Pulmonary mechanic and lung histology induced by Crotalus durissus cascavella snake venom

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Short communication

Pulmonary mechanic and lung histology induced by

Crotalus durissus

cascavella

snake venom

Joselito de Oliveira Neto

a,*

, Jo

~

ao Alison de Moraes Silveira

b

, Daniel Silveira Serra

c

,

Daniel de Araújo Viana

a

, Diva Maria Borges-Nojosa

d

, C

elia Maria Souza Sampaio

e

,

Helena Serra Azul Monteiro

b

, Francisco Sales

Avila Cavalcante

c

,

Janaina Serra Azul Monteiro Evangelista

a a

Postgraduate Program in Veterinary Sciences, Faculty of Veterinary, State University of Ceara, Fortaleza, Ceara, Brazil b_{Department of Physiology and Pharmacology, Faculty of Medicine, Federal University of Cear}_{a, Fortaleza, Cear} _{a, Brazil} c_{Postgraduate Program in Physics, State University of Cear}_{a, Fortaleza, Cear}_{a, Brazil}

d_{Department of Biology, Federal University of Cear}_{a, Fortaleza, Ceara, Brazil} e_{Department of Biology, State University of Cear}_{a, Fortaleza, Cear}_{a, Brazil}

a r t i c l e

i n f o

Article history:

Received 15 September 2016 Received in revised form 23 July 2017

Accepted 26 July 2017 Available online 28 July 2017

Keywords:

Crotalus durissus cascavella

Pulmonary mechanics Histopathology Venom Balb/C

a b s t r a c t

This study have analyzed the pulmonary function in an experimental model of acute lung injury, induced by theCrotalus durissus cascavellavenom(C. d. cascavella)(3.0mg/kg -i.p), in pulmonary mechanic and histology at 1 h, 3 h, 6 h, 12 h and 24 h after inoculation. TheC. d. cascavellavenom led to an increase in Newtonian Resistance (RN), Tissue Resistance (G) and Tissue Elastance (H) in all groups when compared to the control, particularly at 12 h and 24 h. The Histeresivity (h) increased 6 h, 12 h and 24 h after

inoculation. There was a decrease in Static Compliance (CST) at 6 h, 12 h and 24 h and inspiratory capacity (IC) at 3 h, 6 h, 12 h and 24 h.C. d. cascavellavenom showed significant morphological changes such as atelectasis, emphysema, hemorrhage, polymorphonuclear inflammatory infiltrate, edema and conges-tion. After a challenge with methacholine (MCh),RNdemonstrated significant changes at 6, 12 and 24 h. This venom caused mechanical and histopathological changes in the lung tissue; however, its mecha-nisms of action need further studies in order to better elucidate the morphofunctional lesions.

1. Introduction

The subspecies Crotalus durissus cascavella(C. d. cascavella) is found in the Caatinga from Northeastern of Brazil (Pinho and Pereira, 2001; Barraviera, 1989). Its venom is presented as a toxic-enzymatic complex, mainly composed of phosphodiesterase, L-amino oxidase, 5-nucleotidase and toxins such as crotoxine, con-vulxin, crotalin and gyroxin (Rangel-Santos et al., 2004; Fonseca et al., 2006; Spinosa et al., 2008). This complex substance leads to systemic functional changes, being responsible for the primary cause of death after a snakebite (Evangelista et al., 2008), among which the acute respiratory failure related to neuromuscular pa-ralysis can be highlighted (Damico et al., 2005).

Mechanical properties of the respiratory system are well

deﬁned; however, the characterization of lung structures and

quantification of inflammation generated byC. d. cascavellavenom are little known. Therefore, it was necessary to study the effects of possible changes in lung architecture so that, in any involvement, therapeutic intervention may be the earliest and most efficient as possible. This study aimed to analyze the pulmonary function in an experimental model of acute lung injury induced byC. d. cascavella

venom.

2. Methods

2.1. Venom, chemicals and reagents

TheC. d. cascavellavenom was obtained and kindly provided by Professor Dr. Diva Maria Borges Nojosa, from Núcleo Regional de

Oﬁologia (NUROF) of the Department of Biology from Federal

University of Ceara, Fortaleza, Brazil. The venom was lyophilized and diluted in saline (0.9% w/v NaCl solution) at the time of use. All

*Corresponding author.

E-mail address:joselitoneto@yahoo.com.br(J. Oliveira Neto).

