Pathophysiological and Clinical Significance of Crotalus durissus cascavella Venom-Induced Pulmonary Impairment in a Murine Model

Crotalus venom has broad biological activity, including neurotoxic, myotoxic, hematologic, and cytotoxic compounds that induce severe systemic repercussions. We evaluated the pathophysiological and clinical significance of Crotalus durissus cascavella (Cdc) venom-induced pulmonary impairment in mice. We conducted a randomized experimental study, involving 72 animals intraperitoneally inoculated with saline solution in the control group (CG), as well as venom in the experimental group (EG). The animals were euthanized at predetermined intervals (1 h, 3 h, 6 h, 12 h, 24 h, and 48 h), and lung fragments were collected for H&E and Masson histological analysis. The CG did not present inflammatory alterations in pulmonary parenchyma. In the EG, interstitial and alveolar swelling, necrosis, septal losses followed by alveolar distensions, and areas of atelectasis in the pulmonary parenchyma were observed after three hours. The EG morphometric analysis presented pulmonary inflammatory infiltrates at all time intervals, being more significant at three and six (p = 0.035) and six and 12 h (p = 0.006). The necrosis zones were significant at intervals of one and 24 h (p = 0.001), one and 48 h (p = 0.001), and three and 48 h (p = 0.035). Crotalus durissus cascavella venom induces a diffuse, heterogeneous, and acute inflammatory injury in the pulmonary parenchyma, with potential clinical implications for respiratory mechanics and gas exchange. The early recognition and prompt treatment of this condition are essential to prevent further lung injury and to improve outcomes.

After three hours, we observed intense polymorphonuclear inflammatory infiltrates (two-headed arrow), congested veins (keys), and swelling (circle). (E,F): after three hours, the presence of intense inflammatory infiltrates (arrow) with perivascular polymorphonuclear cells, septal loss (two-headed arrow), congested veins (key), interstitial swelling (circle), and atelectasis (asterisk) was observed. (HE, 20× objective). Table 1. Semi-quantitative analysis of the action of Crotalus durissus cascavella venom on the pulmonary parenchyma at different time exposures in Swiss mice. Semi-quantitative representation of lesion intensity and inflammatory infiltrates (mild (+), moderate (++), and intense (+++)). Exposure time in the pulmonary parenchyma. After three hours, we observed intense polymorphonuclear inflammatory infiltrates in the peribronchial and perivascular regions and extensive breakdown of the pulmonary architecture associated with small areas of necrosis and hyperplasia in the bronchialassociated lymphoid tissue-(BALT). Moderate septal rupture and alveolar swelling, interstitial swelling, congested veins, ciliary alteration in the respiratory epithelium, hemorrhagic spots, and atelectasis were also observed (Table 1; Figure 1E,F).

Exposure
At six hours, there was a decrease in peribronchial and perivascular inflammatory infiltrates. However, there was a significant increase in vascular congestion, necrosis, septal ruptures and loss, alveolar distension, and hemorrhagic spots (Table 1; Figure 2A,B). After 12 h, an increase in inflammatory activity and intense diffuse distributed areas of coagulopathy, vascular congestion, extensive necrosis, septal thickening, and rupture, as well as alveolar and bronchial smooth muscle distension, was observed (Table 1; Figure 2C,D).
At 24 h, a diffuse and peribronchial inflammatory process persisted with emphysematous areas, vasoconstriction, intense necrosis zones, septal wall thickening and rupture, bronchial epithelial hyperplasia, and bronchial and vascular muscle distension (Table 1; Figure 2E,F). At 48 h, the inflammatory infiltrates remained with areas of accentuated necrosis, vascular congestion, and the appearance of atelectasis (Table 1; Figure 3A,B).
Masson's trichrome staining revealed matrix alterations with multifocal collagen deposits in the pulmonary parenchyma, which are more intense in the perivascular and bronchial regions ( Figure 3C,D). Significant fibroblastic infiltration was noted after six hours. Morphometric analysis of the inflammatory kinetic in the pulmonary parenchyma of the control group did not show significant alterations.
At six hours, there was a decrease in peribronchial and perivascular inflammatory infiltrates. However, there was a significant increase in vascular congestion, necrosis, septal ruptures and loss, alveolar distension, and hemorrhagic spots (Table 1; Figure 2A,B). After 12 h, an increase in inflammatory activity and intense diffuse distributed areas of coagulopathy, vascular congestion, extensive necrosis, septal thickening, and rupture, as well as alveolar and bronchial smooth muscle distension, was observed (Table 1; Figure  2C,D).  At 24 h, a diffuse and peribronchial inflammatory process persisted with emphysematous areas, vasoconstriction, intense necrosis zones, septal wall thickening and rupture, bronchial epithelial hyperplasia, and bronchial and vascular muscle distension (Table 1; Figure 2E,F). At 48 h, the inflammatory infiltrates remained with areas of accentuated necrosis, vascular congestion, and the appearance of atelectasis (Table 1; Figure 3A,B). Masson's trichrome staining revealed matrix alterations with multifocal collagen deposits in the pulmonary parenchyma, which are more intense in the perivascular and bronchial regions ( Figure 3C,D). Significant fibroblastic infiltration was noted after six hours. Morphometric analysis of the inflammatory kinetic in the pulmonary parenchyma of the control group did not show significant alterations.

