Investigation of the effects of hydroxychloroquine on rats with pulmonary contusion caused by blunt thoracic trauma

ABSTRACT

Purpose:  To investigate the early and late effects of hydroxychloroquine (HCQ) on inflammation, fibrosis, and apoptosis in an experimental rat model of pulmonary contusion.

Methods:  Thirty-five male Wistar albino rats were randomly allocated into five groups (n = 7): control, pulmonary contusion evaluated on day 3 or day 10 (PC-3 and PC-10), and pulmonary contusion treated with HCQ at 100 mg/kg/day by gavage and evaluated on day 3 or day 10 (PCHCQ-3 and PCHCQ-10). Lung injury was assessed histopathologically by alveolar edema, congestion, leukocyte infiltration, and fibrosis scores and immunohistochemically by caspase-3, inducible nitric oxide synthase (iNOS), and endothelial nitric oxide synthase (eNOS) expression.

Results:  No alveolar edema was observed. Congestion and leukocyte infiltration were significantly greater in all contusion groups than in the control group. Fibrotic changes were detected on day 10 in both treated and untreated contusion groups. Caspase-3 and iNOS expression were significantly higher in the contusion groups than in the control group. HCQ did not significantly reduce leukocyte infiltration, fibrosis, apoptosis, or iNOS expression compared with the corresponding untreated groups.

Conclusion:  HCQ administered at 100 mg/kg/day for three or 10 days did not demonstrate significant anti-inflammatory, anti-apoptotic, or antifibrotic effects in this rat model of pulmonary contusion.

Key words
Contusions; Hydroxychloroquine; Inflammation; Fibrosis; Apoptosis; Caspase 3; Models, Theoretical

Introduction

Pulmonary contusion is one of the most common injuries resulting from blunt chest trauma, occurring in 30–75% of cases and associated with a mortality rate ranging from 10–25%¹. The injury disrupts the alveolar-capillary barrier, leading to pulmonary edema, alveolar congestion, and ventilation-perfusion mismatch². Additionally, increased alveolar-capillary permeability and intrapulmonary shunting can cause severe respiratory complications³,⁴. As the injury progresses, intraluminal fibroplastic plaque formation, congestion foci, and interstitial fibrosis may develop around respiratory bronchioles, further complicating the clinical picture⁵-⁷. These fibrotic changes may contribute to persistent reductions in lung compliance and long-term respiratory morbidity beyond the acute phase. Animal models can be used to investigate the pathophysiology of lung contusion to explore potential therapeutic interventions. The effects of bleomycin on pulmonary contusion were examined in a rat model of lung trauma to investigate inflammatory and fibrotic processes, which closely resemble those observed in pulmonary contusion, particularly the disruption of alveolar integrity and subsequent fibrotic changes⁸.

To reduce lung damage caused by contusion, it is important to use treatments that inhibit the inflammatory response, restore oxidant–antioxidant balance, and prevent the development of fibrosis. Hydroxychloroquine (HCQ), a drug commonly used for its antimalarial and immunomodulatory properties, has shown promise in modulating inflammatory pathways in diseases such as rheumatoid arthritis and systemic lupus erythematosus⁹. The anti-inflammatory effects of HCQ are thought to result from its inhibition of antigen-presenting cell activity, suppression of T-cell activation, reduction of arachidonic acid release, and inhibition of eicosanoid production¹⁰,¹¹. Hydroxychloroquine, widely recognized for these properties, was also investigated during the SARS-CoV-2 pandemic for its potential antiviral effects. Several studies evaluated its efficacy in mitigating viral replication and reducing immune-mediated complications¹²,¹³. It was used during the pandemic because of its potential benefits in lung injury, as it was thought to possess anti-inflammatory and immunomodulatory effects that could reduce cytokine release and mitigate immune-mediated damage. Studies examined its impact on viral replication and inflammatory processes, aiming to evaluate its role in reducing lung damage associated with severe COVID-19¹²,¹³.

Hydroxychloroquine may have potential benefits in treating pulmonary contusion by modulating the underlying inflammatory response. However, there are no published data on the effects of HCQ in pulmonary contusion. This study aimed to evaluate the early and late effects of HCQ on inflammation, fibrosis, and apoptosis in a rat model of experimental lung contusion using histopathological and immunohistochemical methods. The findings are expected to contribute to the current body of knowledge and may help inform future therapeutic strategies for the management of pulmonary contusion.

Methods

Study design and animal model

This experimental study was conducted between March 2024 and August 2024 at the Animal Research Laboratory of Istanbul Medeniyet University, Istanbul, Turkey, in collaboration with the Department of Pediatric Surgery and the Department of Pathology. Histopathological and immunohistochemical analyses were performed at the Department of Pathology. The study was approved by the Istanbul Medeniyet University Animal Experiments Local Ethics Committee (IMU-HADYEK) in March 2023 with protocol number 2023/1-2. There was no time limitation specified in the ethics committee approval. The interval between ethical approval and study execution was due to laboratory scheduling and the procurement and standardization of materials and equipment; the approved protocol remained unchanged and valid during the study period.

