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    Calculation of normalization factors by a specialized software program from an increasing number of genes evaluated to allow assessment of mRNA expression of Th1, Th2, and Th17 cell–associated inflammatory mediators in cells in BAL fluid samples collected from healthy horses exposed to HBO. The software uses a pairwise comparison model that calculates 2 parameters (M and V) for optimal stability and normalization of the data set. The V value determines the optimal number of reference genes required for accurate normalization by analyzing pairwise variation between sequential normalization factors containing an increasing number of reference genes (2, 3, and 4 in this instance). The data shown represent the pairwise variations between 2 sequential normalizations factors. Values of V < 0.15 are most suitable for data analysis. V3/4 indicates that addition of the fourth (least stable) reference gene to the previous 3 (more stable) reference genes increases the V to > 0.15. Therefore the combination of the 3, but not 4, reference genes was suitable for data analysis.

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    Box-and whisker plot of the ratios of pretreatment and posttreatment mRNA expressions of eotaxin-2, IL-1β, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p35, interferon (IFN)-γ, and TNF-α in lung cells isolated from BAL fluid samples collected from 8 healthy horses before (day 0) and after once-daily HBO therapy for 10 days (treatment beginning day 1). Horses were exposed to 100% oxygen in a horizontally oriented pressurized hyperbaric chamber. For each chamber session, horses were unsedated. Pressure in the chamber was increased to 3 ATA over a 60-minute period. This pressure was maintained for 20 minutes and then decreased to ambient pressure over a period of 15 minutes. With once-daily treatments, the horses were exposed to HBO conditions (100% oxygen at > 1 ATA) for a total of 90 minutes over a period of 10 consecutive days. A BAL fluid sample was collected from each horse for quantitative PCR analysis on days 0 and 10; on day 10, the BAL fluid sample was collected immediately following the completion of the hyperbaric chamber session. Expression of each mRNA was compared with the expression of a combination of the reference genes GAPDH, HPRT, and RPL-32. A value of 1 indicates no change in expression; values < 1 indicate a downregulation in gene expression, and values > 1 indicate an upregulation in gene expression. For each box, the horizontal line represents the median and the upper and lower boundaries represent the third and first quartiles, respectively. Whiskers represent the maximum and minimum values. *Eotaxin-2 mRNA expression (relative to expression of the reference genes GAPDH, HPRT, and RPL-32) was significantly (P = 0.031) lower in BAL fluid cells isolated from the horses following HBO therapy, compared with findings before treatment.

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Expression of T helper cell–associated inflammatory mediator mRNAs in cells of bronchoalveolar lavage fluid samples and oxygen concentration in arterial blood samples from healthy horses exposed to hyperbaric oxygen

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  • 1 Department of Veterinary Clinical and Diagnostic Sciences, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada.
  • | 2 Department of Veterinary Clinical and Diagnostic Sciences, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada.
  • | 3 Department of Veterinary Clinical and Diagnostic Sciences, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada.
  • | 4 Department of Veterinary Clinical and Diagnostic Sciences, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada.
  • | 5 Moore Equine Veterinary Services Ltd, 260048A Writing Creek Crescent, Rocky View County, AB, T4A 0M9, Canada.
  • | 6 Moore Equine Veterinary Services Ltd, 260048A Writing Creek Crescent, Rocky View County, AB, T4A 0M9, Canada.
  • | 7 Department of Veterinary Clinical and Diagnostic Sciences, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada.
  • | 8 Department of Veterinary Clinical and Diagnostic Sciences, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada.
  • | 9 Moore Equine Veterinary Services Ltd, 260048A Writing Creek Crescent, Rocky View County, AB, T4A 0M9, Canada.

Abstract

OBJECTIVE To evaluate the mRNA expression of T helper (Th)1, Th2, and Th17 cell–associated inflammatory mediators in cells of bronchoalveolar lavage fluid samples collected from healthy horses exposed to hyperbaric oxygen (HBO) and to monitor blood oxygen concentration during and following HBO therapy.

ANIMALS 8 healthy horses.

PROCEDURES In a randomized controlled crossover design study, each horse was exposed (beginning day 1) to 100% oxygen at a maximum of 3 atmospheres absolute (304 kPa) daily for 10 days or ambient air at atmospheric pressure in the HBO chamber for an equivalent amount of time (control). Bronchoalveolar lavage fluid samples were collected on days 0 and 10. After validation of candidate reference genes, relative mRNA expressions of various innate inflammatory, Th1 cell–derived, Th2 cell–derived (including eotaxin-2), Th17 cell–derived, and regulatory cytokines were measured by quantitative PCR assays. For 3 horses, arterial blood samples were collected for blood gas analysis during a separate HBO session.

RESULTS The optimal combination of reference genes was glyceraldehyde-3-phosphate dehydrogenase, hypoxanthine ribosyltransferase, and ribosomal protein L32. Compared with day 0 findings, expression of eotaxin-2 mRNA was significantly lower (0.12-fold reduction) and the percentage of neutrophils in bronchoalveolar lavage fluid samples was significantly lower on day 10 when horses received HBO therapy. Values of Pao2 rapidly increased (> 800 mm Hg) but immediately decreased to pretreatment values when HBO sessions ended.

CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that HBO therapy does not increase mRNA expression of inflammatory cytokines, but reduces eotaxin-2 mRNA transcription. The Pao2 increase was transient with no cumulative effects of HBO.

Abstract

OBJECTIVE To evaluate the mRNA expression of T helper (Th)1, Th2, and Th17 cell–associated inflammatory mediators in cells of bronchoalveolar lavage fluid samples collected from healthy horses exposed to hyperbaric oxygen (HBO) and to monitor blood oxygen concentration during and following HBO therapy.

ANIMALS 8 healthy horses.

PROCEDURES In a randomized controlled crossover design study, each horse was exposed (beginning day 1) to 100% oxygen at a maximum of 3 atmospheres absolute (304 kPa) daily for 10 days or ambient air at atmospheric pressure in the HBO chamber for an equivalent amount of time (control). Bronchoalveolar lavage fluid samples were collected on days 0 and 10. After validation of candidate reference genes, relative mRNA expressions of various innate inflammatory, Th1 cell–derived, Th2 cell–derived (including eotaxin-2), Th17 cell–derived, and regulatory cytokines were measured by quantitative PCR assays. For 3 horses, arterial blood samples were collected for blood gas analysis during a separate HBO session.

RESULTS The optimal combination of reference genes was glyceraldehyde-3-phosphate dehydrogenase, hypoxanthine ribosyltransferase, and ribosomal protein L32. Compared with day 0 findings, expression of eotaxin-2 mRNA was significantly lower (0.12-fold reduction) and the percentage of neutrophils in bronchoalveolar lavage fluid samples was significantly lower on day 10 when horses received HBO therapy. Values of Pao2 rapidly increased (> 800 mm Hg) but immediately decreased to pretreatment values when HBO sessions ended.

CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that HBO therapy does not increase mRNA expression of inflammatory cytokines, but reduces eotaxin-2 mRNA transcription. The Pao2 increase was transient with no cumulative effects of HBO.

Hyperbaric oxygen therapy involves breathing 100% oxygen at higher-than-normal atmospheric pressure, which is 1 ATA (101.3 kPa).1 The pressurized environment creates a greater oxygen molecule density in the air, which increases the partial pressure gradient of oxygen between the alveoli and capillaries. Breathing 100% oxygen at sea-level pressure (1 ATA) results in complete saturation of hemoglobin as well as an increase in the amount of oxygen dissolved in the plasma. For each increase in absolute pressure > 1 ATA, more oxygen becomes dissolved in the plasma.1–3

Results of several studies1,3–5 have indicated that HBO therapy exerts various effects that impact immunity and oxygen and cellular metabolism. Indeed, HBO causes vasoconstriction,1 reduces neutrophil adherence to endothelial cells,6 and inhibits proinflammatory cytokine production in mononuclear cells.7–9 On the other hand, it is also known that macrophages isolated from the blood, spleen, and lungs of rats exposed to HBO produce more proinflammatory mediators (both spontaneously and following lipopolysaccharide stimulation) than do macrophages from rats exposed to air at atmospheric pressure.10 In leukocytes, HBO therapy enhances bacterial-killing capacity.1,11 Furthermore, high concentrations of oxygen increase the production of reactive oxygen species, which can cause lipid peroxidation, protein and DNA oxidation, and enzyme inactivation.1,12 At pressures > 2 ATA (202.6 kPa), HBO therapy induces oxidative stress on humans and laboratory animals, causing adverse systemic effects; the extent of damage increases with prolonged exposure.12–14 Toxic effects of oxygen often manifest in the CNS or the pulmonary system.15–17 Pulmonary toxic effects often develop after prolonged exposure to HBO.15 High concentrations of oxygen cause pulmonary damage, including thickening of the alveolar wall, development of interstitial and intra-alveolar edema, and extensive infiltration of inflammatory cells into the lungs.15–17 However, at levels < 2 ATA, these effects are typically not observed unless the individual's intrinsic antioxidant defense mechanisms are either compromised or overwhelmed.13,18

Hyperbaric oxygen therapy has been used in a variety of clinical cases where hyperoxic conditions would likely provide positive effects. In human medicine, HBO therapy is beneficial under conditions of ischemia or hypoxia, promotes wound healing, and protects against certain infections.1,3 However, the use of HBO as a therapeutic intervention in equine medicine has only gained interest in more recent years.2 Consequently, evidence to support most of the proposed applications in horses is lacking. To date, HBO therapy studies in horses have been limited to assessments of its effects on skin grafts, wound healing, endotoxemia, platelet function, and stem cell proliferation.19–22

Although HBO therapy is currently used in equine practice, its physiologic effects are poorly understood and may actually be deleterious for the cells lining the lungs. In addition, the appropriate duration and levels of blood oxygenation are unknown for HBO therapy in horses. Therefore, the purpose of the study reported here was primarily to assess the effects of HBO on the expression (at the mRNA level) of Th1, Th2, and Th17 cell–specific cytokine profiles in pulmonary cells of healthy horses as a means to determine whether the HBO protocol used altered inflammatory responses in the lungs. Concurrently, a second aim was to monitor blood oxygenation in a small number of horses during and after HBO therapy. Our hypotheses were that HBO therapy would increase blood oxygen concentration when horses were within the hyperbaric chamber but only for a short period after cessation of each treatment session and that HBO therapy would induce lung inflammation in healthy horses.