Morphometric Analysis of Lung Parenchyma
The morphometric analysis of lung parenchyma in the experimental groups identified polymorphonuclear diffuse and focal inflammatory infiltrates, specifically in the bronchial and perivascular topography. We noticed significant differences in inflammatory changes at three and six hours (p = 0.035) and six and 12 h (p = 0.006) ( Figure 4A). The intensity of the inflammatory response was higher at 12 h. The morphometric quantification of pulmonary parenchyma necrosis areas indicated extensive tissue destruction, being statistically significant at the times of one and 24 h (p = 0.001), one and 48 h (p = 0.001), and three and 48 h (p = 0.035) ( Figure 4B). The other times evaluated did not demonstrate any statistical significance.
bronchial and perivascular topography. We noticed significant differences in inflammatory changes at three and six hours (p = 0.035) and six and 12 h (p = 0.006) ( Figure 4A). The intensity of the inflammatory response was higher at 12 h. The morphometric quantification of pulmonary parenchyma necrosis areas indicated extensive tissue destruction, being statistically significant at the times of one and 24 h (p = 0.001), one and 48 h (p = 0.001), and three and 48 h (p = 0.035) ( Figure 4B). The other times evaluated did not demonstrate any statistical significance.  Alveolar distensions and ruptures in diverse areas of the pulmonary parenchyma were quantified and compared throughout the inflammatory process and from the moment of exposure to the venom, with significant differences being observed between all experimental groups and the control group. There was a significant difference between the control and experimental group in relation to the times one and 12 h (p = 0.027), one and 24 h (p = 0.001), and three and 24 h (p = 0.027) ( Figure 4C). The quantification of collagen deposits at six, 12, 24, and 48 h did not demonstrate any statistical significance ( Figure 4D).