All procedures were performed in accordance with institutional guidelines for the care and use of laboratory animals, the ARRIVE guidelines, the Guide for the Care and Use of Laboratory Animals, and the American Veterinary Medical Association Guidelines for the Euthanasia of Animals.

A total of 35 male Wistar Albino rats, weighing between 300 and 500 grams, were randomly assigned to one of five groups:

• Control group (C) (n = 7): No surgical intervention; fed ad libitum;

• Pulmonary contusion group (PC-3) (n = 7): Contusion model, fed ad libitum and sacrificed after three days;

• Pulmonary contusion group (PC-10) (n = 7): Contusion model, fed ad libitum and sacrificed after 10 days;

• Pulmonary contusion + HCQ group (PCHCQ-3) (n=7): Contusion model: HCQ 100 mg/kg/day via gavage was administered starting on the day of contusion for three days; fed ad libitum and sacrificed after three days;

• Pulmonary contusion + HCQ group (PCHCQ-10) (n = 7): Contusion model: HCQ 100 mg/kg/day via gavage was administered starting on the day of contusion for 10 days; fed ad libitum and sacrificed after 10 days.

Induction of pulmonary contusion

Pulmonary contusion was induced using a modified drop-weight method. Anesthesia was achieved with an intraperitoneal injection of 50 mg/kg ketamine HCl (Ketalar®, Eczacıbaşı) and 15 mg/kg xylazine (Rompun^®, Bayer) to ensure spontaneous respiration during the procedure¹⁴. The contusion model and impact parameters were adapted from the isolated bilateral lung contusion model described by Raghavendran et al.¹⁵. A cylindrical aluminum weight (400 g) was dropped from a height of 60 cm through a vertical steel tube onto the right lateral chest wall of the anesthetized rats, delivering an impact energy of 2.35 J. At the end of the experimental period (day 3 or day 10), rats were euthanized by gradual carbon dioxide (CO₂) inhalation using a controlled displacement method. CO₂ was introduced into the euthanasia chamber at a fill rate of approximately 50% of the chamber volume per minute until respiratory arrest occurred. Gas flow was maintained for at least 2 minutes after cessation of breathing.

Death was confirmed by the absence of heartbeat and respiratory movements for at least 5 minutes, together with loss of pupillary reflex. All procedures were conducted in accordance with the American Veterinary Medical Association Guidelines for the Euthanasia of Animals and institutional regulations for laboratory animal care.

Histopathological and immunohistochemical analyses

Macroscopically, pulmonary contusion was observed in all contusion groups during tissue sampling after sacrifice (Fig. 1). The samples were fixed in 10% formaldehyde, embedded in paraffin, and sectioned for staining with hematoxylin-eosin (H&E) and Masson’s trichrome (MT). Alveolar edema, congestion, and leukocyte infiltration were examined in H&E-stained paraffin blocks (Fig. 2). The presence of alveolar edema and congestion was established by quantifying the percentage of positive findings in the paraffin blocks⁸,¹⁶. Histopathological evaluation for alveolar edema, congestion, leukocyte infiltration, and fibrosis was performed by a pathologist blinded to the study groups using scoring systems.

Figure 1
Macroscopic appearance of pulmonary contusion following experimental blunt thoracic trauma. The dashed line outlines the patchy contusion area in the right upper lobe observed during tissue sampling after the experiment.

Figure 2
Hematoxylin and eosin staining of lung tissue. (a) Control group showing normal alveolar architecture without significant congestion or inflammatory cell infiltration; (b) pulmonary contusion plus hydroxychloroquine day 10 group showing prominent vascular and alveolar congestion and neutrophil infiltration; (c) pulmonary contusion day 10 group showing vascular and alveolar congestion and leukocyte infiltration. Arrows indicate the relevant histopathological findings. Scale bar = 50 µm.

The levels of alveolar edema and congestion were scored from 0 to 3 as follows:

• 0: No pathology (0–5%) (maximum pathology ≤ 5%);

• 1: Mild (6–15%);

• 2: Moderate (16–20%);

• 3: Severe (21–25%).

Leukocyte infiltration was evaluated by dividing each section into 10 subregions at 40x magnification, with infiltration levels scored from 0 to 4 in each subregion as follows:

• 0: No extravascular leukocytes;

• 1: ≤ 10 leukocytes;

• 2: 11–45 leukocytes;

• 3: > 45 leukocytes.

Pulmonary fibrosis was analyzed in the paraffin blocks stained with MT (Fig. 3). Each section was divided into 10 subregions, and fibrosis was scored from 0 to 4 at 20x magnification as follows:

Figure 3
Masson’s trichrome staining of lung tissue. (a) Control group showing normal interalveolar septa without fibrosis; (b) pulmonary contusion day 10 group showing interalveolar septal thickening and fibrotic changes; (c) pulmonary contusion plus hydroxychloroquine day 10 group showing similar fibrotic changes. Arrows indicate areas of interalveolar septal thickening and fibrosis. Scale bar = 50 µm.

