Endotoxemia is a leading cause of morbidity and death in horses and is a frequent sequel to diseases of the digestive tract such as colitis.1,2 The adverse effects of endotoxemia are induced by LPS, a component of the outer cell membrane of gram-negative bacteria.1
Treatment of horses for endotoxemia generally consists of treating the underlying cause, supportive treatment (eg, IV administered fluids), colloidal treatment, and administration of an NSAID and polymyxin B.3 Reducing lipid-derived, proinflammatory mediators by administering an NSAID is a proven method of ameliorating the clinical and biochemical changes associated with administration of LPS.3 However, in a study4 of human patients with severe sepsis, administration of an NSAID did not reduce mortality rate, although the blood concentrations of arachidonic acid metabolites were reduced. Furthermore, administration of an NSAID may cause toxicosis, especially to the renal and alimentary systems.5 Nonsteroidal anti-inflammatory drugs, such as flunixin meglumine, can retard healing of injured intestine by inhibiting production of prostaglandin, which in turn can result in increased absorption of endotoxin.6 Polymyxin B, an antimicrobial that binds to the lipid A component of the LPS molecule, is used at a subantimicrobial dose to treat horses for endotoxemia.7 Nevertheless, polymyxin B failed to substantially ameliorate clinical signs of endotoxemia in a carbohydrate-overload model of endotoxemia.8 Although administration of an NSAID or polymyxin B may reduce the clinical signs and hematologic derangements associated with endotoxemia, additional treatments warrant investigation.
Hyperbaric oxygen treatment, in which a subject inspires oxygen at values close to 100% and at pressures greater than atmospheric,9 has been investigated as a treatment for ameliorating experimentally induced endotoxemia in rodents. Lipopolysaccharide-treated mice had decreased production of proinflammatory cytokines and a marked increase in the production of anti-inflammatory cytokine interleukin-10 after HBOT,10,11 and HBOT prevented fever in LPS-treated rabbits by reducing circulating TNF-α and hypothalamic prostaglandin E2.12 In addition, rats repeatedly exposed to HBOT after they were administered LPS had a significant decrease in inflammatory mediators, free radicals, and mortality rate.13 Prior exposure to HBOT attenuated the effects of LPS on rabbits and rodents.12,14–16 Hyperbaric oxygen treatment has been used in clinical practice to treat horses for various conditions, such as anemia, fungal pneumonia, thermal burns, carbon monoxide poisoning, smoke inhalation, ileus, edema of the CNS, perinatal asphyxia, peripheral neuropathies, exertional rhabdomyolysis, cellulitis, compartmental syndrome, and ischemic injuries.17
The purpose of the study reported here was to determine whether HBOT could ameliorate the effects of experimentally induced endotoxemia in horses. We hypothesized that LPS would induce clinical and hematologic changes and that treating the horses with HBOT before administration of LPS would ameliorate these changes.
Materials and Methods
Animals—Eighteen adult mares ranging in age from 6 to 16 years (median, 11 years) and weighing 450 to 568 kg (median, 504 kg) were used in the study. The horses had not been exposed to experimentally administered LPS for at least 1 year. The horses had received routine vaccinations, had been dewormed regularly, and were determined to be healthy on the basis of physical examination and evaluation of a CBC. Horses had been kept on pasture, and their diet had been supplemented with grass hay. The day prior to the study, the horses were housed in individual stalls and a 14-gauge, 10-cm cathetera was placed into each jugular vein.
Experimental protocol—The study was approved by the University of Tennessee Institutional Animal Care and Use Committee. The horses were randomly assigned to 1 of 3 groups, and 2 horses were studied each day. Six horses were kept in a stall at ambient temperature before they were administered LPS (negative control group), 6 horses were treated with HBOT before they were administered LPS (treatment group), and 6 horses were treated with HBOT before they were administered PSS (positive control group). Experiments were started at 8:00 am to avoid the effects of diurnal variation, except on the days when 2 horses were administered HBOT; on these days, the experiment for the second horse started at 9:30 am. During treatment with HBOT, horses were kept in the same custom-made, monoplace hyperbaric chamber,b which was 12 feet high with an internal diameter of 10 feet and an interior volume of 23.36 m3 (23,360 L). The horses were neither sedated nor restrained and were observed continuously through a viewing window. During a period of 15 minutes, the pressure in the chamber was increased to 2.6 atmospheres. This pressure was maintained for 60 minutes and then decreased to ambient pressure over 15 minutes. The percentage of oxygen in the chamber was between 86.4% and 93.4%. After HBOT, the horse was returned to its stall, and 15 minutes later, LPS (0.2 μg of Escherichia coli 055 B5c/kg in 0.5 L of PSS) or PSS (0.5 L) was administered over 30 minutes by use of a volumetric infusion pump.d Lipopolysaccharide or PSS was administered into the left jugular catheter, and blood samples were collected from the right jugular catheter. Horses in the negative control group were kept in their stalls during the experiment and administered LPS at the same time interval as the horses in the treatment group. All horses were kept in the same stalls before, during, and after the experiment, each for the same period, and were offered free-choice water and grass hay.