Materials and Methods

The study protocol was approved by the Animal Care Committee of the Health Science Centre at the University of Calgary. The study had a randomized controlled crossover design and was conducted and presented in accordance with the REFLECT statement guidelines.23 The data were collected at Bar None Ranches, De Winton, AB, Canada (altitude, approx 1,000 m); owner consent was obtained for use of horses on the premises. The number of horses for the mRNA experiments (n = 8) was calculated with a power of 80% on the basis of the effects of HBO therapy on cells in BAL fluid samples from the first 3 horses. Horses had no history of respiratory tract disease and were kept in the same environment with the same management and diet. The study was performed over the minimum number possible of consecutive days, which was 76. For each horse, results of a general physical examination, CBC, serum biochemical analysis, and cytologic examination of a BAL fluid sample24–26 performed within a 2-week period prior to the study were all within reference ranges. Exclusion criteria were any abnormality revealed by those assessments including evidence of lung inflammation in the BAL fluid sample.

Study procedures

Horses were randomly assigned to receive HBO therapy or no HBO therapy (control) once daily for 10 days (beginning on day 1). When horses did not receive HBO therapy, they were exposed to ambient air at atmospheric pressure in the same hyperbaric chamber for a period equivalent to that needed to complete the HBO therapy. After a washout period of 8 weeks, each horse underwent the other experimental protocol. For each horse, a BAL fluid sample was collected the day before each 10-day experimental period (day 0) and after the last chamber session on day 10. For all 8 horses, arterial blood gas analysis was performed prior to and immediately after exiting the hyperbaric chamber on days 1 and 10, respectively. Arterial blood gas monitoring was performed for 3 horses during HBO therapy on days 1 and 10. Horses were returned to the breeding program after the study.

Assessment of the effect of HBO therapy on blood oxygen concentration

For the blood oxygenation experiments, arterial blood gas analysis was performed on all 8 horses prior to and immediately after exiting the hyperbaric chamber (within 10 minutes after coming out of the HBO chamber) on days 1 and 10, respectively, of the HBO and control treatments. By use of an ultrasound-guided technique, a sample of arterial blood (2 mL) was collected from the right or left (alternate side was used on day 10) common carotid artery with a 23-gauge, 1.5-inch needle. Analysis was immediately performed on-site with a handheld analyzer.a In addition, arterial blood gases were monitored in 3 other horses while they were undergoing HBO therapy within the chamber during a separate session. For these horses, an arterial catheter was placed in the left transverse facial artery without sedation before the HBO therapy session. A threaded plug on one of the HBO vessel ports was replaced by a threaded adapter (sterilized prior to the experiment) machined with arterial line connectors on both the outside and inside of the chamber. The arterial line was flushed with saline (0.9% NaCl) solution before being connected to the arterial catheter. An arterial blood sample (2 mL after priming the line by removal of 25 mL of blood) was withdrawn through the port from outside the HBO chamber prior to pressurization (baseline), when O2 pressure reached 3 ATA (303.9 kPa; 0 minutes), 10 and 20 minutes later, during decompression when O2 pressure reached 2 ATA and again at 1 ATA, and 10 minutes after the horse was exposed to ambient air. Blood gas analysis was performed immediately after collection of samples with a handheld analyzera following manufacturer's recommendations. The arterial catheter was removed immediately after collection of the last sample when horses had been exposed to ambient air for 10 minutes. Owing to the technical challenges of this method, this extensive data collection was only performed on 3 horses.

Assessment of the effect of HBO therapy on inflammatory mediators

For the inflammatory mediator experiments, the primary outcomes measured were the findings of cytologic examination of BAL fluid samples and inflammatory gene expressions in the BAL fluid samples. Healthy horses underwent exposure to HBO or no exposure (control treatment) once daily for 10 days and subsequently received the alternate treatment after an 8-week interval. Horses receiving HBO therapy were exposed to 100% oxygen in a horizontally oriented pressurized hyperbaric chamber.b Horses were not sedated for each chamber session. Over a period of 60 minutes, a slow ramping pressurization protocol was used to increase the pressure in the chamber to 3 ATA. This pressure was maintained for 20 minutes and then decreased to ambient pressure over a period of 15 minutes. With once-daily treatments, the horses were exposed to HBO conditions (100% oxygen at > 1 ATA) for a total of 90 minutes over a period of 10 consecutive days. Previously published reports describing HBO therapy protocols for horses typically have a total pressurization time of 60 to 90 minutes at a maximum pressure of 1.5 to 2.6 ATA (152 to 263 kPa).19–21 Thus, a long exposure time at > 1 ATA pressure was chosen for the present study protocol to determine whether a change in inflammatory factors would be elicited under HBO therapy conditions that exceed those of standard protocols. For the control treatment, horses were exposed to ambient air at atmospheric pressure in the same HBO chamber for a period equivalent to that needed to complete the HBO therapy. Each horse received the HBO and control treatment (8-week interval). A BAL fluid sample was collected from each horse before (day 0) and after the HBO or control treatment (day 10) for cytologic examination and quantitative PCR analysis; on day 10, the BAL fluid sample was collected immediately following the completion of the hyperbaric chamber session. For collection of the BAL fluid samples, horses were sedated, as previously described by Wasko et al.27 Briefly, two 250-mL volumes of sterile endotoxin-free saline (0.9% NaCl) solution were delivered and aspirated via an appropriately positioned video-endoscope.c A 250-µL aliquot of fluid underwent cytologic examination, and a differential count was performed on 400 nucleated cells after staining the slides with a modified Wright-Giemsa solution28; the percentage of each cell type was calculated. Fifty-milliliter aliquots were centrifuged at 2,200 × g for 10 minutes; supernatants were discarded, and the cell pellets were resuspended in 1.5 mL of RNA preservative solution.d Samples were stored at −80°C until analyzed.