• 0: Normal lung structure;

• 1: Thickening of ≤ 50% of the interalveolar septa;

• 2: Thickening of > 50% of the interalveolar septa without fibrotic foci;

• 3: Thickening in the form of isolated fibrotic foci;

• 4: Multiple fibrotic foci with partial or complete disruption of the parenchymal structure.

For immunohistochemical staining, caspase 3 (Cell Signaling, Danvers, MA, United States of America, CST 9664T), an apoptosis marker; inducible nitric oxide synthase (iNOS) (Sigma, St. Louis, Missouri, United States of America, ZRB1449-25UL), an inflammation marker; and endothelial nitric oxide synthase (eNOS) (Sigma, St. Louis, Missouri, United States of America, ZRB1252-25UL), an endothelial function marker, were evaluated using semi-quantitative scoring. Caspase 3 and iNOS were scored from 0 to 4, while eNOS was scored from 0 to 3⁸,¹⁷.

Statistical analysis

Frequencies and percentages were used in the descriptive analysis of the data. The χ² test was used to analyze qualitative independent data, and Fisher’s exact test was used when the assumptions of the χ² test were not met. Statistical Package for the Social Sciences version 28.0 was used for statistical analysis. P < 0.05 were considered statistically significant.

Results

Histopathological findings

The histopathological and immunohistochemical results of all groups are shown in Table 1. No alveolar edema was observed in any group. Congestion scores were significantly higher in the contusion groups (PC-3, PC-10, PCHCQ-3, and PCHCQ-10) compared to the control group (p < 0.001) (Table 2). Moderate congestion (grade 2) was observed at 57.1% in both the PC-3 and PCHCQ-3 groups and at 100% in the PC-10 and PCHCQ-10 groups. Severe congestion (grade 3) was observed only in the PC-3 and PCHCQ-3 groups, at a rate of 42.9% in each. The number of leukocytes outside the vascular system was significantly higher (p < 0.05) in all experimental groups compared to the control group (Table 3). Assessment of the fibrosis distribution score revealed a disruption of the normal lung structure in the PC-10 and PCHCQ-10 groups compared with the other groups (p < 0.05) (Table 4).

Table 1
Histopathological and immunohistochemical findings.

		n	%
Alveolar edema
No pathology (0–5%)		35	100
Mild (6–15%)		0	0
Moderate (16–20%)
Severe (21–25%)
Congestion
No pathology		7	20
Moderate		22	62.9
Severe		6	17.1
Leukocyte infiltration
No extravascular leukocytes		5	14.3
≤ 10 leukocytes		8	22.9
10–45 leukocytes		13	37.1
> 45 leukocytes		9	25.7
Pulmonary fibrosis
Normal lung structure		28	80
Thickening of ≤ 50% of the interalveolar septa		7	20
Caspase 3
No expression		5	14.3
≤ % 10 cell expression		13	37.1
% 11–20 cell expression		9	25.7
% 21–40 cell expression		5	14.3
>% 40 cell expression		3	8.6
iNOS
No expression		5	14.3
≤ % 10 cell expression		1	2.9
% 11–20 cell expression		9	25.7
% 21–40 cell expression		20	57.1
eNOS
No expression		6	17.1
Mild expression		3	8.6
Moderate expression		11	31.4
Severe expression		15	42.9
Groups	PCHCQ-3	7	20
	PC-3	7	20
	PCHCQ-10	7	20
	PC-10	7	20
	C	7	20

• PCHCQ-3: pulmonary contusion + hydroxychloroquine group; PC-3: pulmonary contusion group; PCHCQ-10: pulmonary contusion + hydroxychloroquine group; PC-10: pulmonary contusion group; C: control group; iNOS: inducible nitric oxide synthase; eNOS: endothelial nitric oxide synthase.
• Source: Elaborated by the authors.

Table 2
Distribution of congestion among the study groups.

Congestion		PCHQ-3	PC-3	PCHQ-10	PC-10	C	p -value
No (0–5%)	n	0*	0*	0*	0*⁵	7	p < 0.05	χ2
	%	0	0	0	0	100
Moderate (16–20%)	n	4	4	7	7	0
	%	57.1	57.1	100	100	0
Severe (21–25%)	n	3	3	0	0	0
	%	42.9	42.9	0	0	0

• ^*
  Difference compared with the control group, p < 0.05; PCHCQ-3: pulmonary contusion + hydroxychloroquine group; PC-3: pulmonary contusion group; PCHCQ-10: pulmonary contusion + hydroxychloroquine group; PC-10: pulmonary contusion group; C: control group.
• Source: Elaborated by the authors.

Table 3
Distribution of leukocyte infiltration among the study groups.

Leukocyte infiltration		PCHQ-3	PC-3		PCHQ-10		PC-10	C	p -value
No extravascular leukocytes	n	0*	0*	0*	0*	0*	0*	5	p < 0.05	χ2
	%	0	0		0		0	71.4
< 10 leukocytes	n	0	0		0		6	2
	%	0	0		0		85.7	28.6
10–45 leukocytes	n	2	3		7		1	0
	%	28.6	42.9		100		14.3	0
> 45 leukocytes	n	5	4		0		0	0
	%	71.4	57.1		0		0	0

• ^*
  Difference compared with the control group, p < 0.05; PCHCQ-3: pulmonary contusion + hydroxychloroquine group; PC-3: pulmonary contusion group; PCHCQ-10: pulmonary contusion + hydroxychloroquine group; PC-10: pulmonary contusion group; C: control group.
• Source: Elaborated by the authors.