Data collection—Time 0 was considered the time at which infusion of LPS or PSS was finished. Baseline clinical data, including rectal temperature, heart and respiratory rates, color (pink, hyperemic, or congested) and moistness of the mucous membranes (tacky or moist), and CRT (< 2 seconds = reference range; 2 to 3 seconds = prolonged), were collected at time −165 minutes (30 minutes before the horses were exposed to HBOT or ambient atmosphere) and again 15 minutes after the horse exited the chamber before administration of LPS or PSS. The LPS or PSS was administered over 30 minutes (start of LPS infusion, −30 minutes). Clinical data were again collected immediately after LPS or PSS was infused (0 minutes) and at 30, 60, 90, 120, 180, 240, and 360 minutes thereafter. At the end of the study, phenylbutazonee (4.4 mg/kg, IV) was administered to LPS-treated horses.
At each time point, the horses were evaluated by 2 unmasked observers for clinical signs of endotoxemia and signs were scored subjectively on a scale of 0 to 3 (0 = no signs, 1 = mild signs, 2 = moderate signs, and 3 = severe signs).18 Mild signs of endotoxemia included yawning, muscle fasciculations, signs of mild depression, and signs of mild abdominal pain such as stretching and swishing of the tail. Moderate signs of endotoxemia included, in addition to the mild signs of endotoxemia, signs of moderate depression, diarrhea, and signs of moderate abdominal pain, such as pawing. Severe signs of endotoxemia included, in addition to mild and moderate signs of endotoxemia, signs of severe depression and signs of severe abdominal pain, such as recumbency.
Blood sampling and tests—Blood was collected from the right jugular catheter of each horse immediately after clinical data were obtained. Four milliliters of blood was placed into each of 2 tubesf containing calcium EDTA for determination of CBC, PCV, and total plasma protein. Complete blood countsg were performed at −165, −30, 0, 60, 120, 180, and 360 minutes. Six milliliters of blood was placed in a tubeh containing sodium heparin at −165, −30, 0, 30, 60, 90, 120, 180, 240, and 360 minutes for determination of concentrations or activity of blood glucose, blood lactate, and plasma TNF-α. The PCV and concentrations or activity of total plasma protein, blood glucose, and blood lactate were determined within 5 minutes after blood was collected. The tubes containing sodium heparin were centrifugedi at 1,302 × g for 5 minutes to separate RBCs from plasma, and the plasma was placed in polypropylene cryotubesj and stored at −80°C, as in previous studies.18–20 Concentration of blood glucose was determined by use of a biosensor systemk that used an electrochemical detection technique involving a disposable, dry reagent strip. The reference range for the blood glucose concentration determined by use of this instrument is 70.8 to 111.2 mg/dL. The activity of blood lactate was determined by use of an enzymatic test and reflectance photometry.l The reference range for blood lactate activity by use of this instrument is 0.32 to 1.23 mmol/L. The concentration of TNF-α in plasma was determined by use of a commercially available ELISA kit.17,m Analysis of TNF-α concentration was performed by use of a human TNF-α assay and conducted in accordance with the manufacturer's protocol. The assay was a solid-phase, 2-site, chemiluminescent, immunometric assay, and the limit of detection was 4 pg/mL.
Statistical analysis—Continuous data were summarized as mean ± SEM or median and range, depending on whether the data conformed to a normal distribution. A mixed-model, repeated-measures ANOVAn was used to determine the effect of treatment, time, and the treatment by time interaction on the following dependent variables: blood lactate activity, heart and respiratory rates, score of endotoxemia, rectal temperature, mucous membrane color, CRT, PCV, total protein concentration, blood glucose concentration, WBC count, neutrophil count, and plasma TNF-α concentration. Treatment, time, and horse were included as class variables, and horse was included as a random factor in the model. The multiple range test, according to the Tukey method, was used to distinguish among the various levels of time and treatment. The fit of the model to the data was evaluated by comparing the residuals with a normal distribution by use of the Shapiro-Wilk test statistic. When necessary, independent variables were transformed to normalize the residuals from the model. The effect of treatment on the presence or absence of moisture on the oral mucous membrane was evaluated by use of a χ2 test for 2 × 1 tables° and applying a Bonferroni correction factor to adjust for multiple comparisons. Values of P ≤ 0.05 were considered significant.
