The efficacy of N‐acetylcysteine in decreasing airway inflammation and mucus accumulation in horses with 18 hours of head confinement

Abstract

Background

During transportation many horses develop post‐transportation infection, which can be life‐threatening and end their sport career. Preventing mucus accumulation and inflammation during transportation is vital, emphasizing the need for effective strategies to enhance overall horse health welfare.

Objectives

Assess the impact of N‐acetylcysteine (NAC) on mucus accumulation and inflammation in horses subjected to 18 hours of head confinement.

Animals

Six healthy crossbred horses, 5.3 ± 2.1 years of age and weighing 387 ± 30 kg.

Methods

Prospective placebo‐controlled cross‐over design study. The horses' heads were restrained in their stalls for a period of 18 hours. They were studied under 4 conditions: Not confined (NC): before head confinement, placebo (P), and confined head (CH): 18 hours of head confinement without treatment, and N‐Acetylcysteine (NAC): 18 hours of head confinement treated with NAC before confinement (15 mg/kg/day NAC PO for 3 days). Bronchoalveolar lavage (BAL) was performed in each condition. Mucus accumulation along the trachea was evaluated by endoscopy.

Results

Endoscopic scores were significantly different between CH and other conditions, whereas no significant differences were found among NC, P, and NAC. The BAL cell count (34 291 ± 2624 cells/μL), neutrophil and lymphocyte count (18 601 ± 3193 cells/μL and 3337.4 ± 593 cells/μL, respectively) in CH were significantly higher compared to NAC. Neutrophil percentage was significantly higher in CH (53.8 ± 8%) compared to horses that received NAC (20.08 ± 8%). Conversely, in comparison to NAC (66.33 ± 9%), the percentage of macrophages was significantly lower in CH (35.7 ± 10%).

Conclusions

N‐acetylcysteine was found to significantly decrease mucus accumulation and inflammatory cell counts in horses with head confinement.

Abbreviation

BAL

bronchoalveolar lavage

CH

confined head

FEV1

forced expiratory volume in 1 second

NAC

N‐acetylcysteine

NC

not‐confined

P

placebo

VC

vital capacity

1. INTRODUCTION

The transportation of horses for various purposes, such as competitions, breeding, and veterinary services, is a common practice. During transportation, it is essential to restrain the horses properly to prevent physical injuries. However, long term confinement of the head and neck can cause disorders of the mucociliary function and consequently accumulation of mucus and bacteria in lower airways. Studies have shown that restraining the head for 6‐12 hours during transportation causes bacteriological and cytological changes in the airways. ¹ , ² Short‐distance transportation (2.5 hours) also can result in an increase in bronchoalveolar lavage (BAL) neutrophils, ³ and a study showed that transportation caused health problems in 2.8% of horses, in which fatality occurred in 0.24% of them. ⁴ Both long‐term and short‐term movement can lead to health disorders in horses, ⁵ including pneumonia, ⁶ , ⁷ colic (which mostly originates from large colon impaction ⁴ ), and Salmonella spp enterocolitis. ⁸ In addition, transportation stress can result in increases in cortisol concentration ⁹ and heart rate, ¹⁰ as well as alterations in immune function. ¹¹ These alterations can lead to increased risk of respiratory diseases. ¹² Equine herpes virus‐1 (EHV‐1), EHV‐2, EHV‐4, EHV‐5 or some combination of these was detected in half of the 167 horses that were brought into the United States by long‐haul transportation. ¹³

Several studies have been conducted to find ways to prevent post‐transportation pneumonia. One suggestion has been to lower the head below the withers during transportation to decrease the occurrence of airway inflammation. ¹⁴ The use of penicillin procaine to prevent airway inflammation in horses the heads of which were confined for 24‐48 hours was not effective. ¹⁴ In another study, 12 horses were examined after a short‐haul transportation period of 1.5‐2 hours. These horses were given daily antioxidant supplement for 3 weeks before and after the study. The results showed a decrease in interleukin‐1β (IL‐1β) and serum amyloid A‐1 (SAA1) concentrations in the horses that received the supplement, indicating that it may have anti‐inflammatory effects. ¹¹