Table 4
Distribution of fibrosis score among the study groups.

Fibrosis score		PCHQ-3	PC-3	PCHQ-10	PC-10	C	p -value
Normal lung structure	n	7	7	4	3*,**,***	7	p < 0.05	χ2
	%	100	100	57.1	42.9	100
≤ 50% thickening of interalveolar septa	n	0	0	3	4	0
	%	0	0	42.9	57.1	0

• PCHCQ-3: pulmonary contusion + hydroxychloroquine group; PC-3: pulmonary contusion group; PCHCQ-10: pulmonary contusion + hydroxychloroquine group; PC-10: pulmonary contusion group; C: control group;
• ^*
  difference with PCHQ-3 p < 0.05;
• ^**
  difference with PC-3 p < 0.05;
• ^***
  difference with C p < 0.05.
• Source: Elaborated by the authors.

Immunohistochemical findings

The caspase 3 staining was found to be significantly higher in all experimental groups compared to the control group (p < 0.05). Caspase 3 expression above 40% was seen exclusively in the PCHCQ-10 group at a rate of 42.9% (Fig. 4). The PCHCQ-10 group demonstrated moderate apoptosis levels, which were not significantly different from those in the non-treated PC-10 group (Table 5).

Figure 4
Caspase-3 immunohistochemical staining of lung tissue. (a) Control group showing minimal caspase-3 expression; (b) pulmonary contusion day 10 group showing increased caspase-3-positive cells; (c) pulmonary contusion plus hydroxychloroquine day 10 group showing persistent caspase-3 expression. Arrows indicate caspase-3-positive cells. Scale bar = 50 µm.

Table 5
Distribution of caspase 3 staining among the study groups.

Caspase 3		PCHQ-3	PC-3	PCHQ-10	PC-10	C	p -value
No expression	n	0*	0*	0*	0⁵	5	p < 0.05	χ2
	%	0	0	0	0	71.4
≤ 10% expression	n	5	3	0	3	2
	%	71.4	42.9	0	42.9	28.6
11–20% expression	n	2	0	4	3	0
	%	28.6	0	57.1	42.9	0
21–40% expression	n	0	4	0	1	0
	%	0	57.1	0	14.3	0
> 40% expression	n	0	0	3	0	0
	%	0	0	42.9	0	0

• PCHCQ-3: pulmonary contusion + hydroxychloroquine group; PC-3: pulmonary contusion group; PCHCQ-10: pulmonary contusion + hydroxychloroquine group; PC-10: pulmonary contusion group; C: control group;
• ^*
  difference with C p < 0.05.
• Source: Elaborated by the authors.

aInducible nitric oxide synthase was expressed at significantly higher levels in all contusion groups compared with the control group (Fig. 5). Severe iNOS expressions were present in 100% of the PCHCQ-3 group, whereas it was 42.9% in the PC-3 group. It was observed at a rate of 85.7% in the PC-10 group. There was no statistically significant difference in iNOS expression among the contusion groups (Table 6). Endothelial nitric oxide synthase expression scores were high in all groups (Fig. 6).

Figure 5
Inducible nitric oxide synthase immunohistochemical staining of lung tissue. (a) Control group showing minimal inducible nitric oxide synthase expression; (b) pulmonary contusion day 10 group showing increased inducible nitric oxide synthase expression; (c) pulmonary contusion plus hydroxychloroquine day 10 group showing persistente inducible nitric oxide synthase expression. Arrows indicate positively stained cells. Scale bar = 50 µm.

Figure 6
Endothelial nitric oxide synthase immunohistochemical staining of lung tissue. (a) Control group showing endothelial nitric oxide synthase expression; (b) pulmonary contusion day 10 group showing a staining pattern similar to that of the control group; (c) pulmonary contusion plus hydroxychloroquine day 10 group showing comparable endotelial nitric oxide synthase expression. Arrows indicate positively stained endothelial structures. Scale bar = 50 µm.

Table 6
Inducible nitric oxide synthase (iNOS) scores among the study groups.

iNOS		PCHQ-3	PC-3	PCHQ-10	PC-10	C	p -value
No expression	n	0*	0*	0*	0*	5	p < 0.05	χ2
	%	0	0	0	0	71.4
Mild expression	n	4	0	0	0	1
	%	0	0	0	0	14.3
Moderate expression	n	0	4	3	1	1
	%	0	57.1	42.9	14.3	14.3
Severe expression	n	7	3	4	6	0
	%	100	42.9	57.1	85.7	0

• PCHCQ-3: pulmonary contusion + hydroxychloroquine group; PC-3: pulmonary contusion group; PCHCQ-10: pulmonary contusion + hydroxychloroquine group; PC-10: pulmonary contusion group; C: control group;
• ^*
  difference with C p < 0.05.
• Source: Elaborated by the authors.