Results
All horses had an endotoxemia score of 0 prior to exposure to LPS. Clinical scores for endotoxemia for horses in the negative control and treatment groups increased significantly (P < 0.05) over baseline scores (−165 minutes) after LPS was administered. Mean ± SEM clinical score for horses in the negative control group over all time points was 1.03 ± 0.11, and this was significantly (P = 0.009) greater than the score of 0.74 ± 0.09 for horses in the treatment group. Mean score for horses in the positive control group over all time points was 0 ± 0 and was significantly (P = 0.001) different from that of horses in the negative control and treatment groups (Figure 1).
Mean clinical score of horses in 3 groups at specified time points in a study of the effects of hyperbaric oxygen treatment on horses with experimentally induced endotoxemia. Diamonds = Treatment group (HBOT + LPS). Asterisks = Negative control group (ambient air + LPS). Circles = Positive control group (HBOT + PSS).
Citation: American Journal of Veterinary Research 72, 9; 10.2460/ajvr.72.9.1266
Mean clinical score of horses in 3 groups at specified time points in a study of the effects of hyperbaric oxygen treatment on horses with experimentally induced endotoxemia. Diamonds = Treatment group (HBOT + LPS). Asterisks = Negative control group (ambient air + LPS). Circles = Positive control group (HBOT + PSS).
Citation: American Journal of Veterinary Research 72, 9; 10.2460/ajvr.72.9.1266
Mean clinical score of horses in 3 groups at specified time points in a study of the effects of hyperbaric oxygen treatment on horses with experimentally induced endotoxemia. Diamonds = Treatment group (HBOT + LPS). Asterisks = Negative control group (ambient air + LPS). Circles = Positive control group (HBOT + PSS).
Citation: American Journal of Veterinary Research 72, 9; 10.2460/ajvr.72.9.1266
Exposure of horses in the negative control and treatment groups to LPS was associated with an increase in respiratory rate over baseline values, but the increase was only significant (P = 0.02) for horses in the negative control group at 60 minutes. Administration of HBOT to horses in the positive control group was not associated with a significant change in respiratory rate. Overall mean respiratory rate of horses in the negative control (19.8 ± 1.3 breaths/min) and treatment (19.8 ± 1.5 breaths/min) groups was significantly (P = 0.009 and 0.013, respectively) greater than overall mean respiratory rate of horses in the positive control group (13.1 ± 0.5 breaths/min). However, overall mean respiratory rate for horses in the negative control group did not differ significantly from that of horses in the treatment group.
Exposure of horses in the negative control and treatment groups to LPS was associated with an increase in heart rate over baseline values, but the increase was significantly different only among horses in the treatment group at 30, 60, 90, 120, 180, 240, and 360 minutes (P = 0.04, < 0.001, = 0.002, = 0.019, = 0.117, = 0.049, and = 0.028, respectively). Administration of HBOT to horses in the positive control group was not associated with a significant change in heart rate. The overall mean ± SEM heart rate of horses in the negative control group (42.9 ± 0.9 beats/min) did not differ significantly from the heart rate of horses in the treatment (49.4 ± 1.6 beats/min) and positive control (39.4 ± 0.8 beats/min) groups. The overall mean heart rate of horses in the treatment group was significantly (P = 0.001) greater than the overall mean heart rate of horses in the positive control group. The mean heart rate of horses in the treatment group at 60 minutes was significantly (P < 0.001) greater than that of horses in the positive control group (Figure 2).
Mean heart rate (beats/min) of the same horses as in Figure 1. See Figure 1 for key.
Citation: American Journal of Veterinary Research 72, 9; 10.2460/ajvr.72.9.1266
Mean heart rate (beats/min) of the same horses as in Figure 1. See Figure 1 for key.
Citation: American Journal of Veterinary Research 72, 9; 10.2460/ajvr.72.9.1266
Mean heart rate (beats/min) of the same horses as in Figure 1. See Figure 1 for key.
