammatory response leading to declined cellular energetics and organ dysfunction remains unknown. A plausible explanation may be that decreased intracellular oxygen availability triggers a progressive shut down of mitochondrial oxidation thereby decreasing the production of ATP, increasing mitochondrial stress, and an exacerbated inflammatory response. In support of this explanation is the reports indicating that factors such as HIF-1a that control metabolic processes may also control immune cell function (59, 60). These studies show that intracellular metabolism is an important factor in the regulation of immune responses. Therefore our studies interlinking multiple biological processes and functions to the insult due to severe hemorrhage show the significance of understanding the role of metabolic perturbations in the maintenance of cellular homoeostasis and organ function following hemorrhagic shock. In summary, the results demonstrate that mitochondrial functional decline is among the key causative factors in the splenic functional dysregulation observed following HI and potentially contribute to outcome following HI.

Highlights.

  • Hemorrhagic shock results in mitochondrial dysregulation.

  • Mitochondrial oxygen consumption rate is decreased following hemorrhagic shock in a rat model.

  • Mitochondrial permeability and citrate synthase activity are decreased in splenocytes following hemorrhagic shock.

  • Hypoxia is likely to be a major cause for the splenic mitochondrial dysfunction and inflammatory response in the splenocytes following hemorrhagic shock.

Acknowledgments

Authors acknowledge the assistance of Sumin Lu in ATP and lactate determination. RR acknowledges the support of the National Institute of Health (R01 GM101927 and R01 GM122059) and laboratory start up assistance from the Augusta University, Augusta, GA, USA.

Raghavan Raju acknowledges the support of the National Institute of Health (R01 GM 101927) and laboratory start up assistance from the Augusta University, Augusta, GA, USA.

Footnotes

CONFLICT OF INTEREST

AUTHORS DECLARE NO CONFLICT OF INTEREST

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