*

Discussion

The pathophysiological mechanisms underlying pulmonary contusion include disruption of the alveolar-capillary barrier, excessive inflammatory response, and subsequent fibrotic development²,¹⁷-¹⁹. Understanding these mechanisms is crucial for developing effective therapeutic strategies.

This study investigated the effects of HCQ in an experimental rat model of pulmonary contusion. The contusion model was successfully established, as evidenced by increased congestion and leukocyte infiltration in the contusion groups compared with the control group and by disruption of lung architecture and fibrosis at day 10. However, HCQ treatment did not produce statistically significant improvements in leukocyte infiltration, fibrosis distribution, iNOS expression, or apoptosis compared with the untreated contusion groups at the evaluated time points.

In the present study, short-term HCQ administration (three days) did not reduce leukocyte infiltration compared with the untreated pulmonary contusion groups. A numerical reduction in leukocyte infiltration was observed in the long-term HCQ treatment group (10 days), but this difference did not reach statistical significance. Therefore, an anti-inflammatory effect of HCQ could not be demonstrated under the experimental conditions of this study.

Similarly, there was no significant reduction in fibrosis scores between the HCQ-treated and untreated groups at day 10, suggesting a limited antifibrotic effect of HCQ in this model. Although HCQ has anti-inflammatory and immunomodulatory properties, its limited efficacy in pulmonary contusion may be due to the complex, multifactorial pathophysiology of the injury. The extent of mechanical tissue damage, the rapid onset of inflammation, and the presence of redundant inflammatory pathways may diminish the impact of a single-agent intervention. Furthermore, HCQ alone may not be sufficiently potent to counteract the acute and severe inflammatory response triggered by blunt chest trauma.

The HCQ dose (100 mg/kg/day) used in this study was selected based on prior preclinical studies evaluating its anti-inflammatory and immunomodulatory effects in rodent models of various inflammatory and fibrotic diseases²⁰,²¹. Although no previous studies have investigated HCQ in the context of pulmonary contusion, doses ranging from 50 to 200 mg/kg/day have been reported to be effective and well-tolerated in similar models. Given this evidence, 100 mg/kg/day was chosen to balance efficacy and safety. Future research should include multiple dose groups to evaluate dose–response relationships and determine the optimal therapeutic range for HCQ in pulmonary contusion.

Caspase 3 is a critical effector enzyme in apoptosis and is commonly used as a marker of programmed cell death. While HCQ has been reported to modulate apoptosis in other experimental models, we did not detect a significant effect of HCQ on caspase 3 staining in this pulmonary contusion model³,²². The observed numerical differences between early and late time points should therefore be interpreted cautiously; without additional measures of regeneration or proliferation, these patterns cannot be attributed to a reparative response. Future studies should incorporate complementary assays to better characterize the balance between apoptosis and regeneration after contusion.

Inducible nitric oxide synthase is an enzyme that produces large amounts of nitric oxide (NO) during inflammatory responses²³. Elevated iNOS expression is associated with increased oxidative stress and tissue damage in inflammatory conditions²⁴-²⁷. In our study, we found that iNOS levels were significantly elevated in all contusion groups compared to the control group, indicating an ongoing inflammatory response. However, there was no significant reduction in iNOS expression following HCQ treatment. Thus, the effects of HCQ on iNOS-mediated oxidative stress and inflammation appeared to be limited in our model.

Endothelial nitric oxide synthase is involved in the regulation of vascular tone and endothelial function. Unlike iNOS, eNOS produces low levels of NO that are essential for maintaining vascular homeostasis²⁸. The present study demonstrated that eNOS expression was elevated in all experimental groups, including the control group. This observation may be related to the potential effects of the sacrifice procedure. Even though eNOS staining intensity did not change markedly in the untreated groups, the treated groups showed a decrease in staining intensity over time. However, this difference was not statistically significant. These results are consistent with previous studies demonstrating that HCQ has a neutral or protective effect on endothelial cells under certain conditions²⁹.

Hydroxychloroquine has theoretical anti-inflammatory and immunomodulatory properties that could be relevant to lung injury. However, given the lack of significant improvements in our histopathological and immunohistochemical outcomes, any potential clinical benefit of HCQ in pulmonary contusion remains uncertain. Future studies should explore alternative dosing strategies, longer follow-up periods, and combination approaches (e.g., with established anti-inflammatory or antifibrotic agents) to target better both the acute injury phase and its chronic sequelae¹⁵,²⁵-²⁷.

Several limitations must be considered when interpreting the results of this study. First, oxidative stress was not assessed using biochemical markers (e.g., malondialdehyde or superoxide dismutase), which might have provided a more comprehensive understanding of the injury mechanism and treatment response. Second, functional respiratory parameters (e.g., oxygen saturation, arterial blood gases, or lung mechanics) were not measured to correlate histological findings with physiologic impairment; serial functional monitoring would have required additional instrumentation and repeated anesthesia and handling, which could introduce confounding effects and were therefore outside the scope of the approved protocol. Third, the absence of a comparator (e.g., steroid-treated) group limits comparison with established anti-inflammatory therapies²⁹-³². Future studies should incorporate biochemical oxidative stress markers, functional respiratory outcomes, and appropriate comparator groups to provide a more comprehensive evaluation.