Discussion
Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), induced by Crotalus durissus snake venom, are severe medical conditions that require prompt and appropriate clinical management. Treating these conditions involves supportive care, mechanical ventilation, and antivenom therapy. Few studies have examined pulmonary involvement in snakebite accidents [4,7,19], and the pathogenesis of respiratory failure by Crotalus durissus cascavella venom has not been fully elucidated. However, respiratory failure is frequently associated with ALI, mechanical distress, and respiratory muscle paralysis [20]. In snake accidents, involving Crotalus genus, this manifestation has been reported most frequently in the subspecies Crotalus durissus [6,21,22]. This study showed patterns of lung injury induced by Crotalus durissus cascavella venom with extensive necroinflammatory changes in the entire pulmonary parenchyma, including bronchi and bronchial damage, interstitial and alveolar swelling, matrix alterations, vascular congestion, coagulopathy with vascular rupture, and diffuse alveolar hemorrhage. These findings are compatible with the pathophysiologic subtract for ALI and the clinical expression of ARDS.
In crotalic accidents, the crotoxin can compromise neuromuscular function, blocking the transmission of neurotransmitters and impacting diaphragm muscle function, which may result, clinically, in muscle paralysis and respiratory failure [4,23,24]. Other toxins, such as lectins [25,26] and serine proteases [27], as well as hypotensive components [28,29], can act synergetically to potentialize diffuse coagulation in the small and larger veins, as observed in the present study. These coagulopathies can also increase thromboplastin [17,30] and induce progressive ischemic lesions, necrosis, and inflammatory changes. This phenomenon was observed and quantified at all time intervals. In addition, it is necessary to consider the extracellular effect of cytokines and signal leukocytes, consisting of plateletactivating inductor molecules and inflammatory mediators [31,32].
Early epithelial hyperplasia, as well as bronchial obliteration, atelectasis, vasoconstriction, and swelling, suggest significant impairment of respiratory mechanics and pulmonary structural damage induced by Crotalus durissus venom [6,23]. We observed septal ruptures, as well as alveolar distensions, capillary, and bronchial obliteration, at 24 and 48 h after venom exposure. Our data suggest a loss of integrity of the alveolar-capillary membrane, accentuating tissue acidity and necrosis, which may lead to respiratory failure, hemorrhage, and pulmonary infarction. These findings were confirmed by morphometric analysis.
Pulmonary emphysema is characterized by alveolar elastolysis, generally induced by extensive and significant alterations in the distal structure of the terminal bronchiole, which provokes dilation of the airways or destruction of the alveolar wall, resulting in loss of respiratory surface and a reduction in elastic tissue [33]. The emphysematous areas increase lung compliance, respiratory work, and anteroposterior thoracic diameter of the mice. In addition, the resultant ventilation-perfusion disturbance is directly related to the magnitude of the pulmonary inflammatory damage and subsequent fibroblast proliferation, increase in collagen synthesis, and critical alterations in the matrix of the emphysema pathogenesis [34,35]. Furthermore, the activity of inflammatory proteases secreted by leukocytes and neutrophilic elastase contributes to the destruction of elastic fibers and the inhibition of alpha1-antitrypsin (AAT). Under physiological conditions, type II pneumocytes synthesize AAT in sufficient concentrations to prevent alveolar elastolysis [36,37].
The clinical expression of histopathological lesions was detected early at three hours, including high respiratory rates and signs of respiratory distress. Progressive atelectasis after three hours is especially worrying, as ventilation-perfusion mismatch generates pulmonary shunt and impairment in respiratory mechanics [38,39].
Distal matrix alterations in the small airways and alveoli at six, 12, 24, and 48 h indicate inflammatory damage in several areas of the pulmonary parenchyma. These early matrix alterations suggest an elastance loss, usually seen in late and chronic respiratory failure [36], alerting the need for rapid clinical interventions in patients bitten by Crotalus durissus to avoid extensive and irreversible pulmonary damage. Similar findings were reported for bronchial muscle distension [21].
The progression of inflammatory infiltrates and bronchus-associated lymphoid tissue (BALT) hyperreactivity can be explained by systemic toxin injury. An exacerbated response of liberating inflammatory mediators and cytokines, principally polymorphonuclears, occurs in response to leukocyte recruitment and tissue damage [21,40,41].
The increase in pulmonary inflammatory injury, mostly related to neutrophilic proliferation, especially at one, three, 24, and 48 h, is partly explained by the release of macrophages chemotactic factors, epithelium disruption, and proinflammatory effects of the venom related to crotoxin component phospholipase [42,43]. However, inflammatory modulation with decreased neutrophilic infiltrates at six and 12 h might be related to transient early neutrophil senescence and apoptosis induced by extensive free radicals' production due to intense phagocyte activity [44][45][46][47][48].
Supportive care is the cornerstone of the treatment of ALI induced by Crotalus durissus venom. Clinical interventions include oxygen therapy, fluid management, and correcting electrolyte disturbances and coagulopathy [49]. Supplemental oxygen therapy is essential to ensure adequate arterial oxygen content in hypoxemic patients. Our data showed early extensive necroinflammatory changes in lung parenchyma and clinical signs of respiratory distress, suggesting a potential risk of respiratory failure in the first hours after venom inoculation (Supplementary Material). Since we observed intense inflammatory infiltrates in the perivascular regions, vascular congestion, and interstitial swelling, fluid management might be critical to prevent fluid overload and pulmonary edema, which can worsen lung injury.
We showed an early extensive breakdown of the pulmonary architecture associated with atelectasis. Lung ultrasound and computerized tomography of the thorax (Chest CT) are accurate diagnostic tools for assessing the severity of lung injury. Non-invasive ventilation (NIV) could reduce respiratory muscle workload and prevent atelectrauma in high-risk patients with ALI. Mechanical ventilation is often required in patients with severe ALI induced by Crotalus durissus venom. The goal of mechanical ventilation is to provide adequate oxygenation and ventilation while minimizing further lung injury. The use of positive end-expiratory pressure (PEEP) might prevent alveolar collapse and maintain adequate lung recruitment [48].
Other therapies may be used to manage ALI induced by Crotalus durissus venom. The morphometric analysis of lung parenchyma identified extensive polymorphonuclear inflammatory infiltrates, specifically in the bronchial and perivascular topography. Corticosteroids have been shown to reduce inflammation and improve oxygenation in some patients with ARDS, which may improve airway resistance and ventilation. However, the use of these therapies is controversial, and their efficacy and safety in the management of ALI induced by Crotalus durissus venom are unclear [49]. Preclinical findings of the efficacy of varespladib, a secreted phospholipase A2 inhibitor, was found to effectively inhibit the neurotoxic, anticoagulant, and myotoxic effects of snake venom [50][51][52][53]. Additionally, it prevented any further decreases in neuromuscular block. An ongoing phase 2 clinical trial (BRAVO study) aims to evaluate the safety, tolerability, and efficacy of oral varespladibmethyl in snakebite envenoming [54]. In vitro and in vivo preclinical venom inhibition assays of metalloproteinase inhibiting drugs also showed encouraging results [55]. Marimastat, batimastat, and prinomastat have been shown to be highly potent in their ability to inhibit SVMPs [52,56].
In Crotalus durissus accidents, patients can develop respiratory distress due to repercussions on the structure of pulmonary parenchyma and gas exchange, with potentially irreversible sequelae [46]. Our findings demonstrate the urgent need for further investigations into the respiratory repercussions and clinical conduction of patients bitten by Crotalus durissus cascavella. Early clinical interventions during the first 6 h of hospitalization, guided by point-of-care biomarkers, are critical to improve patient outcomes in high-risk subgroups and avoid long-term pulmonary sequelae [7,57]. Antivenom should be administered as early as possible, ideally within the first few hours after the snakebite [12]. Respiratory muscle paralysis and pulmonary edema remain the primary causes of death, with current treatments being based on clinical and respiratory support [49]. Thus, experimental histopathological analyses can provide valuable insights for clinical trials in this field.