Derived from the amino acid L‐cysteine, N‐acetylcysteine (NAC) is a sulfhydryl‐containing compound with mucolytic activity. It was used over 40 years ago to treat respiratory diseases such as chronic bronchitis in human medicine. ¹⁵ Today, NAC primarily is recognized for its antioxidant properties. ¹⁶ The anti‐inflammatory effects of NAC are a result of preventing the activity of pro‐inflammatory cytokines, including IL‐8, IL‐6, and tumor necrosis factor alpha (TNF‐α). ¹⁷ , ¹⁸ Studies on NAC efficacy in humans however might not directly apply to horses, and more research is required to determine the effectiveness of NAC in horses. N‐acetylcysteine is available in various pharmaceutical forms, with the PO form being primarily used for respiratory diseases with dosage ranging from 5 to 25 mg/kg/day. ¹⁹ In a study conducted on 123 patients with chronic obstructive pulmonary disease (COPD), patients receiving a higher dose of NAC showed more improvement in forced expiratory volume in 1 second (FEV1), along with a decrease in IL‐8 concentration. ²⁰

We aimed to assess the impact of NAC on decreasing mucus accumulation and inflammation in airways of horses experiencing prolonged head confinement. We hypothesized that prolonged head restraint would lead to mucus and inflammatory cell accumulation in the lower airways, and that NAC would be effective in decreasing inflammation and mucus accumulation because of its mucolytic and anti‐inflammatory properties.

2. MATERIALS AND METHODS

2.1. Animals

The study was conducted on 6 healthy crossbred research horses, owned by the University of Tehran. They were 5.3 ± 2.1 years old and weighed 387 ± 30 kg. Before the study, thorough physical examinations were performed, along with upper airway endoscopy and a CBC, to eliminate the possibility of concurrent medical conditions.

Before each part of the study, clinical examinations were conducted, including lung auscultation, rectal temperature, heart rate, respiratory rate, and signs of nasal discharge, which were recorded.

2.2. Study design

We evaluated the effects of NAC in a placebo‐controlled experimental study using a randomized cross‐over design. The study was approved by the faculty of Veterinary Medicine of the University of Tehran, Research Council (approval date: November 2022).

The horses were studied under 4 conditions, as follows:

• NC: Non‐confined head

• P: Placebo

• CH: 18 hours of head confinement without treatment

• NAC: 18 hours of head confinement treated with NAC

Data were first recorded in the NC condition, followed by the P condition. Horses then were allocated to the CH and NAC conditions in a cross‐over design, with a 1‐month washout period between each condition. The order in which they underwent CH vs NAC was randomized. In the NC condition, data were recorded before any intervention. In P condition (ie, horses the heads of which were not confined), normal saline (Darou Pakhsh Pharmaceutical Mfg. Co., Tehran, Iran) was given PO to horses twice (a day before and 2 hours before sampling), and the same procedures were performed. In CH and NAC conditions, the horses' heads were restrained, with the nostrils above the level of the withers for 18 hours in their stalls. Horses in the NAC group received N‐acetylcystein (Zambon Co., Fluimucil, 600 mg tablet) PO at a dosage of 15 mg/kg once per day, diluted in water, starting 3 days before restraining. The last dose was given 2 hours before BAL sampling. The next morning, BAL samples were taken. Horses were sedated using xylazine (0.6‐1.0 mg/kg IV; Alfasan Inc., The Netherlands), and an endoscope was inserted through a nostril, directed into the trachea and down into a lung. The airways were topically anesthetized during the advancement of the endoscope using 30‐50 mL of 0.5% lidocaine (2% lidocaine; Nasr Pharma Co., Iran) diluted with sterile pyogen‐free water. A total 300 mL of sterile, pre‐warmed (37°C), isotonic saline solution was infused sequentially, and aspirated samples were kept on ice and processed within 1 hour.