Contents lists available atScienceDirect

Toxicon

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / t o x i c o n

http://dx.doi.org/10.1016/j.toxicon.2017.07.023

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chemicals were purchased from Sigma-Aldrich®

(St Louis, MO, USA).

2.2. Animals

The Local Ethics Committee on the Use of Animals approved the experimental protocol (12773584-4). 36 Balb/C mice (males,

20e30 g) were randomly divided into 6 groups. According toMiller

and Tainter (1944) apud Feitosa (1996), the calculation of the lethal dose was done according to the method of the probit in which it was observed that the LD50 for venom of C.d. Casvella is 3.0±_{0.34 mg/kg.}

All experimental groups received inoculation of 3.0

m

g/kg of

venom, diluted in 0.1 mL of saline (NaCl 0.9%) (i.p.). The animals were analyzed after 1 h of inoculation (group 1 h, n¼6), 3 h after

inoculation (group 3 h, n¼6), 6 h after inoculation (group 6 h,

n¼6), 12 h after inoculation (12 h group, n¼6) and 24 h after

inoculation (group 24 h, n¼6). The Ctrl group (n¼6) received only 0.1 mL of saline (i.p).

2.3. Respiratory system mechanics

After the inoculation period of the venom in each group, the animals were sedated (diazepam, 1 mg/kg, i.p.), and anesthetized

with sodium pentobarbital (50 mg/kg, i.p., Hypnol®

3%, Syntect, Brazil) and tracheotomized. The animals were intubated with a 18-gauge cannula (Eastern Medikit, Delhi, India) that was then con-nected to a computer-controlled ventilator for small animals (Scirec©

-ﬂexVent®

, Montreal, QC, Canada). The animals were ventilated at baseline settings: respiratory frequency of 150 breaths/min, tidal volume of 10 mL/kg, limiting pressure of 30 cmH2O, and positive end-expiratory pressure (PEEP) of 3 cmH2O. Mices were then paralyzed with pancuronium bromide (0.5 mL/kg, i.p., Cristalia, Lindoia, MG, Brazil).

Initially we standardized the mechanical history of the respi-ratory system with two deep inﬂations (DI, 6-s long, peak pressure: 30 cmH2O), followed by a period of 5min of ventilation. Soon after, the impedance of the respiratory system (Zrs) was measured with

the forced oscillation technique (Hantos et al., 1992), 12 sequential 10-s sampling intervals, for a total of 2 min.

The experimentalZrswasﬁtted to the constant phase model as

previously described (Hirai et al., 1999):

Zrs¼ RNþIð2

p

fÞiþ G Hi

ð2

p

fÞa

(1)

a

¼2

p

tan

1H

G

(2)

whereRNis the Newtonian resistance, which represents the central

airways resistance,i ¼ ffiffiffiffiffiffiffi 1 p

,fis the frequency (Hz),Irepresents airway inertance, andGandHare respectively the dissipative and elastic properties of lung tissue (Hantos et al., 1992). Hysteresivity (

h

¼G=H) was also calculated (Fredberg, 2001).

Thereafter, starting at the functional residual capacity (FRC) deﬁned by the PEEP, theﬂexiVent delivered 7 inspiratory pressure steps for a total pressure of 30 cmH2O, followed by 7 expiratory steps, pausing at each step for 1 s. At each step plateau pressure (P)

was recorded and related to the total volume (V) delivered to

produce a quasi-static PV (pressure-volume) curve. Static compli-anceðCSTÞwas calculated as the slope of the curve (Salazar and

Knowles, 1964). Two quasi-static PV curves were obtained to measureCST, an estimate of inspiratory capacity (IC), and PV loop

area.

2.4. Hyperresponsiveness of airway smooth muscle

Immediately after measurements of respiratory system me-chanics, two DIs (Deep Inﬂation) were done, followed by 5 min of ventilation with baseline settings. Airway smooth muscle hyper-responsiveness was evaluated by inhalation of methacholine (MCh) (Sigma-Aldrich, St. Louis, MI, USA) delivered by aerosol produced by an ultrasonic nebulizer (Inalasonic, NS Indústria de Aparelhos Medicos, S~ao Paulo, SP, Brazil) coupled to the inspiratory line of the ventilator. For such purpose, 4 mL of MCh solution (3, 6, 12,5, 25 and 50 mg/mL) were added to the nebulizer container. The nebulization was carried out during 30 s under mechanical ventilation (Xue et al., 2008).