Messenger RNA analyses

The RNA extraction and cDNA synthesis were performed as previously described.29,30 Briefly, BAL-isolated cells were thawed on ice and homogenized,e and RNA was then extracted.f Synthesis of cDNA was done with 500 ng of RNA mixed in solution with reverse transcriptase,g RNase inhibitor,h and oligo (dT) primers.i Yield and purity of RNA and cDNA were assessed with a spectrophotometer.j Only RNA samples with ratios of absorbance at a wavelength of 260 nm to absorbance at a wavelength of 280 nm between 1.80 and 2.00 were used for DNA synthesis.

Expressions of mRNA transcripts were determined by quantitative PCR procedures.k The reaction solution contained 13 μL of Taq DNA polymerase solution,l 1.5 μL (40nM) of forward and reverse primers, 2 μL of cDNA, and 7 μL of nuclease-free water. Negative control samples contained sterile water. The cycling conditions were as follows: initial denaturation (95°C for 5 minutes), denaturation (45 cycles at 95°C for 1 minute), annealing (64°C for 30 seconds), and extension (70°C for 30 seconds), followed by a melting curve (60° to 95°C). Reactions were always executed in triplicate. Reaction specificity was also verified by gel electrophoresis of the PCR products.

The primer sequences of the candidate reference genes and 9 of the 10 inflammatory genes used in this study have been described.29,30 The primer set for equine TNF-α was designed according to Giguère et al.31 Reference genes were GAPDH, RPL-32, HPRT, and SDHA. Target genes were innate inflammatory (TNF-α and IL-8), Th1 cell–derived (interferon-γ and IL-1β), Th2 cell–derived (IL-4, IL-5, IL-6, and eotaxin-2), Th17 cell–derived (IL-6 and IL-12p35), and regulatory (IL-10) cytokines.

Stability and relative quantification analyses of target genes were assessed with a primer efficiency correction, with a baseline correction, by use of previously validated32 window-of-linearity method software.m Validation of the reference genes was performed with a software program.n The latter software uses a pairwise comparison model that calculates 2 parameters (M and V) for optimal stability and normalization of the data set. The M value ranks the candidate reference genes according to their stability; a low M value represents high expression stability. The V value determines the optimal number of reference genes required for accurate normalization by analyzing pairwise variation between sequential normalization factors containing an increasing number of reference genes. A value of V < 0.15 is required for accurate normalization.33 A relative expression software tool was used to assess the relative amounts of target inflammatory genes in horses under control and HBO-exposure conditions by performing a pairwise comparison; those relative amounts which were then compared between treatments. The relative expression software tool calculates expression ratios (ratios of mRNA expression before and after HBO therapy in this study) with 95% confidence intervals and determines significance with a statistical randomization algorithm.34 Differences in arterial blood gas variables and percentages of cells in BAL fluid samples before and after HBO therapy were assessed with a Wilcoxon signed rank test. A value of P < 0.05 was considered significant for all measurement comparisons.

Results

Of 14 horses examined within a 2-week period prior to the study, only 8 had no notable physical examination findings, and the count and cytologic appearance of cells in a BAL fluid sample were considered normal. The 8 horses were therefore included in the study. The horses were Thoroughbred broodmares (mean age, 14 years; age range, 12 to 16 years).

Effect of HBO therapy on arterial blood gas concentrations

The results of the arterial blood gas analyses for all 8 horses were summarized (Table 1). Among the horses, there was no difference in any blood gas analysis variable prior to and immediately after exiting the hyperbaric chamber on days 1 and 10 of the HBO or control treatment. Values of Paco2 generally decreased after HBO therapy, but the changes were not significant.

Table 1—

Median (25th to 75th percentile) arterial blood pH, Paco2, and Pao2 in 8 healthy horses before and after once-daily HBO therapy or control treatment for 10 days (treatment beginning day 1).