Moreover, the study duration was relatively short for evaluating longer-term fibrosis and tissue remodeling. Longer observation periods, additional time points, and integration of functional respiratory assessments are necessary to fully characterize the chronic effects of HCQ on lung injury and recovery after contusion.

Conclusion

In this rat model of pulmonary contusion, HCQ administered at 100 mg/kg/day for three and 10 days did not demonstrate significant anti-inflammatory, anti-apoptotic, or antifibrotic effects based on the evaluated histopathological and immunohistochemical outcomes at days 3 and 10. Further studies incorporating functional respiratory parameters, additional biochemical oxidative stress markers, longer follow-up periods, alternative dosing strategies, and combination regimens are needed.

Acknowledgements

The authors would like to thank the staff of the Animal Research Laboratory of Istanbul Medeniyet University for their technical assistance during the experimental procedures. The authors also acknowledge the Department of Pathology for their support in the histopathological and immunohistochemical analyses.

• Research performed at the Animal Research Laboratory, Istanbul Medeniyet University, Istanbul, Turkey.
• Funding
  Scientific Research Projects Coordination Unit of Istanbul Medeniyet University
  Grant No.: 2024-GAP-Tıp-0002
• Declaration of use of artificial intelligence tools
  During the preparation of this work, the authors used artificial intelligence tools solely for language editing and grammar improvement. The authors reviewed and edited the content as needed and take full responsibility for the content of the publication.