Conclusions
Crotalus durissus venom induces extensive pathophysiological changes, resulting in impaired pulmonary mechanics, poor gas exchange, and respiratory distress. The EG morphometric analysis presented pulmonary inflammatory infiltrates at all time intervals, being more significant between three and twelve hours. The necrosis zones were significant at intervals of one and 24 h, one and 48 h, and three and 48 h.
The early recognition and prompt treatment of this condition are essential to prevent further lung injury and to improve outcomes. Clinicians should be aware of the risk of extensive alveolar and vascular damage, which potentially lead to hypoxemia in the first hours after venom exposure. Assessment of alveolar collapse and severity of venominduced lung injury evaluated by lung imaging might be clinically helpful. Further studies are needed to better understand the pathophysiology of ALI induced by Crotalus durissus venom and to develop novel biomarkers in ophidian envenomation.

Biological Material
The venom of Cdc was obtained from twenty adult snakes of both sexes, which was kept at the Venomous Animals and Herpetology Laboratory of the Feira de Santana State University (LAPH-UEFS). Venom was extracted, vacuum dried, and grouped into a "pool" at −20 • C for later analysis. The Laboratory is homologated by Instituto Brasileiro do Meio Ambiente e dos Recursos Renováveis (IBAMA), with a Federal Registration Number 480922, and it is administered by Sistema Nacional do Patrimônio Genético e do Conhecimento Tradicional Associado (SisGen), with its protocol number being ABC319C. Venom protein concentration was determined using bovine serum albumin from Sigma-Aldrich (St. Louis, MO, USA) as the protein standard [58].
A total of 72 male Swiss mice, weighing between 18 and 22 g, were randomly allocated into experimental and control groups (four experimental groups, four control groups, seven mice per group) and kept at controlled temperatures (22 • and 25 • C), lighting (12-h light/dark cycle), humidity, and airflow conditions. All mice have also received water and food ad libitum. The animals in the experimental group received a 1.0 µg dose of venom per animal via intraperitoneal injection (i.p), diluted in 500 µL sterile saline solution (NaCl 0.9%), as well as the control group sterile saline solution (NaCl 0.9%), via i.p. [59]. The animals were monitored during the study interval. We euthanized animals with an overdose of xylazine chloride 2% (Xilazin ® , Syntec, Santana do Parnaíba, SP, 06513-010, Brazil) and ketamine chloride 10% (Vetanarcol ® , Konig, Santana do Parnaíba, SP, 06500-000, Brazil) to collect tissue samples. Lung tissue was initially conditioned in formaldehyde 10% for a maximum of 48 h ( Figure 5). The behavior of the animals and the clinical manifestations were monitored during the experimental period. The study was conducted in accordance with the ethical principles for animal experimentation approved by the Animal Ethics Committee of the Feira de Santana State University (006/2018).