Time pointVariableBefore HBO therapy sessionAfter HBO therapy sessionBefore control sessionAfter control session
Day 1pH7.454 (7.421–7.472)7.458 (7.434–7.487)7.417 (7.398–7.434)7.455 (7.406–7.468)
 Paco2 (mm Hg)43 (38.5–47.3)38.1 (34.4–40.3)43.1 (39.7–45.8)36.2 (35.7–44.3)
 Pao2 (mm Hg)79.0 (75–84)76.0 (70–86)76.5 (62.7–82)75.0 (65–85)
Day 10pH7.472 (7.435–7.49)7.472 (7.442–7.484)7.427 (7.409–7.452)7.446 (7.423–7.451)
 Paco2 (mm Hg)40.3 (39.43–42.6)37.5 (37.2–42)41.1 (39.4–47)43.3 (41.1–45)
 Pao2 (mm Hg)76.5 (69.7–91.5)79.0 (66–87)79.0 (64.2–84.5)78.0 (74–95)

Horses were randomly assigned to receive HBO therapy or no HBO therapy (control) once daily for 10 days (treatment beginning day 1) in a crossover study. When horses did not receive HBO therapy, they were exposed to ambient air at atmospheric pressure in the same hyperbaric chamber for a period equivalent to that needed to complete the HBO therapy. After a washout period of 8 weeks, each horse underwent the other experimental protocol. For HBO therapy, horses were exposed to 100% oxygen in a horizontally oriented pressurized hyperbaric chamber. For each chamber session, horses were unsedated. Pressure in the chamber was increased to 3 ATA over a 60-minute period; this pressure was maintained for 20 minutes and then decreased to ambient pressure over a period of 15 minutes. With once-daily treatments, the horses were exposed to HBO conditions (100% oxygen at > 1 ATA) for a total of 90 minutes over a period of 10 consecutive days. Arterial blood gas analysis was performed on all 8 horses prior to and immediately after exiting the hyperbaric chamber (within 10 minutes after coming out of the HBO chamber) on days 1 and 10, of the HBO and control treatments. By use of an ultrasound-guided technique, a sample of arterial blood was collected from the right or left (alternating side) common carotid artery and analyzed on-site with a handheld analyzer. There was no difference in pH, Paco2, and Pao2 prior to and immediately after exiting the hyperbaric chamber on days 1 and 10 of the HBO or control treatments. Reference values for arterial blood variables in horses (at room temperature, not corrected for altitude [study was performed at an altitude of approx 1,000 m]) are as follows: pH, 7.394 to 7.442; Paco2, 39.5 to 44.4 mm Hg; and Pao2, 85.8 to 104.9 mm Hg.

The 3 horses monitored within the hyperbaric chamber during a separate HBO session had similar changes in Pao2 (Table 2). At 3 ATA (0 minutes), 2 of the 3 horses had values of Pao2 greater than the maximum limit of the analyzer (800 mm Hg); this change was detected in the other horse at the 10-minute time point at 3 ATA. These high values of Pao2 persisted until decompression was started. Values of Pao2 were measurable in all 3 horses at 1 ATA, and the decrease in Pao2 to baseline values was extremely rapid (within 10 minutes) once horses were exposed to ambient air. Therefore, no cumulative effects of oxygenation were observed. The values by the end of the decompression period were not significant. Similarly, no significant effect of HBO therapy on Paco2 or blood Hco3 concentration was evident from the data collected on these 3 horses.

Table 2—

Individual temperature-corrected values of arterial blood pH, Paco2, and Pao2 for 3 of the 8 horses in Table 1 obtained during an HBO therapy session.

VariableBaseline3 ATA (0 minutes)3 ATA (10 minutes)3 ATA (20 minutes)Decompression (2 ATA)End of decompression (1 ATA)Ambient air conditions (10 minutes)
pH7.467.487.497.507.577.457.45
 7.467.487.497.527.527.457.45
 7.407.427.437.437.437.447.44
Paco241.745.951.64935.645.740.5
 45.852.651.846.744.841.843.7
 50.953.441.842.548.138.544.7
Pao296530> 800> 800> 80038189
 76> 800> 800> 800> 80036887
 87> 800> 800> 80061436377

For these horses, an arterial catheter was placed in the left transverse facial artery without sedation before the HBO therapy session. A threaded plug on one of the HBO vessel ports was replaced by a threaded adapter (sterilized prior to the experiment) machined with arterial line connectors on both the outside and inside of the hyperbaric chamber. The arterial line was flushed with saline (0.9% NaCl) solution before being connected to the arterial catheter. An arterial blood sample was withdrawn through the port from outside the hyperbaric chamber prior to pressurization (baseline), when O2 pressure reached 3 ATA (0 minutes), 10 and 20 minutes later, during decompression when O2 pressure reached 2 ATA and again at 1 ATA (end of decompression), and 10 minutes after each horse was exposed to ambient air. Arterial blood samples were analyzed on-site with a handheld analyzer; the analyzer's maximum limit for Pao2 < was 800 mm Hg.

See Table 1 for remainder of key.

Effect of HBO therapy on cells in BAL fluid samples

When the 8 horses underwent HBO therapy, the mean neutrophil cell count in BAL fluid was significantly (P = 0.042) lower at day 10 than at day 0 (Table 3). Determination of differential cell counts on the BAL fluid samples collected before and immediately after the last day of treatment (HBO therapy or control) revealed no therapy-induced changes in the percentages of macrophages, lymphocytes, eosinophils, and mast cells.

Table 3—

Mean ± SD differential cell counts (%) in BAL fluid samples obtained from the 8 healthy horses in Table 1 before (day 0) and after once-daily HBO or control treatment for 10 days (beginning day 1).