Data availability statement

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

References

• ¹ Haxhija EQ, Nöres H, Schober P, Höllwarth ME. Lung contusion-lacerations after blunt thoracic trauma in children. Pediatr Surg Int. 2004;20(6):412–4. https://doi.org/10.1007/s00383-004-1165-z
  » https://doi.org/10.1007/s00383-004-1165-z
• ² Hamrick MC, Duhn RD, Carney DE, Boswell WC, Ochsner MG. Pulmonary contusion in the pediatric population. Am Surg. 2010;76(7):721–4. https://doi.org/10.1177/000313481007600727
  » https://doi.org/10.1177/000313481007600727
• ³ Dewar DC, Mackay P, Balogh Z. Epidemiology of post-injury multiple organ failure in an Australian trauma system. ANZ J Surg. 2009;79(6):431–6. https://doi.org/10.1111/j.1445-2197.2009.04968.x
  » https://doi.org/10.1111/j.1445-2197.2009.04968.x
• ⁴ Raghavendran K, Notter RH, Davidson BA, Helinski JD, Kunkel SL, Knight PR. Lung contusion: inflammatory mechanisms and interaction with other injuries. Shock. 2009;32(2):122–30. https://doi.org/10.1097/SHK.0b013e31819c385c
  » https://doi.org/10.1097/SHK.0b013e31819c385c
• ⁵ Cohn SM, Dubose JJ. Pulmonary contusion: an update on recent advances in clinical management. World J Surg. 2010;34(8):1959–70. https://doi.org/10.1007/s00268-010-0599-9
  » https://doi.org/10.1007/s00268-010-0599-9
• ⁶ Raghavendran K, Davidson BA, Woytash JA, Helinski JD, Marschke CJ, Manderscheid PA, Notter RH, Knight PR. The evolution of isolated bilateral lung contusion from blunt chest trauma in rats: cellular and cytokine responses. Shock. 2005;24(2):132–8. https://doi.org/10.1097/01.shk.0000169725.80068.4a
  » https://doi.org/10.1097/01.shk.0000169725.80068.4a
• ⁷ Rendeki S, Molnár TF. Pulmonary contusion. J Thorac Dis. 2019;11(Suppl.2):S141–51. https://doi.org/10.21037/jtd.2018.11.53
  » https://doi.org/10.21037/jtd.2018.11.53
• ⁸ Gercel G, Aksu B, Ozkanli S, Uzun H, Aksu F, Ozatman E, Durakbaşa ÇU. Investigation of Bosentan’s effects on pulmonary contusion created by blunt thoracic trauma in rats. Eur J Pediatr Surg. 2020;30(1):71–8. https://doi.org/10.1055/s-0039-1697908
  » https://doi.org/10.1055/s-0039-1697908
• ⁹ Ben-Zvi I, Kivity S, Langevitz P, Shoenfeld Y. Hydroxychloroquine: from malaria to autoimmunity. Clin Rev Allergy Immunol. 2012;42(2):145–53. https://doi.org/10.1007/s12016-010-8243-x
  » https://doi.org/10.1007/s12016-010-8243-x
• ¹⁰ Griese M, Kappler M, Stehling F, Schulze J, Baden W, Koerner-Rettberg C, Carlens J, Prenzel F, Nährlich L, Thalmeier A, Sebah D, Kronfeld K, Rock H, Ruckes C, HCQ-study group, Wetzke M, Seidl E, Schwerk N. Randomized controlled phase 2 trial of hydroxychloroquine in childhood interstitial lung disease. Orphanet J Rare Dis. 2022;17(1):289. https://doi.org/10.1186/s13023-022-02399-2
  » https://doi.org/10.1186/s13023-022-02399-2
• ¹¹ Gies V, Bekaddour N, Dieudonné Y, Guffroy A, Frenger Q, Gros F, Rodero MP, Herbeuval JP, Korganow AS. beyond anti-viral effects of chloroquine/hydroxychloroquine. Front Immunol. 2020;11:1409. https://doi.org/10.3389/fimmu.2020.01409
  » https://doi.org/10.3389/fimmu.2020.01409
• ¹² Gautret P, Lagier JC, Parola P, Hoang VT, Meddeb L, Mailhe M, Doudier B, Courjon J, Giordanengo V, Vieira VE, Dupont HT, Honoré S, Colson P, Chabrière E, La Scola B, Rolain JM, Brouqui P, Raoult D. Retraction notice to “Hydroxychloroquine and azithromycin as a treatment of COVID-19: results of an open-label non-randomized clinical trial” [International Journal of Antimicrobial Agents 56 (2020), 105949]. Int J Antimicrob Agents. 2025;65(1):107416. https://doi.org/10.1016/j.ijantimicag.2024.107416
  » https://doi.org/10.1016/j.ijantimicag.2024.107416
• ¹³ Rosenberg ES, Dufort EM, Udo T, Wilberschied LA, Kumar J, Tesoriero J, Weinberg P, Kirkwood J, Muse A, DeHovitz J, Blog DS, Hutton B, Holtgrave DR, Zucker HA. Association of treatment with hydroxychloroquine or azithromycin with in-hospital mortality in patients with COVID-19 in New York State. JAMA. 2020;323(24):2493–502. https://doi.org/10.1001/jama.2020.8630
  » https://doi.org/10.1001/jama.2020.8630
• ¹⁴ Irwin MR, Curay CM, Choi S, Kiyatkin EA. Basic physiological effects of ketamine-xylazine mixture as a general anesthetic preparation for rodent surgeries. Brain Res. 2023;1804:148251. https://doi.org/10.1016/j.brainres.2023.148251
  » https://doi.org/10.1016/j.brainres.2023.148251
• ¹⁵ Raghavendran K, Davidson BA, Helinski JD, Marschke CJ, Manderscheid P, Woytash JA, Notter RH, Knight PR. A rat model for isolated bilateral lung contusion from blunt chest trauma. Anesth Analg. 2005;101(5):1482–9. https://doi.org/10.1213/01.ANE.0000180201.25746.1F
  » https://doi.org/10.1213/01.ANE.0000180201.25746.1F
• ¹⁶ Guzel A, Basaran UN, Aksu B, Kanter M, Yalcin O, Aktas C, Guzel A, Karasalihoglu S. Protective effects of S-methylisothiourea sulfate on different aspiration materials-induced lung injury in rats. Int J Pediatr Otorhinolaryngol. 2008;72(8):1241–50. https://doi.org/10.1016/j.ijporl.2008.05.001
  » https://doi.org/10.1016/j.ijporl.2008.05.001