Citation: American Journal of Veterinary Research 72, 9; 10.2460/ajvr.72.9.1266
Exposure of horses in the negative control and treatment groups to LPS was associated with a significant increase in rectal temperature over baseline values. The increase over baseline for horses in the negative control group occurred at 120, 180, 240, and 360 minutes (P = 0.006, = 0.003, < 0.001, and < 0.018, respectively), and for horses in the treatment group, the increase over baseline occurred at 90, 120, 180, 240, and 360 minutes (P = 0.012, < 0.001, = 0.002, < 0.001, and < 0.001, respectively). Mean rectal temperature of horses in the positive control group did not increase significantly from the group's mean baseline temperature. Overall mean rectal temperature of horses in the treatment group (38.5°C; range, 38.3 to 38.7°C) was significantly greater than the mean rectal temperature of horses in the negative (37.9°C; range, 37.7° to 38.1°C; P = 0.001) and positive (37.6°C; range, 37.4° to 37.8°C; P < 0.001) control groups. Mean rectal temperature of horses in the treatment group was significantly greater than that of horses in the positive control group at 90, 120, 180, 240, and 360 minutes (P = 0.014, < 0.001, < 0.001, < 0.001, and < 0.001, respectively). Mean rectal temperature of horses in the negative control group was significantly greater than that of horses in the positive control group at 180 and 240 minutes (P = 0.018 and 0.014, respectively). There was no significant (P = 0.13) difference in the overall mean rectal temperature between horses in the negative control group and horses in the positive control group (Figure 3).
Mean rectal temperature of the same horses as in Figure 1. See Figure 1 for key.
Citation: American Journal of Veterinary Research 72, 9; 10.2460/ajvr.72.9.1266
Mean rectal temperature of the same horses as in Figure 1. See Figure 1 for key.
Citation: American Journal of Veterinary Research 72, 9; 10.2460/ajvr.72.9.1266
Mean rectal temperature of the same horses as in Figure 1. See Figure 1 for key.
Citation: American Journal of Veterinary Research 72, 9; 10.2460/ajvr.72.9.1266
Exposure of horses in the negative control and treatment groups to LPS was associated with hyperemia of the oral mucous membranes, compared with baseline values (P = 0.03), and oral mucous membranes of horses in the negative control and treatment groups were significantly (P = 0.04) more hyperemic than the oral membranes of horses in the positive control group. Administration of HBOT to horses in the positive control group was not associated with a change in color of the oral mucous membranes over time. The difference in the color of the mucous membranes between horses in the negative control group and horses in the treatment group was not significant (P = 0.98). Exposure of horses in the negative control and treatment groups to LPS was associated with an increase in CRT. Administration of HBOT to horses in the positive control group was not associated with a significant change in CRT. The CRT for horses in the negative control (range, 1.4 to 1.9 seconds) and treatment (range, 1.3 to 1.8 seconds) groups was significantly (P = 0.003 and 0.01, respectively) longer than the CRT of horses in the positive control group (range, 0.7 to 1.3 seconds). The overall difference in the CRT between horses in the negative control group and horses in the treatment group was not significant (P = 0.72). Mean CRTs for horses in all groups were within the reference range of 1 to 2 seconds. Exposure of horses in the negative control and treatment groups to LPS was associated with a decrease in moistness of the mucous membranes. Administration of HBOT to horses in the positive control group was not associated with a change in moistness of the mucous membranes. The oral mucous membranes of horses in the negative control group were significantly (P < 0.001) more tacky than those of horses in the positive control group. There was no significant difference in the moistness of the oral mucous membranes between horses in the treatment and negative control groups (P = 0.054) and between horses in the treatment and positive control groups (P = 0.081).
Hematologic, lactate, and glucose values—Mean PCV and plasma total protein concentration did not differ significantly among groups (Table 1). Administration of LPS to horses in the negative control and treatment groups was associated with an increase in PCV and plasma total protein concentration over baseline, but the increase was not significant. Administration of HBOT to horses in the positive control group was not associated with a change in PCV and plasma total protein concentration.
Least squares means for variables measured at various points in 6 horses kept in an ambient atmosphere in a stall before administration of LPS (negative control group), 6 horses treated with HBOT before administration of LPS (treatment group), and 6 horses treated with HBOT before administration of PSS (positive control group).