Cell type (%)Day before first HBO therapy sessionAfter tenth HBO therapy sessionDay before first control sessionAfter tenth control session
Macrophage56.2 ± 9.559.0 ± 14.563.4 ± 9.962.9 ± 7.9
Lymphocyte35.6 ± 10.836.6 ± 14.531.3 ± 11.933.2 ± 7.5
Neutrophil5.3 ± 3.11.8 ± 0.8*2.1 ± 1.62.1 ± 1.5
Mast cell2.7 ± 1.52.4 ± 2.23.1 ± 1.51.7 ± 1.2
Eosinophil0.2 ± 0.30.2 ± 0.30.1 ± 0.10.1 ± 0.2

A BAL fluid sample was collected from each horse before (day 0) and after (day 10) the HBO or control treatment; on day 10, the BAL fluid sample was collected immediately following the completion of the hyperbaric chamber session. For collection of the BAL fluid samples, horses were sedated, and two 250-mL volumes of sterile endotoxin-free saline (0.9% NaCl) solution were delivered and aspirated via an appropriately positioned video-endoscope.c A 250-µL aliquot of fluid underwent cytologic examination, and a differential count was performed on 400 nucleated cells after staining the slides with a modified Wright-Giemsa solution; the percentage of each cell type was calculated.

Value before the first HBO therapy session was significantly (P = 0.042) different from the value after the tenth HBO therapy session. Reference values for BAL fluid cell counts in horses are as follows: macrophages, 44.4% to 74%; lymphocytes, 16.8% to 49.2%; neutrophils, ≤ 5%; mast cells, ≤ 2%; and eosinophils, ≤ 1%.

See Table 1 for remainder of key.

Validation of reference genes

Data analysis with the specialized software model indicated that GAPDH and SDHA were the most and least stable reference genes, respectively. The software program ranked the most to least stable genes as follows: GAPDH (0.307) then HPRT (0.344), RPL-32 (0.361), and SDHA (0.547). The software program indicated that the top gene combination for generating optimal normalization was GAPDH, HPRT, and RPL-32, yielding the lowest V value (0.124; Figure 1). The addition of SDHA to this combination increased the V value to > 0.15, indicating that the inclusion of this gene did not allow for accurate normalization.

Figure 1—
Figure 1—

Calculation of normalization factors by a specialized software program from an increasing number of genes evaluated to allow assessment of mRNA expression of Th1, Th2, and Th17 cell–associated inflammatory mediators in cells in BAL fluid samples collected from healthy horses exposed to HBO. The software uses a pairwise comparison model that calculates 2 parameters (M and V) for optimal stability and normalization of the data set. The V value determines the optimal number of reference genes required for accurate normalization by analyzing pairwise variation between sequential normalization factors containing an increasing number of reference genes (2, 3, and 4 in this instance). The data shown represent the pairwise variations between 2 sequential normalizations factors. Values of V < 0.15 are most suitable for data analysis. V3/4 indicates that addition of the fourth (least stable) reference gene to the previous 3 (more stable) reference genes increases the V to > 0.15. Therefore the combination of the 3, but not 4, reference genes was suitable for data analysis.

Citation: American Journal of Veterinary Research 77, 10; 10.2460/ajvr.77.10.1148

Effect of HBO therapy on mRNA transcription of inflammatory mediator genes in pulmonary cells

The control treatment (atmospheric air) had no significant effect on the mRNA expression of the tested genes (data not shown). Eotaxin-2 mRNA expression (relative to expression of the reference genes GAPDH, HPRT, and RPL-32) was significantly (P = 0.031) lower (0.12-fold decrease; SE range, 0.76 to 1.02) in BAL fluid cells isolated from the horses following HBO therapy, compared with findings before treatment (Figure 2). There were no significant effects of HBO therapy on mRNA transcription of TNF-α, IL-1β, IL-4, IL-5, IL-6, IL-8, IL-10, or IL-12p35.

Figure 2—
Figure 2—

Box-and whisker plot of the ratios of pretreatment and posttreatment mRNA expressions of eotaxin-2, IL-1β, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p35, interferon (IFN)-γ, and TNF-α in lung cells isolated from BAL fluid samples collected from 8 healthy horses before (day 0) and after once-daily HBO therapy for 10 days (treatment beginning day 1). Horses were exposed to 100% oxygen in a horizontally oriented pressurized hyperbaric chamber. For each chamber session, horses were unsedated. Pressure in the chamber was increased to 3 ATA over a 60-minute period. This pressure was maintained for 20 minutes and then decreased to ambient pressure over a period of 15 minutes. With once-daily treatments, the horses were exposed to HBO conditions (100% oxygen at > 1 ATA) for a total of 90 minutes over a period of 10 consecutive days. A BAL fluid sample was collected from each horse for quantitative PCR analysis on days 0 and 10; on day 10, the BAL fluid sample was collected immediately following the completion of the hyperbaric chamber session. Expression of each mRNA was compared with the expression of a combination of the reference genes GAPDH, HPRT, and RPL-32. A value of 1 indicates no change in expression; values < 1 indicate a downregulation in gene expression, and values > 1 indicate an upregulation in gene expression. For each box, the horizontal line represents the median and the upper and lower boundaries represent the third and first quartiles, respectively. Whiskers represent the maximum and minimum values. *Eotaxin-2 mRNA expression (relative to expression of the reference genes GAPDH, HPRT, and RPL-32) was significantly (P = 0.031) lower in BAL fluid cells isolated from the horses following HBO therapy, compared with findings before treatment.