Histopathological Analysis of Pulmonary Parenchyma
The tissue fragments were processed, diaphonized, and sliced into 4 µm thick sections onto slides, which were later stained in a solution of hematoxylin-eosin (HE) and Masson's Trichrome Sigma-Aldrich (St. Louis, MO, USA). The cuts were examined, and the images were captured using an Olympus BX51 microscope (Tokyo, Japan), coupled with a digital camera (DP25), and digitalized on cellSens software.

Morphometric Analysis of Inflammatory Infiltrates Necrosis Areas, Alveolar Distensions, and Septal Loss
We conducted the morphometric evaluation in five randomly selected microscopic fields on slides containing parenchymal sections of the mice from the control and experimental groups. We evaluated inflammatory cell counting by square millimeters using a 20× eyepiece and 20× objective lens in an area of 60 mm 2 . The same methodology was used to quantify the necrosis areas, collagen deposits, alveolar distension, and septic loss by measuring and calculating alveolar saccule distensions and areas in the septic region that presented compromised integrity. All data obtained were processed and analyzed using ImageJ (USA) software. The graphics were generated using GraphPad Prism 6.0 (GraphPad, San Diego, CA, USA).

Statistical Analysis
After verifying the absence of normal distribution using the Kolmogorov-Smirnov test, the Mann-Whitney test was used to compare the experimental and control groups, and the Friedman test, followed by the Wilcoxon test (with Bonferroni correction), were used to compare time intervals. Values of p < 0.05 were considered statistically significant. Statistical analyses were conducted using GraphPad Prism, version 6.00 (GraphPad, San Diego, CA, USA).

Supplementary Materials:
The following supporting information can be downloaded at: www.mdpi.com/xxx/s1, Video S1: Clinical signs of respiratory distress in mice.

Histopathological Analysis of Pulmonary Parenchyma
The tissue fragments were processed, diaphonized, and sliced into 4 µm thick sections onto slides, which were later stained in a solution of hematoxylin-eosin (HE) and Masson's Trichrome Sigma-Aldrich (St. Louis, MO, USA). The cuts were examined, and the images were captured using an Olympus BX51 microscope (Tokyo, Japan), coupled with a digital camera (DP25), and digitalized on cellSens software.

Morphometric Analysis of Inflammatory Infiltrates Necrosis Areas, Alveolar Distensions, and Septal Loss
We conducted the morphometric evaluation in five randomly selected microscopic fields on slides containing parenchymal sections of the mice from the control and experimental groups. We evaluated inflammatory cell counting by square millimeters using a 20× eyepiece and 20× objective lens in an area of 60 mm 2 . The same methodology was used to quantify the necrosis areas, collagen deposits, alveolar distension, and septic loss by measuring and calculating alveolar saccule distensions and areas in the septic region that presented compromised integrity. All data obtained were processed and analyzed using ImageJ (USA) software. The graphics were generated using GraphPad Prism 6.0 (GraphPad, San Diego, CA, USA).

Statistical Analysis
After verifying the absence of normal distribution using the Kolmogorov-Smirnov test, the Mann-Whitney test was used to compare the experimental and control groups, and the Friedman test, followed by the Wilcoxon test (with Bonferroni correction), were used to compare time intervals. Values of p < 0.05 were considered statistically significant. Statistical analyses were conducted using GraphPad Prism, version 6.00 (GraphPad, San Diego, CA, USA).