| Time (min) | ||||||||
|---|---|---|---|---|---|---|---|---|
| Variable | −165 | −30 | 0 | 60 | 90 | 120 | 180 | 360 |
| Blood glucose concentration (mg/dL) | ||||||||
| Negative control group | 84.6 | 89.3 | 82.3 | 99.7 | 98.8 | 77.2 | 75.3 | 81.7 |
| Treatment group | 93.7 | 104.2 | 108.3 | 111.5 | 103.3 | 88.5 | 77.8 | 79.5 |
| Positive control group | 91.5 | 95.7 | 109.7 | 104.8 | 107.8 | 105.3 | 109.0 | 103.7 |
| Blood lactate concentration (mmol/L) | ||||||||
| Negative control group | 0.77 | 0.88 | 1.10 | 1.48 | 1.85* | 1.63 | 1.12 | 1.12 |
| Treatment group | 0.72 | 0.73 | 1.27 | 1.02 | 1.42 | 1.72* | 1.4 | 1.32 |
| Positive control group | 0.72 | 1.25 | 0.87 | 0.82 | 1.02 | 0.85 | 0.75 | 0.72 |
| PCV (%) | ||||||||
| Negative control group | 36.2 | 35.8 | 39.5 | 45.0* | NA | 44.6* | 42.0 | 41.2 |
| Treatment group | 33.6 | 34.2 | 36.2 | 43.2* | NA | 44.8* | 41.8* | 41.0 |
| Positive control group | 36.5 | 37.2 | 34.9 | 36.3 | NA | 36.7 | 36.2 | 34.5 |
| Total protein concentration (g/dL) | ||||||||
| Negative control group | 7.52 | 7.33 | 7.54 | 7.97 | NA | 7.86 | 7.59 | 7.41 |
| Treatment group | 7.41 | 7.43 | 7.58 | 7.94 | NA | 7.91 | 7.61 | 7.64 |
| Positive control group | 7.42 | 7.39 | 7.26 | 7.59 | NA | 7.59 | 7.46 | 7.42 |
| WBC count (1 × 103 cells/μL) | ||||||||
| Negative control group | 7.85 | 8.30 | 5.47* | 2.14*† | NA | 1.99*† | 2.50*† | 6.56 |
| Treatment group | 8.61 | 9.21 | 6.89 | 1.69*† | NA | 1.67*† | 2.60*† | 7.82 |
| Positive control group | 7.01 | 8.32 | 8.47 | 9.61§‡ | NA | 9.43§‡ | 9.36§‡ | 8.6 |
| Neutrophil count (1 × 103 cells/μL) | ||||||||
| Negative control group | 4.7 | 5.2 | 3.7 | 0.5*† | NA | 0.5*† | 1.5*† | 5.7 |
| Treatment group | 5.7 | 6.2 | 5.3 | 0.54*† | NA | 0.46*† | 1.6*† | 7.0 |
| Positive control group | 4.5 | 5.5 | 6.2 | 7.5ठ| NA | 7.2ठ| 7.3ठ| 6.2 |
Significantly (P ≤ 0.05) different from baseline (−165 minutes).
Significantly (P ≤ 0.05) different from the positive control group.
Significantly (P ≤ 0.05) different from the treatment group.
Significantly (P ≤ 0.05) different from the negative control group.
NA = Not applicable.
Administration of LPS to horses in the negative control and treatment groups was associated with a significant increase in the activity of blood lactate over baseline values. Mean activity of blood lactate increased significantly over baseline for horses in the treatment group only at 120 minutes (P = 0.036) and for horses in the negative control group only at 90 minutes (P = 0.02; Table 1). The greatest activity of blood lactate in the treatment group occurred 30 minutes later than that of the negative control group. Administration of HBOT to horses in the positive control group was not associated with a significant change in the activity of blood lactate. Overall mean activity of blood lactate of horses in the positive control group (0.85 ± 0.05 mmol/L) was significantly less than that of horses in the negative control (1.24 ± 0.07 mmol/L) and treatment (1.18 ± 0.06 mmol/L) groups (P = 0.002 and 0.005, respectively). Overall mean activity of blood lactate of horses in the negative control group was significantly (P = 0.002) greater than the overall mean activity of blood lactate of horses in the positive control group but did not differ significantly (P = 0.83) from that of horses in the treatment group. Overall mean blood lactate activities among horses in each group were within the reference range.
Overall mean concentration of blood glucose did not differ significantly among groups. Horses in the positive control, treatment, and negative control groups had an overall mean ± SEM blood glucose concentration of 102.9 ± 2.0 mg/dL, 85.7 ± 2.4 mg/dL, and 96.4 ± 2.8 mg/dL, respectively (Table 1). Mean concentrations of blood glucose of horses in the negative control and treatment groups increased initially from the baseline concentrations, then decreased from 120 minutes to the end of the study (360 minutes). The initial increase in blood glucose concentration among the negative control and treatment groups was not significant. Overall mean values for horses in each group were within the reference range.
Administration of LPS to horses in the negative control and treatment groups was associated with a significant decrease in the mean WBC and neutrophil counts from baseline values. A decrease in the WBC count from baseline for horses in the negative control group was observed at 0, 60, 120, and 180 minutes (P = 0.02, < 0.001, < 0.001, and < 0.001, respectively). For horses in the treatment group, a decrease in the WBC count from baseline occurred at 60, 120, and 180 minutes (P < 0.001, 0.001, and 0.001, respectively). A decrease in the neutrophil count from baseline was observed at 60, 120, and 180 minutes for horses in the negative control (P < 0.001, 0.001, and 0.012, respectively) and treatment (P < 0.001, 0.001, and 0.001, respectively) groups. The WBC and neutrophil counts of horses in the positive control group did not differ significantly from baseline values. The WBC and neutrophil counts of horses in the negative control group did not differ significantly from that of horses in the treatment group (Table 1). Administration of LPS to horses in the negative control and treatment groups was associated with an increase in the mean concentration of plasma TNF-α of horses in these 2 groups over baseline; however, the increase was not significant. Mean concentration of plasma TNF-α among horses in the negative control and treatment groups was highest at 60 minutes and then gradually decreased; however, the overall mean concentration of plasma TNF-α did not differ significantly among the groups (Figure 4). Administration of HBOT to horses in the positive control group was not associated with a change in overall mean concentration of plasma TNF-α.