After nebulization, the same previous analysis was repeated (forced oscillation, 10-s sequential intervals for 2 min). Data regarding airway hyperresponsiveness collected after nebulization

of MCh are presented as

D

RN, where

D

means the parameter after

nebulization minus its value before MCh challenge. All

D

values

were normalized by the pre-nebulization values.

2.5. Histological analysis

After euthanasia or natural death of animals from the

experi-ments, lungs were collected and stored in ﬁxative solution (10%

formaldehyde) for 72 h. Then tissues were washed and progres-sively dehydrated in an ascending series of alcohols (70%, 80%, 90%, 100%, 100%; 1 h at each concentration); diaphanized in xylene (2x, 1 h each); and bathed in parafﬁn (2x, 1h30 each bath). Then, tissues

were embedded in parafﬁn and 4

m

m sections were obtained using

a manual microtome (Leica RM2025®

). After a deparafﬁnization process (56_{C for 24 h), tissues were mounted on slides, rehydrated} with decreasing concentration of alcohol (90%, 80% and 70%; 3 min at each solution) and stained by hematoxylin-eosin (HE) for further analysis in light microscopy (Microscope Nikon Eclipse Nis, Nis Software 4.0®

).

2.6. Statistical analysis

Statistical analyses were performed using GraphPad Prism version 5.00 (GraphPad, San Diego, CA, USA). To compare the re-sults gathered from each group, initially the normality of the data (KolmogoroweSmirnov test with Lilliefor's correction), and the homogeneity of variances (Levene median test) were evaluated. If

both conditions were satisﬁed, one-way ANOVA was used. In the

negative case, KruskaleWallis ANOVA was selected instead. The signiﬁcance level was always set at 5%.

3. Results and discussion

3.1. Respiratory system mechanics

In RN, a good estimate of the total airway resistance (Bates,

2009), statistical signiﬁcance values were observed in all experi-mental groups, being higher in groups 12 h and 24 h (Fig. 1A). These results represent a greater narrowing or an increase in the stiffness of airways smooth muscle in these experimental groups when compared to the control group. By reducing the lung elastic recoil,

breakingﬁbers and destroying alveolar attachments, there was a

limitation of the airﬂow, leading to increased airway resistance. The air trapping and new lung morphology acted reducing the surface area available for gas exchange (Saetta et al., 2001; Ishizawa et al., 2004).

GandHare related to intrinsic properties of the tissue and both

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groups, particularly in groups 12 h and 24 h (Fig. 1B and C). There

are some hypotheses to explain the observed inG: one is changes

on the rheological properties of the tissue (Bates, 2009); another

would be the inﬂuence of the airways narrowing on these

param-eters, which could result in a lung parenchyma distortion, with

small airways closure, providing an effectively lower lung, with aH

proportionally higher (Wagers et al., 2004).

The

h

grows as the lung becomes mechanically heterogeneous.

This parameter had increased and signiﬁcant average values in

groups 6 h, 12 h and 24 h (Fig. 1D). Thus we may assume that the

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increase of

h

values is due to the presence of ventilatory hetero-geneities resulting from the increase in Nr.

TheCSTwas signiﬁcantly reduced in groups 6 h, 12 h and 24 h

(Fig. 1E). The inspiratory capacity (IC) showed signiﬁcant and reduced mean values in groups 3 h, 6 h, 12 h and 24 h (Fig. 1F). These data were consistent with the effective stiffening of lung tissue, reﬂecting the increase ofH.

Nonaka et al. (2008)found changes in experimental groups 3 h (signiﬁcant increase in tissue elastance), 6 h (signiﬁcant increase in

tissue resistance) and 12 h (increase in viscoelastic properties) after intramuscular inoculation ofCrotalus durissus terriﬁcus(0,6

m

g/g). After 24 h values returned to baseline parameters.

After the challenge with methacholine, Nr had a signiﬁcant in-crease in their average values in groups 6 h, 12 h and 24 h (Fig. 1G). The bronchoconstriction caused by methacholine made small air-ways shut completely (Evans et al., 2003), preventing its opening only with the smooth muscle relaxation. Bronchial hyper-responsiveness mechanisms were multiple, being associated with

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acute and chronic inﬂammation and airways remodeling (Boulet et al., 1997; Martin et al., 2000). The biggest tendency for contraction was due to the loss of factors that oppose the short-ening of smooth muscle by intrinsic changes to smooth muscle and loss of elastic recoil of the lung parenchyma (Fredberg, 2001).