The endoscopic scoring of the trachea was visually assessed and determined by 2 of the authors (HT and ZA). The scoring system was based on the following grades:

• Grade 0: No mucus

• Grade 1: Hyperemia

• Grade 2: Local mucus accumulation

• Grade 3: Diffuse mucus accumulation

2.3. Bronchoalveolar fluid evaluation

For cellular analysis, bronchoalveolar fluid (BALF) collected from each horse (minus 5 mL set aside), was centrifuged at 1500 × g for 5 minutes. The supernatant was discarded and the cell pellets were resuspended in 200 μL of BALF. Total cell count was determined manually under a microscope using a hemocytometer and cell counter.

Depending on estimated cellularity, samples were diluted 1:1 to 1:20 with Marcano solution to facilitate cell counting. Marcano solution was used to dilute the blood to count white blood cells. The composition of Marcano solution includes 4% acetic acid and a few drops of 1% methylene blue solution. The equation used to calculate cell concentration was:

$$\text{Total cells}/\mathrm{\mu L}=\left(\text{Total cells counted}\times \text{Dilution factor}\right)/0.1\times \text{Number of squares counted}.$$

Additionally, for cytology examination and differential count, a cytology smear was prepared from each sample. To do so, 10 μL of each BAL sample after centrifugation were used to prepare a cytology smear. Afterward, the sample was air‐dried, fixed with methanol fixative solution, and stained with Giemsa. A clinical pathologist blinded to the group assignments (HM), performed a differential count of 400 cells and calculated the percentage and total count of each cell type.

2.4. Complete blood count and plasma fibrinogen analysis

To assess the hematology profile of each horse, CBC and plasma fibrinogen concentrations were analyzed under all conditions. Blood samples were drawn into EDTA blood collection tubes for CBC and processed using an automated counter (Celltacα MEK‐6450K, Nihon Kohden, Tokyo). Differential cell counting also was performed. Plasma fibrinogen concentration was measured using a heat precipitation micromethod.

2.5. Statistical analysis

Statistical analyses were performed using SAS v.9.4 software (SAS Institute Inc., Cary, North Carolina). Normality of distribution for all continuous variables was assessed visually using the PROC BOXPLOT and the PROC UNIVARIATE qqplot statement. The Shapiro‐Wilk test also was calculated using the PROC UNIVARIATE normal test statement. Because data were normally distributed, the PROC MIXED procedure of SAS was used for analysis. The Tukey‐Kramer method was employed to compare means and to adjust for multiple comparisons. The experimental unit was the individual animal, and the model included the fixed effect of treatment. Endoscopic scores were treated as ordinal data and analyzed using the Kruskal‐Wallis test, using the Dwass, Steel, Critchlow‐Fligner multiple comparison test in the PROC NPAR1WAY procedure of SAS. Median and range for ordinal data, as well as mean and standard deviation (SD) for continuous variables, were reported. All plots were created with ggplot2 3.4.1 using R Studio version 4.2.2. Statistical significance was set at P ≤ .05.

3. RESULTS

3.1. Endoscope scoring

Horses in all conditions had normal clinical findings during the study.

A Kruskal‐Wallis test showed strong evidence for the difference in endoscopy scores among the 4 conditions (n = 24, P = .0003; Table 1). Specifically, CH had a significantly higher score than NC, P, and NAC (P = .01, P  = .01, and P = .03, respectively). However, NAC did not differ significantly from NC and P (P = .44; Table 1).

TABLE 1.

Endoscopic mucus scores recorded in 6 horses after a period of 18 hours under conditions.

Condition	Median	Range	χ 2	df	P‐value
NC1	0a	0‐0	18.55	3	.0003*
P2	0a	0‐0
CH3	3b	2‐3
NAC4	0a	0‐2

Note: Effect of intervention (P, CH, NAC) on endoscopic score compared to NC as the reference. ¹Not confined, ²Placebo, ³Confined head, ⁴N‐acetylcysteine. Medians with different superscript differ significantly. *P ≤ .05.