Mean plasma TNF-α concentrations of the same horses as in Figure 1. See Figure 1 for key.
Citation: American Journal of Veterinary Research 72, 9; 10.2460/ajvr.72.9.1266
Mean plasma TNF-α concentrations of the same horses as in Figure 1. See Figure 1 for key.
Citation: American Journal of Veterinary Research 72, 9; 10.2460/ajvr.72.9.1266
Mean plasma TNF-α concentrations of the same horses as in Figure 1. See Figure 1 for key.
Citation: American Journal of Veterinary Research 72, 9; 10.2460/ajvr.72.9.1266
All horses in the negative control and treatment groups had hematologic and clinical signs of endotoxemia, including leukopenia, neutropenia, signs of depression, tachycardia, hyperemic mucous membranes, and signs of abdominal pain. Two mares in the negative control group and 5 mares in the treatment group became febrile (rectal temperature > 39.0°C). Four mares in the negative control group and 4 mares in the treatment group developed diarrhea. Three mares in the negative control group and 3 mares in the treatment group had blood lactate activity > 2 mmol/L. No adverse effects of HBOT were observed.
Discussion
All LPS-treated horses had clinical signs and biochemical and hematologic changes consistent with endotoxemia. Signs of endotoxemia included tachycardia, muscle fasciculations, stretching, kicking at the abdomen, sternal recumbency, diarrhea, signs of depression, hyperemic oral mucosa, prolonged CRT, and fever. Biochemical and hematologic changes in LPS-treated horses included increased activity of blood lactate, leucopenia, and neutropenia. These findings were consistent with those of previous studies18–22 in horses with experimentally induced endotoxemia.
Marked leucopenia and neutropenia were evident 60 minutes after LPS was administered in the treatment and negative control groups, and the leukocyte and neutrophil counts returned to baseline values by 360 minutes. Neutropenia, which is observed after administration of LPS, is caused primarily by margination of neutrophils and migration of neutrophils from blood into the peripheral tissues23 and coincided with the peak plasma concentration of TNF-α, which is consistent with the findings of another study.20 We are unaware of any reports that describe the effect of HBOT on WBC count in experimentally induced endotoxemia in any species. In the present study, there was no beneficial effect observed in WBC or neutrophil counts of horses treated with HBOT.
The effect of LPS on blood glucose concentration appears to be dose dependent.24,25 In 2 studies,24,25 administration of a high dose of LPS (125 μg/kg) to ponies induced marked hyperglycemia, which was followed by hypoglycemia. Hyperglycemia subsequent to the administration of LPS has been attributed to an adrenergic response that induces a marked increase in serum cortisol concentration.24–26 Although the blood glucose concentration of horses in the negative control and treatment groups increased transiently after LPS administration, the increase was not significant and the values for blood glucose concentration remained close to the reference range. This temporal response in blood glucose concentration was similar to that reported in another study20 in horses that received the same dose of LPS (0.2 μg/kg) that horses in the present study received.
The activity of blood lactate of horses in the negative control group increased significantly over baseline values after LPS administration and was significantly greater than that of horses in the positive control group. This increase has been observed in other studies20,21,26–28 of horses. However, blood lactate activity of horses in the negative control group only increased from 0.77 to 1.86 mmol/L, which was within our laboratory's reference range. In a clinical study29 of horses evaluated for acute colic, horses with plasma lactate activity > 7.0 mmol/L had a poor prognosis, indicating that the peak activity of blood lactate in the horses in the present study was not clinically important. The activity of blood lactate of horses in the negative control group did not differ significantly (P = 0.83) from that of horses in the treatment group. Nevertheless, the highest measured mean activity of blood lactate of horses in the treatment group occurred 30 minutes later than that of horses in the negative control group. Hyperbaric oxygen treatment is associated with a delay in the increase in blood lactate activity in humans when administered after intensive exercise.30 In addition, HBOT administration during intensive exercise delays the increase in blood lactate activity in dogs.31 This delay has been attributed to an enhanced rate of removal of lactate from peripheral blood vessels by HBOT. Because blood lactate activity of horses in the negative control group did not increase substantially, it was difficult to detect a beneficial effect of HBOT on activity of blood lactate in horses treated with LPS in the present study.