3.2. Histopathological results

Histological architecture of lungs (bronchial tree, bronchial and alveolar) had alterations in its organization, caused by atelectasis and emphysema. Atelectasis, present in all groups, had moderate intensity in groups 6 h, 12 h and 24 h (Fig. 2A). Discrete emphysema was present in all experimental groups (Fig. 2B). These changes occurring in a lung segment region might distort the pulmonary parenchyma and its adjacent subsegment, thus affecting the local

tissue mechanics. This was probably related to the increased n

(Correa et al., 2001).

A mild edema was observed only in the group 24 h (Fig. 2C). This was well represented since there was no occurrence of this path-ological change in the literature, and it may be related to the

in-direct injury factor generated by the C. d. cascavellavenom and

attributed to the systemic action of PLA2. There was a report of an

edematous response induced by the Crotalus d. terriﬁcusvenom

which was not dose-dependent, of fast and transient course (Santoro et al., 1999). There were several studies reporting that the PLA2induces edema, an effect that in some cases are dependent of PLA2s which bind to speciﬁc membrane proteins (Iglesias et al., 2005).

In all experimental groups inﬂammatory inﬁltration of

lym-phocytes and plasmocytes was observed, although discrete. There

was a greater concentration of inﬁltrate in groups 3 h and 6 h

(Fig. 2D). There was an increase in the bronchus-associated lymphoid tissue (BALT) in groups 6 h and 12 h (Fig. 2E), associ-ated with an increased activation of these animals' immune system.

Inﬂammatory inﬁltrate with lymphocytes and plasmocytes in all

experimental groups was suggestive of an acute tissue injury

caused by C. d. cascavella venom. This probably resulted in

chemotaxis from the activity of released inﬂammatory chemical

mediators such as prostaglandins, histamine, bradykinin or nitric

oxide and the subsequent diapedesis of inﬂammatory cells to the

injury site (Braga et al., 2007). In other studies withC. d. terriﬁcus

venom there was an increase in lymphonuclear cells in the airways of animals from the group 6 h (Nonaka et al., 2008). Other studies have considered that this response to acute lung injury occurred due to the presence of mononuclear cells (Tong et al., 2006).

Hemorrhage was observed in all treated groups ranging from mild to severe and moderate in the group 3 h (Fig. 2G). This was explained by the existence of crotamine associated with a high activity of PLA2, making this substance to be characterized by its myotoxicity and may thus be responsible for the observed hem-orrhagic process. Its neurotoxic potential was also reported in the literature (Santoro et al., 1999; Nonaka et al., 2008; Beghini et al., 2000). Another possibility for this increase was the presence of hemorrhage in all groups tested, being moderate in the group 12 h and severe in the group 24 h, in airways of small caliber. The occurrence of edema only in the group 24 h, promoted occlusions of these pathways and the production of atelectasis areas, present in all experimental groups, with moderate intensity in groups 6 h, 12 h and 24 h. This process has been found in clinical practice of animals subjected to mechanical ventilation.

Due to these characteristics of ventilatory heterogeneities it was possible to associate with the presence of moderate hemorrhage in the group 12 h and severe in the group 24, atelectasis of moderate intensity in groups 6 h, 12 h and 24 h, mild emphysema present in all groups tested and edema in the group 24 h. The presence of

secretions from the peripheral airways may affect the distribution of ventilation and thus increase the mechanical inhomogeneity. Changes in contractile tone of smooth muscles in the lung paren-chyma should not be discarded (Correa et al., 2001).

More speciﬁc studies on pulmonary complications are necessary in order to establish whether the acute injury process correlates to muscle paralysis and systemic myonecrosis. Consequently, it leads to triggering factors of an inﬂammatory process; if there is a direct action of toxic factors of the venom on the lung tissue, it may lead to morphostructural and mechanical properties changes.

Conﬂict of interest

The authors of this article declare that there is no potential

conﬂicts of interest including employment, consultancies, stock

ownership, honoraria, paid expert testimony and patent applica-tions/registrations related to the current manuscript.

This manuscript is submitted on behalf of all authors.

Ethical statement

This study was approved by the Ethics Committee for Animals' Use of State University of Ceara (Protocol 12773584-4 protocol), following all criteria required for the safety of animals and re-searchers involved.

Acknowledgments

We would like to thank CNPq, CAPES, FINEP and FUNCAP by the

ﬁnancial support of this research.

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