3.2. Bronchoalveolar fluid analysis

No significant differences were observed between the NC and P conditions. However, when comparing horses with confined head (CH) to both the NC and P groups, all the BAL findings were significantly different. The total cell count and total neutrophil count were significantly higher in the CH compared to the NC and P groups (P ≤ .0001). Additionally, the total lymphocyte and macrophage counts were significantly higher in the CH group in relation to the NC and P groups (P ≤ .05; Table 2; Figure 1). In terms of percentages, the neutrophil percentage was higher in the CH (P ≤ .0001), whereas the percentages of lymphocytes and macrophages were significantly lower compared to both the NC and P groups (P ≤ .0001 and P ≤ .05). These results indicate substantial inflammation in horses after head restraint (Table 2; Figure 2).

TABLE 2.

Comparison of total (cells/μL in final sediment) and differential (%) cell count in BAL fluids after a period of 18 hours under 4 conditions.

	Groups of treatment
	NC	P	CH	NAC	P‐value
BAL cc	138.8 ± 23.37A	142.6 ± 24.58AB	34 291 ± 10 662C	16 865 ± 1542D	≤.0001
BAL pmn	6.2 ± 6.16A	5.6 ± 1.5AB	18 601 ± 13 039Cb	1459.33 ± 1027c	.001
BAL lym	58.6 ± 9.26a	62.8 ± 9.92a	3337.4 ± 2205b	1048.67 ± 1032ac	.003
BAL mac	74 ± 11.2a	74.2 ± 18.47a	8996 ± 8811b	7310.67 ± 5598b	.04
BAL pmn %	4.39 ± 0.45a	4.02 ± 0.97ab	53.8 ± 31.25c	20.08 ± 18.07ab	.003
BAL lym %	42.21 ± 0.82A	44.36 ± 4.23AB	10.5 ± 6.81C	13.58 ± 13.78C	≤.0001
BAL mac %	53.37 ± 0.61	51.59 ± 4.51	35.7 ± 33.88a	66.33 ± 23.05b	.21

Note: Data are expressed as the means and standard deviations, with P‐value determined by linear mixed model and Tukey post‐hoc testing. Means with different superscript differ significantly (UPPERCASE letters P ≤ .001; lowercase letters P ≤ .05).

Abbreviations: CH, confined head; NAC, N‐acetylcysteine; NC, not confined; P, placebo.

FIGURE 1.

Dot Plots illustrating the actual values (cells/μL in the final sediment) of total cell count (A), neutrophil count (B), lymphocyte count (C), and macrophage count (D) in BAL fluids after an 18‐hour period for four conditions: NC (Not‐confined head), P (Placebo), CH (18 hours of head confinement without treatment), and NAC (18 hours of head confinement treated with NAC) (n = 6). Means with standard deviations are represented by bars. The y‐axes are presented on an exponential scale to better visualize the range of cell counts. **P ≤ .001; *P ≤ .05.

FIGURE 2.

Dot Plots illustrating the percentage (%) of total cell count (A), neutrophil count (B), lymphocyte count (C), and macrophage count (D) in BAL fluids after an 18‐hour period for four conditions: NC (Not‐confined head), P (Placebo), CH (18 hours of head confinement without treatment), and NAC (18 hours of head confinement treated with NAC) (n = 6). Means with standard deviations are represented by bars. **P ≤ .001; *P ≤ .05.

Treatment with NAC led to a significant decrease in the total cell count compared to CH group (P = .0001). The neutrophil count was significantly lower in the NAC compared to the CH group (P = .001). The total lymphocyte count was significantly higher in the CH group compared to NAC (P = .01). The total macrophage count was not different between CH and NAC (P = .62; Table 2; Figure 1). The neutrophil percentage was significantly higher in the CH group compared to the NAC group (P = .01). Administration of NAC did not result in any significant changes in the percentage of lymphocytes compared to the CH group (P = .56). A significant difference in macrophage percentage was observed between the CH and NAC groups, with a higher percentage observed in the treated condition (P = .03; Table 2; Figure 2).