Citation: American Journal of Veterinary Research 77, 10; 10.2460/ajvr.77.10.1148

Discussion

Numerous studies14–18,35,36 have revealed that oxidative stress secondary to HBO therapy can trigger localized pulmonary inflammation in various species; however, little is known about the effects of HBO therapy in horses. In addition, the biological effect of HBO therapy, including the duration of blood hyperoxygenation, in horses is unknown. The aim of the present study was therefore primarily to assess the effects of HBO therapy on inflammatory mediator mRNA expression, as a means to determine whether HBO therapy alters inflammatory responses in healthy equine lungs. A second objective was to describe the effect of HBO therapy on arterial blood gases in horses. With quantitative PCR methods, the mRNA expression of a variety of Th1-, Th2-, and Th17-associated cytokines in BAL fluid cells from horses that underwent once-daily HBO therapy for 10 days was evaluated. The study findings indicated that mRNA transcription of a variety of proinflammatory mediators was unaltered by HBO therapy. Interestingly, expression of eotaxin-2 in pulmonary cells isolated from the horses when they were exposed to HBO was significantly reduced following treatment. Differential cell counts of the BAL fluid samples collected indicated a significant decrease in the percentage of neutrophils following HBO therapy; however, there were no significant changes from pretreatment findings for the populations of macrophages, lymphocytes, eosinophils, and mast cells in horses when they received the HBO or control treatment. The mechanism that underlies the actions of HBO therapy on gene expression remains unclear. Collectively, the results of the present study have suggested that the HBO protocol used, which provided a long exposure to 100% oxygen at > 1 ATA, does not activate inflammatory genes in healthy horses. The arterial blood oxygen data obtained indicated that there was no cumulative effect of HBO therapy in horses because the pretreatment values (at day 0) were similar to the posttreatment values (at day 10) when horses were exposed to HBO. In addition, the present study involved an innovative arterial sampling technique that allowed monitoring of arterial blood gases in a small number of horses while they were undergoing HBO therapy in the hyperbaric chamber as well as immediately after the HBO therapy session. Interestingly, there was not only a rapid (and expected) increase in the oxygenation of the blood beyond the measurement capabilities of the analyzer (upper limit, 800 mm Hg) during HBO therapy, but also a rapid decrease to baseline values during decompression. A nonsignificant increase in blood pH was also observed. All changes rapidly returned to baseline values during decompression; measuring blood gases from samples collected following decompression when horses had been in ambient air for 10 minutes revealed no changes on variables, compared with findings prior to HBO therapy.

Eotaxin is a potent eosinophil chemoattractant and chemotactic for basophils, mast cells, and Th2 lymphocytes.37 Additionally, eotaxin promotes degranulation in both eosinophils and basophils.37 Eotaxin-2 is a functional homolog of eotaxin with similar cellular selectivity and actions.38 Data have indicated that eotaxin and eotaxin-2 promote recruitment of equine eosinophils in vitro39,40 and that CCR3, the receptor for eotaxin-2, is expressed in equine lung tissue.41 Both mediators have been implicated in allergic lung diseases in humans and horses.37,39,42 Furthermore, we have previously shown that eotaxin-2 has a role in the pathogenesis of inflammatory airway diseases in horses.30

Other studies8–10,43–45 have revealed attenuation of cytokine expression with HBO therapy in laboratory animals as well as in humans. The HBO therapy protocols used in those studies varied, with pressures from 1.5 to 8 ATA, durations from 60 to 90 minutes, frequencies from once to twice daily, and treatment periods from 3 to 20 days. In contrast, pulmonary and CNS toxic effects have been reported at as little as 1.5 ATA43; severe lung edema as an oxygen-induced toxic effect was observed in rats following 200 to 400 minutes at 4 ATA.45 The protocol in the present study used slow (1 hour) pressure ramping up to a maximum pressure of 3 ATA, which was then maintained for 20 minutes, once daily for 10 days. Such a protocol did not trigger the expression of proinflammatory cytokines in the BAL fluid cells of the study horses. Plafki et al36 suggested that oxygen-related toxic effects in the lungs are due to either very intense or prolonged courses of HBO therapy. It has been proposed that pulmonary damage caused by oxidative stress worsens at higher pressures and shorter exposure times, but with less of an associated inflammatory response.15 In light of a study14 in which oxidative stress variables appeared to be directly proportional to the extent of HBO exposure, it is probable that the duration of the horses’ exposure to pressurized oxygen in the present study was sufficiently short to avoid inducing pulmonary inflammatory responses or damage. This was also supported by the fact that the HBO exposure did not affect the percentages of macrophages, lymphocytes, eosinophils, and mast cells in BAL fluid samples.

An increase in the number of neutrophils in BAL fluid samples from horses is often associated with development of clinical or subclinical pulmonary disease. In the present study, evidence of lung inflammation in the screening BAL fluid sample was an exclusion criterion, and in the study horses, the percentage of neutrophils in BAL fluid samples was within reference range at all times.24,25,27 However, despite randomization of the crossover design, horses seemed to have a higher percentage of neutrophils in BAL fluid samples prior to HBO therapy, compared with findings prior to control treatment. Nevertheless, because there was a significant decrease in the percentage of neutrophil after horses were exposed to HBO, it is possible that HBO therapy could exert a beneficial effect in horses with pulmonary disease, and further research is indicated.