The concentration of plasma TNF-α in the negative control group increased after LPS was administered but was not significantly different from baseline values. The greatest plasma TNF-α concentration was observed at 60 minutes in horses in the negative control and treatment groups. Contrary to the findings in the present study, a similar dose of LPS administered to horses in 2 other studies7,18 caused a significant increase in TNF-α concentration. Failure to detect significant changes in concentrations of TNF-α in the present study may have been attributable to intragroup variability, small sample size, and the fact that the assay has not been validated for use in horses, although it has been used in other studies18–20 of horses with endotoxemia.
Administration of LPS to horses in the negative control group was associated with a significant increase in rectal temperature over baseline values, and this increase was consistent with results of other studies.18–20 Maximum mean rectal temperature occurred at 240 minutes for horses in the negative control (38.4°C) and treatment (39.2°C) groups. Time to maximum rectal temperatures in horses treated with LPS in the present study was similar to that reported in other studies.18–20 The febrile response induced by LPS is caused by the effect of inflammatory cytokines, such as interleukin-1, on the hypothalamic thermoregulatory center.32 It is interesting that the overall mean rectal temperature of horses in the treatment group was significantly greater than that of horses in the control groups, suggesting that HBOT may increase the rectal temperature of LPS-treated horses. This increase in rectal temperature was not clinically important, except perhaps when mean rectal temperature of horses in the treatment group increased briefly to > 39°C. The ambient temperature in the chamber was unlikely to be the cause of the increase in rectal temperature in the treatment group because HBOT had no effect on the temperature of horses that did not receive LPS (positive control group). In addition, mean temperature in the HBOT chamber at 45 minutes was 24°C (range, 23° to 25°C), which did not differ greatly from mean ambient temperature in the stalls (21° to 22°C). Furthermore, maximum rectal temperature of horses in the treatment group did not occur until 285 minutes after the horses exited the chamber. These results differ from those of a study12 in which HBOT significantly decreased LPS-induced fever in rabbits, an effect that was attributed to a decrease in circulating TNF-α and a decrease in production of prostaglandin E2 by the hypothalamus. Horses in the treatment group may have developed an increase in rectal temperature because HBOT causes vasoconstriction.33 Under normal thermoregulatory control, peripheral vasoconstriction reduces heat loss34 by preventing heat loss through convection.35 Vasoconstriction, coupled with the increased body temperature observed after administration of LPS, may have caused the temperature of horses in the treatment group to increase significantly over that of horses that received HBOT or LPS alone.
Although all horses in the negative control group had an increase in heart rate after they received LPS, the increased heart rate was not significantly different from baseline. An increase in heart rate has been observed in horses administered an equivalent or lower dose of LPS,18–20 and this increase has been attributed to an increase in TNF-α concentration.19 Increased heart rate in LPS-treated mice has been attributed to increased blood concentrations of prostaglandin 2α and thromboxane A2.36 Failure to observe a significant effect of LPS on heart rate of horses in the negative control group may have been attributable to the small sample size and intragroup variability. Evaluating the effects of HBOT on heart rate in horses administered LPS was not meaningful because the overall heart rate of horses in the treatment group did not differ significantly from that of horses in the negative control group and because the overall heart rate of horses in the negative control group was not significantly increased over baseline values. Mean heart rate of horses in the treatment group (69 beats/min) was significantly greater than that of horses in the positive control group (39 beats/min) at 60 minutes, but this was because 2 horses in the treatment group had transient heart rates of 80 and 100 beats/min.
The scoring system for endotoxemia was based on a published system, and the signs of endotoxemia were consistent with previous studies18,20 of LPS-treated horses. We observed that HBOT significantly ameliorated the clinical signs of endotoxemia. Mean endotoxemia score over all time periods for horses in the negative control group was 1.03 ± 0.12 and was significantly (P = 0.009) greater than the score for horses in the treatment group (0.74 ± 0.09), suggesting a beneficial effect of HBOT in endotoxemic horses. Observers in the present study were not unaware of the treatment group of each horse, which created potential for bias when evaluating clinical scores of endotoxemia.
The dose of LPS used in the present study (0.2 μg/kg) was selected on the basis of reported concentrations of LPS in the blood of horses with naturally occurring gastrointestinal tract disease37 and from doses of LPS administered to horses in other experimental studies.20,38–41 A beneficial effect from HBOT may have been observed if horses had been administered a larger dose of LPS or if other markers of endotoxemia, such as serum concentrations of nitric oxide, inducible nitric oxide synthase, nitric oxide metabolites, hemeoxygenase-1, or interleukin-10, had been used to determine the response to treatment.10,15,16,42 Results of the present study were determined by measurement of various physiologic and biochemical effects of endotoxemia that resulted in mild, short-term illness, whereas studies10,13 that used greater doses of LPS in mice and rats revealed a beneficial effect of HBOT when death was used as the outcome.