Significant differences were observed in the total cell count between the NAC group and both the NC and P groups (P = .0002). However, no statistical differences were found when comparing the total neutrophil count in the NAC group with the NC and P groups (P = .74). Similarly, no significant differences were found in the lymphocyte count between the NAC group and both the NC (P = .23) and P (P = .24) groups. On the contrary, the macrophage counts were significantly higher in the NAC group compared to both the NC and P groups (P ≤ .05; Table 2; Figure 1). The neutrophil percentage was not significantly different between the NAC group and both the NC group (P = .21) and P (P = .2). In contrast, the lymphocyte percentage was significantly lower in the NAC group compared to the NC and P groups (P ≤ .0001), whereas no differences were noted in the percentage of macrophages among the NAC, NC, and P groups (Table 2; Figure 2).

3.3. Complete blood count, plasma fibrinogen, and cellular analysis

No significant differences were observed in CBC variables and plasma fibrinogen concentration among the 4 conditions. The cytology smears were assessed by a clinical pathologist blinded to treatment group (HM). In cytology smears, differential count and cellular morphology indicative of toxic changes in neutrophils were evaluated. In CH, a considerable number of degenerate neutrophils were found in 2 horses. Conversely, in NAC, based on the total and differential cell counts, the severity of inflammation and cellularity was lower (Figure 3).

FIGURE 3.

Photomicrographs of Giemsa‐stained BAL samples. (A) Control. A few normal ciliated epithelial cells (Green arrow) in a poorly cellular sample (×400 magnification). (B‐D) BAL samples taken from horse with head elevation for 12 hours. (B) A large number of degenerated PMNs in a high cellular sample (×400 magnification). (C) A cluster of degenerated PMNs (Black arrow) in a higher magnification (×1000 magnification). (D) Some bacteria as bacilli (Red arrow) arranged in a chain (×1000 magnification). (E and F) BAL samples taken from horse with head elevation for 12 hours and oral administration of N‐acetylcysteine. (E) Some inflammatory cells as vacuolated macrophages in a moderate cellular sample (×400 magnification). (F) Some vacuolated macrophages (Yellow arrow) and ciliated epithelial cells (Green arrow) in a sample higher magnification (×1000 magnification).

4. DISCUSSION

We found that NAC effectively decreased the accumulation of mucus in the lower airways of horses subjected to 18 hours of head confinement. All horses in the CH group had mucus accumulation (grade 2 and 3) after 18 hours, whereas in the NAC group, only 2 horses had grade 2 mucus accumulation (Table 1). Horses that received 15 mg/kg/day of NAC had a significant decrease in inflammation, supported by a decrease in total neutrophil count and percentage of neutrophils. Head restraining resulted in a notable increase in all variables under the CH conditions. With NAC administration, inflammation was decreased based both on laboratory and endoscopic findings.

It is crucial to secure the head and neck during transportation to prevent injuries. Neglecting this precautionary measure can lead to injuries. Nevertheless, restraining the head and neck can cause health problems, and post‐transportation pneumonia is a commonly observed outcome. According a previous study, after 41 hours of transportation, 3 of 8 horses developed pneumonia in the cranioventral lung fields. ²¹ Previous studies have indicated that relieving head elevation for a short time neither prevents the accumulation of inflammatory secretions nor decreases bacterial counts in the airways. ¹ Restraining the head for 12‐24 hours also affects the cytology and bacterial culture results obtained from guttural pouch lavage in healthy horses. Furthermore, an increase in neutrophil counts was observed in guttural pouch washes. ²² Head confinement above the wither level can cause bacterial overload and inflammatory secretions in BALF, even in the absence of transportation. ¹ Our results are consistent with these findings, because restraining the head without movement led to mucus accumulation and an increase in bacterial and neutrophil counts. We hypothesized that NAC, because of its mucolytic, antioxidant, and anti‐inflammatory properties, could be an effective means of minimizing the risk of mucus accumulation in horses undergoing head restraint.