Quantitative PCR methods were used in the present study to assess changes in gene expression. Interpretation of PCR quantification data can be misled by changes in reference gene expression induced by the treatment46; therefore, a strength of the present study was that we first identified the most suitable reference genes in the cells isolated from the BAL fluid samples. We previously reported29 reference genes and validation methods to assess gene stability. Results of studies29,47–49 have validated reference genes for equine skin and equine sarcoids, peripheral blood mononuclear cells, and BAL fluid in horses with inflammatory airway disease or recurrent airway obstruction; however, the stability of reference genes following HBO therapy had not yet been reported. In the present study, it was demonstrated that GAPDH was the reference gene of choice. However, to increase the validity and robustness of the data, a combination of reference genes is more appropriate; in the present study, GAPDH, HPRT, and RPL-32 were identified as the most stable combination of reference genes by use of the specialized software program. Also, efficiency correction of the PCR reactions was applied as another important methodological precaution32 in the present study. Reaction efficiency varies among samples, and small differences in PCR efficiency can significantly impact the interpretations of the data. A software programm for the analysis of quantitative PCR data that allows calculation of the efficiency of individual PCR reactions without the assumptions involved with other methods, such as extrapolating data from a standard curve, was used in the present study. In addition, a relative expression software tool was used to perform the quantitative PCR quantification analysis and included a correction for the differences in efficiency among PCR reactions.50 An advantage of the relative expression software tool is that it determines whether changes in relative gene expression are significant and provides an SE error and a confidence interval range for calculated relative gene expressions.34

A quantitative PCR assay is a powerful tool for detecting changes at the level of gene transcription. In the present study, we were able to investigate the effects of HBO exposure on inflammatory gene expression in lung cells of healthy horses. The facts that the change in eotaxin-2 expression was moderate and that we did not observe changes for the other inflammatory factors evaluated decrease the relevance of verifying whether these changes in mRNA expression directly reflect changes at the protein level. Another limitation of the study was that the expression of inflammatory factors was measured for the entire lung cell population, without sorting inflammatory cell types. This is a common approach in the study of lung cell inflammatory gene expression that allows screening of inflammatory factor production. Lastly, a better understanding of the effects of HBO therapy on lung physiology would require the study of both antioxidant status and signs of oxidative stress, which were outside the scope of the present study. Although these factors have been investigated in humans and laboratory animals4,14,15,22,51–53 as well as in horses with recurrent airway obstruction,54,55 the effects of HBO therapy in horses have yet to be fully elucidated.

The results of the present study indicated that the HBO protocol used did not lead to development of lung inflammation in horses. To the contrary, expression of eotaxin-2 mRNA was reduced in the pulmonary cells isolated from horses following HBO exposure, whereas the control procedure had no effect on pulmonary cell cytokine expression. A limitation of the study was that these results were not confirmed at the protein level. However, in the context of allergic pulmonary diseases, HBO therapy may be a viable therapeutic option for affected horses.

Acknowledgments

Supported by the Clinical Research Fund of the University of Calgary Faculty of Veterinary Medicine.

The authors declare that there were no conflicts of interest.

Presented in part as an abstract at the 27th Annual American Veterinary Internal Medicine Forum, New Orleans, June 2012.

The authors thank Bar None Ranches, Dewinton, AB, Canada, for providing the horses and hyperbaric chamber service, and Mike Vanin and Drs. Markus Czub and Mike Scott for technical assistance.

ABBREVIATIONS

ATA

Atmosphere absolute

BAL

Bronchoalveolar lavage

GAPDH

Glyceraldehyde-3-phosphate dehydrogenase

HBO

Hyperbaric oxygen

HPRT

Hypoxanthine ribosyltransferase

IL

Interleukin

RPL-32

Ribosomal protein L32

SDHA

Succinate dehydrogenase complex subunit A

Th

T helper

TNF-α

Tumor necrosis factor-α

Footnotes

a.

Abbott-iStat and CG4+ cartridges, Abbott Laboratories, Abbott Park, Ill.

b.

Equineox Technologies Ltd, Maple Ridge, BC, Canada.

c.

Optomed, Les Ulis, France.

d.

Qiagen, Mississauga, ON, Canada.

e.

Onmi International, Kennesaw, Ga.

f.

RNeasy Mini kit, Qiagen, Mississauga, ON, Canada.

g.

OmniscriptRT, Qiagen, Mississauga, ON, Canada.

h.

RNase-OUT, Qiagen, Mississauga, ON, Canada.

i.

Invitrogen, Burlington, Ontario, Canada.

j.

Nanodrop ND-1000, Thermo Fisher Scientific, Wilmington, Del.

k.

MX3005P, Stratagene, La Jolla, Calif.

l.

PerfeCta TM SYBR Green Supermix, Quanta BioSciences, Gaithersburg, Md.

m.

LinRegPCR, version 110, Heart Failure Research Center, Academic Medical Center, Amsterdam, Netherlands. Available at: LinRegPCR.nl.

Accessed Feb 28, 2012.

n.

geNormplus software. Available at: medgen.ugent.be/~jvdesomp/genorm. Accessed Feb 28, 2012

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Contributor Notes

Address correspondence to Dr. Léguillette (rleguill@ucalgary.ca).