The HBOT regimen used in the present study was based on protocols used to treat human patients and on experimental procedures used in laboratory animals and horses. Human patients receiving HBOT are usually treated on more than 1 occasion with 90% to 100% oxygen at pressures of 1 to 3 atmospheres for 30 to 90 minutes.9 Laboratory animals undergoing experimental procedures with HBOT are typically treated with 98% to 100% oxygen at 2 to 3 atmospheres for 60 to 120 minutes.10,11,13,15,16,43,44 We found only 1 experimental study45 that used a protocol for treating horses with HBOT, in which horses were exposed to 2.6 atmospheres of 77% to 80% oxygen for 60 minutes to observe the effect on full-thickness skin grafts. Protocols for HBOT that used 2 to 3 atmospheres for 60 to 90 minutes have been described for treating horses for various diseases.17 An HBOT protocol different from that used in the present study, such as the administration of HBOT after induction of endotoxemia or a different atmospheric pressure or concentration of oxygen, may have produced different results.
In the present study, horses were treated with HBOT prior to administration of LPS because studies12,14–16 that used rabbits and rodents revealed a beneficial effect when HBOT was administered before LPS. In a study46 of myocardial infarction in rats, the partial pressure of oxygen in the myocardium remained significantly increased for 2.5 hours after HBOT was terminated; thus, the effects of HBOT appear to persist after the animal is removed from the hyperbaric oxygen chamber. The fact that we did not find a marked beneficial effect of HBOT in endotoxemic horses could be because horses are exquisitely more sensitive to LPS than are rabbits and rodents.47 The horse is among the most sensitive of all species to the effects of LPS,47 but the reason for this sensitivity has not been elucidated. Studies are warranted to evaluate the effects of HBOT administered after induction of endotoxemia because horses with naturally occurring endotoxemia are likely to be treated with HBOT after, rather than before, the development of endotoxemia.
Although horses in the present study were exposed previously to experimentally administered LPS, none had received LPS for at least 1 year. Repeated exposure to endotoxin can lead to tolerance,48 but early tolerance of humans to endotoxin lasts only for a few days, and even late tolerance extends only for several weeks.49 In 1 study,50 early tolerance to endotoxin developed after horses received 2 doses of LPS administered 24 hours apart, but duration of tolerance was not determined. We are unaware of any reports that document late tolerance to endotoxin in horses. All LPS-treated horses in the present study had characteristic responses to LPS, as indicated by clinical and hematologic assessment.
In the present study, HBOT administered to horses as a single treatment before administration of LPS did not appear to ameliorate the physical signs (eg, fever and increased heart and respiratory rates) and hematologic and serum biochemical effects of endotoxemia; however, there was a significant beneficial effect observed in the endotoxemia score among horses administered LPS and treated with HBOT. The protective effect of HBOT in this study was minimal and does not support its use as a treatment for horses prior to development of endotoxemia. Nevertheless, the data suggest the need for further studies to assess the efficacy of HBOT as a treatment for horses with endotoxemia.
ABBREVIATIONS
| CRT | Capillary refill time |
| HBOT | Hyperbaric oxygen treatment |
| LPS | Lipopolysaccharide |
| PSS | Physiologic saline (0.9% NaCl) solution |
| TNF-α | Tissue necrosis factor-α |
Angiocath, Becton-Dickinson, Sandy, Utah.
Equine oxygen therapy, Versailes, Ky.
Escherichia coli 055 B5, Sigma-Aldrich, St Louis.
Vet/IV 2.2 volumetric infusion pump, Heska Corp, Fort Collins, Colo.
Phenylbutazone, 200mg/mL, Sparhawk Laboratories Inc, Lenexa, Kan.
Vacutainer K2 EDTA, 7.2 mg, BD, Franklin Lakes, NJ.
Advia 120, Siemens, Tarrytown, NY.
Vacutainer sodium heparin, 45 USP units, BD, Franklin Lakes, NJ.
Dynac, Alatamonte Springs, Fla.
Microcentrifuge tubes, Fisher Scientific, Pittsburgh, Pa.
Precision QID, MediSense, Bedford, Mass.
Accutrend, Roche Diagnostics, Mannheim, Germany.
Pierce Biotechnology, Rockford, Ill.
PROC GLIMMIX, SAS, version 9.1, SAS Institute Inc, Cary, NC.
PROC FREQ, SAS, version 9, SAS Institute Inc, Cary, NC.
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