In contrast to human medicine, NAC is not commonly used in equine medicine. A previous study investigated the impact of PO NAC on endometritis in mares. ²³ The prescribed dosage was 10 mg/kg for 4 days, which significantly decreased the number of polymorphonuclear leukocytes (PMN) in endometrial biopsy samples and the intensity of staining for cycloxigenase‐2 (COX2) of the epithelial nuclei, indicating that NAC has anti‐inflammatory and antioxidant effects in horses. ²³ Another study aimed to assess the effects of NAC and coenzyme Q10 supplementation on skeletal muscle in fit Thoroughbred horses. ²⁴ The investigators administered a daily PO dosage of 10 g of NAC for 30 days, which improved the redox status of the muscle without negatively affecting performance. In addition, muscle glutathione concentration increased, and the concentration of oxidized glutathione decreased significantly. ²⁴ In our study, we administered a PO dosage of 15 mg/kg/day of NAC for 3 days, with the final dose given 2 hours before endoscopy and BALF sampling. As previously noted, a significant decrease in the grade of mucus accumulation was noted. Furthermore, in the NAC condition, decreases in both neutrophil and lymphocyte counts were observed, along with a decrease in neutrophil percentage compared to the CH condition.

N‐acetylcysteine can decrease nuclear factor kappa B (NF‐κB) activity by modulating cellular redox status and impacting multiple pathways. ²⁵ In vitro studies confirm that decreasing NF‐κB activity mitigates pro‐inflammatory gene regulation and mediators. ²⁶ In a previous study, it was shown that a concentration of >1 mM NAC in vitro could lead to a decrease in both intercellular adhesion molecule‐1 and IL‐8. ²⁷ Additionally, another study found that NAC can inhibit inflammatory cytokines TNF‐α, IL‐1β, and IL‐6 at both the protein and the mRNA level in a macrophage cell line activated by lipopolysacharide. ²⁸

In our study, horses with induced mucus accumulation were treated with NAC. We hypothesized that NAC would effectively decrease airway inflammation. The laboratory and endoscopic findings of our study provide evidence supporting the effectiveness of NAC in decreasing inflammation. Although our study had some limitations, such as not being conducted on transported horses, we believe that the environmental factors were well‐controlled because all the horses were bedded with wood shavings, and their diet was similar. They were kept under the same conditions for an extended period, and stress was low. Thus, future studies should focus on using NAC in transported horses for clinically relevant results. Furthermore, our study could serve as a starting point for evaluating the potential of NAC as a treatment option for horses suffering from asthma. Our study had an additional limitation in that the authors were unaware at the time that Gerber's mucus scoring ²⁹ had become the reference for scoring mucus accumulation.

5. CONCLUSION

In conclusion, we aimed to investigate the effectiveness of NAC in decreasing mucus accumulation in the airways of horses and its potential effectiveness in preventing post‐transportation pneumonia during long‐haul transportation. Our findings provide evidence that NAC can be administered PO before transportation to substantially decrease mucus accumulation and inflammation in the lower airways of horses. We found notable decreases in total cell count, total neutrophil count, percentage of neutrophil, and mucus accumulation in horses that received a daily dose of 15 mg/kg NAC for 3 days.

CONFLICT OF INTEREST DECLARATION

Authors declare no conflict of interest.

OFF‐LABEL ANTIMICROBIAL DECLARATION

Authors declare no off‐label use of antimicrobials.

INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC) OR OTHER APPROVAL DECLARATION

Study was approved by the Faculty of Veterinary Medicine of the University of Tehran Research Council (approval date: November 2022).

HUMAN ETHICS APPROVAL DECLARATION

Authors declare human ethics approval was not needed for this study.

Tavanaeimanesh H, Alinia Z, Sadeghian Chaleshtori S, Moosavian H, Mohebi Z, Daneshi M. The efficacy of N‐acetylcysteine in decreasing airway inflammation and mucus accumulation in horses with 18 hours of head confinement. J Vet Intern Med. 2024;38(2):1224‐1231. doi: 10.1111/jvim.16976