• ¹⁷ Simon B, Ebert J, Bokhari F, Capella J, Emhoff T, Hayward T 3rd, Rodriguez A, Smith L. Management of pulmonary contusion and flail chest: an Eastern Association for the Surgery of Trauma practice management guideline. J Trauma Acute Care Surg. 2012;73(5):S351–61. https://doi.org/10.1097/TA.0b013e31827019fd
  » https://doi.org/10.1097/TA.0b013e31827019fd
• ¹⁸ Kaminski A, Pohl CB, Sponholz C, Ma N, Stamm C, Vollmar B, Steinhoff G. Up-regulation of endothelial nitric oxide synthase inhibits pulmonary leukocyte migration following lung ischemia-reperfusion in mice. Am J Pathol. 2004;164(6):2241–9. https://doi.org/10.1016/S0002-9440(10)63780-7
  » https://doi.org/10.1016/S0002-9440(10)63780-7
• ¹⁹ Cohn SM, Zieg PM. Experimental pulmonary contusion: review of the literature and description of a new porcine model. J Trauma. 1996;41(3):565–71. https://doi.org/10.1097/00005373-199609000-00036
  » https://doi.org/10.1097/00005373-199609000-00036
• ²⁰ Plantone D, Koudriavtseva T. Current and future use of chloroquine and hydroxychloroquine in infectious, immune, neoplastic, and neurological diseases: a mini-review. Clin Drug Investig. 2018;38(8):653–71. https://doi.org/10.1007/s40261-018-0656-y
  » https://doi.org/10.1007/s40261-018-0656-y
• ²¹ Kolli AR, Semren TZ, Bovard D, Majeed S, van der Toorn M, Scheuner S, Guy PA, Kuczaj A, Mazurov A, Frentzel S, Calvino-Martin F, Ivanov NV, O’Mullane J, Peitsch MC, Hoeng J. Pulmonary delivery of aerosolized chloroquine and hydroxychloroquine to COVID-19: ın vitro experimentation to human dosing predictions. AAPS J. 2022;24(1):33. https://doi.org/10.1208/s12248-021-00666-x
  » https://doi.org/10.1208/s12248-021-00666-x
• ²² Gregório P, Cunha RS, Biagini G, Bosquetti B, Budag J, Ortiz A, Sánchez-Niño MD, Barreto FC, Stinghen AEM. Chloroquine may induce endothelial injury through lysosomal dysfunction and oxidative stress. Toxicol Appl Pharmacol. 2021;414:115412. https://doi.org/10.1016/j.taap.2021.115412.
  » https://doi.org/10.1016/j.taap.2021.115412
• ²³ Gallorini M, Rapino M, Schweikl H, Cataldi A, Amoroso R, Maccallini C. Selective ınhibitors of the ınducible nitric oxide synthase as modulators of cell responses in LPS-stimulated human monocytes. Molecules. 2021;26(15):4419. https://doi.org/10.3390/molecules26154419
  » https://doi.org/10.3390/molecules26154419
• ²⁴ Liu L, Ren J, He Z, Men K, Mao Y, Ye T, Chen H, Li L, Xu B, Wei Y, Wei X. Cholesterol-modified hydroxychloroquine-loaded nanocarriers in bleomycin-induced pulmonary fibrosis. Sci Rep. 2017;7(1):10737. https://doi.org/10.1038/s41598-017-11450-3
  » https://doi.org/10.1038/s41598-017-11450-3
• ²⁵ Savin IA, Zenkova MA, Sen’kova AV. Pulmonary fibrosis as a result of acute lung inflammation: molecular mechanisms, relevant in vivo models, prognostic and therapeutic approaches. Int J Mol Sci. 2022;23(23):14959. https://doi.org/10.3390/ijms232314959
  » https://doi.org/10.3390/ijms232314959
• ²⁶ Bush A, Cunningham S, de Blic J, Barbato A, Clement A, Epaud R, Hengst M, Kiper N, Nicholson AG, Wetzke M, Snijders D, Schwerk N, Griese M. European protocols for the diagnosis and initial treatment of interstitial lung disease in children. Thorax. 2015;70(11):1078–84. https://doi.org/10.1136/thoraxjnl-2015-207349
  » https://doi.org/10.1136/thoraxjnl-2015-207349
• ²⁷ Akca T, Canbaz H, Tataroglu C, Caglikulekci M, Tamer L, Colak T, Kanik A, Bilgin O, Aydin S. The effect of N-acetylcysteine on pulmonary lipid peroxidation and tissue damage. J Surg Res. 2005;129(1):38–45. https://doi.org/10.1016/j.jss.2005.05.026
  » https://doi.org/10.1016/j.jss.2005.05.026
• ²⁸ Fulton D, Church JE, Ruan L, Li C, Sood SG, Kemp BE, Jennings IG, Venema RC. Src kinase activates endothelial nitric-oxide synthase by phosphorylating Tyr-83. J Biol Chem. 2005;280(43):35943–52. https://doi.org/10.1074/jbc.M504606200
  » https://doi.org/10.1074/jbc.M504606200
• ²⁹ Förstermann U, Sessa WC. Nitric oxide synthases: regulation and function. Eur Heart J. 2012;33(7):829–37. https://doi.org/10.1093/eurheartj/ehr304
  » https://doi.org/10.1093/eurheartj/ehr304
• ³⁰ Mokra D, Mikolka P, Kosutova P, Mokry J. Corticosteroids in acute lung injury: the dilemma continues. Int J Mol Sci. 2019;20(19):4765. https://doi.org/10.3390/ijms20194765
  » https://doi.org/10.3390/ijms20194765
• ³¹ Rocco PR, Souza AB, Faffe DS, Pássaro CP, Santos FB, Negri EM, Lima JG, Contador RS, Capelozzi VL, Zin WA. Effect of corticosteroid on lung parenchyma remodeling at an early phase of acute lung injury. Am J Respir Crit Care Med. 2003;168(6):677–84. https://doi.org/10.1164/rccm.200302-256OC
  » https://doi.org/10.1164/rccm.200302-256OC
• ³² Lee M, Rey K, Besler K, Wang C, Choy J. Immunobiology of nitric oxide and regulation of inducible nitric oxide synthase. In: Kloc M, editor. Macrophages: origin, functions and biointervention (Results and Problems in Cell Differentiation, vol 62). Cham: Springer; 2017. p. 181–207. https://doi.org/10.1007/978-3-319-54090-0_8
  » https://doi.org/10.1007/978-3-319-54090-0_8

Edited by

• Section editor:
  Norbert Nemeth https://orcid.org/0000-0002-1162-3778

Publication Dates

• Publication in this collection
  14 Aug 2026
• Date of issue
  2026

History

• Received
  04 May 2026
• Accepted
  09 July 2026