Local and Hematological Alterations Induced by Philodryas Olfersii Snake Venom in Mice
Total Page：16
File Type：pdf, Size：1020Kb
(This is a sample cover image for this issue. The actual cover is not yet available at this time.) This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the author's institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier's archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/authorsrights Author's Personal Copy Toxicon 132 (2017) 9e17 Contents lists available at ScienceDirect Toxicon journal homepage: www.elsevier.com/locate/toxicon Local and hematological alterations induced by Philodryas olfersii snake venom in mice * Juliana S. Oliveira a, Luciana B. Sant'Anna a, , Manoel C. Oliveira Junior b, Pamella R.M. Souza b, Adilson S. Andrade Souza b, Wellington Ribeiro c, Rodolfo P. Vieira b, e, Stephen Hyslop d, Jose C. Cogo e a Laboratory of Histology and Regenerative Therapy, Institute of Research and Development (IP&D), Vale do Paraíba University (UNIVAP), Avenida Shishima Hifumi, 2911, Urbanova, 12244-000, Sao~ Jose dos Campos, SP, Brazil b Laboratory of Pulmonary and Exercise Immunology (LABPEI), Nove de Julho University (UNINOVE) and Brazilian Institute of Teaching and Research in Pulmonary and Exercise Immunology (IBEPIPE), 01504-000, Sao~ Paulo, SP, Brazil c Laboratory of Pharmacology and Biochemistry, Institute of Research and Development (IP&D), Vale do Paraíba University (UNIVAP), Avenida Shishima Hifumi, 2911, Urbanova, 12244-000, Sao~ Jose dos Campos, SP, Brazil d Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas (UNICAMP), Rua Tessalia Vieira de Camargo, 126, Cidade Universitaria Zeferino Vaz, 13083-887, Campinas, SP, Brazil e Department of Bioengineering and Biomedical Engineering, Brazil University, Rua Carolina Fonseca, 584/235 (Campus I and II), Vila Santana, 08230-030, Itaquera, Sao~ Paulo, SP, Brazil article info abstract Article history: Envenomation by the South American opisthoglyphous snake Philodryas olfersii causes local pain, edema, Received 25 November 2016 erythema and ecchymosis; systemic envenomation is rare. In this work, we examined the inflammatory Received in revised form activity of P. olfersii venom (10, 30 and 60 mg) in mouse gastrocnemius muscle 6 h after venom injection. 22 March 2017 Intramuscular injection of venom did not affect hematological parameters such as red cell count, he- Accepted 23 March 2017 moglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin and mean corpuscular Available online 24 March 2017 hemoglobin concentration. The venom caused thrombocytopenia (at all three doses), leukopenia and lymphopenia (both at the two highest doses), as well as neutrophilia (30 mg), monocytosis (30 mg) and Keywords: Acute inflammation basophilia (10 mg). Of the cytokines that were screened [IL-1b, IL-6, IL-10, IL-13, IL-17, TNF-a, IFN-g, MIP-2 fi Cytokines and KC] and IGF-1, only IGF-1 showed a signi cant increase in its circulating concentration, seen with Edema 60 mg of venom; there were no significant changes in the cytokines compared to control mice. Histo- Myonecrosis logical analysis revealed the presence of edema, an inflammatory infiltrate and progressive myonecrosis. Inflammatory infiltrate Edema and myonecrosis were greatest with 60 mg of venom, while the inflammatory infiltrate was Philodryas olfersii venom greatest with 10 mg of venom. All venom doses caused the migration of polymorphonuclear and mononuclear leukocytes into muscle, but with no significant dose-dependence in the response. These findings show that, at the doses tested, P. olfersii venom does not cause hematological alterations and has limited effect on circulating cytokine concentrations. These data also confirm that the principal effects of the venom in mice are local edema, inflammatory cell infiltration and myonecrosis. © 2017 Elsevier Ltd. All rights reserved. 1. Introduction cause of non-front-fanged colubroid envenomations in this conti- nent (Prado-Franceschi and Hyslop, 2002; Weinstein et al., 2011, The back-fanged colubroid snake genus Philodryas (Dipsadidae, 2013), with the main species involved in human envenomations Xenodontinae), commonly referred to as racers, consists of ~20 being P. chamissonis (Otero et al., 2007), P. olfersii (Ribeiro et al., species with a widespread distribution throughout South America 1999; Correia et al., 2010) and P. patagoniensis (Medeiros et al., (Zaher et al., 2008, 2014). Snakes of this genus are the principal 2010); species less commonly involved include P. aestivus (Fowler and Salomao,~ 1994), P. baroni (Küch and Jesberger, 1993) and P. viridissima (Means, 2010). * Corresponding author. The venoms of P. olfersii (Assakura et al., 1992; Acosta de Perez E-mail address: [email protected] (L.B. Sant'Anna). et al., 2003; Rodríguez-Acosta et al., 2006) and P. patagoniensis http://dx.doi.org/10.1016/j.toxicon.2017.03.013 0041-0101/© 2017 Elsevier Ltd. All rights reserved. Author's Personal Copy 10 J.S. Oliveira et al. / Toxicon 132 (2017) 9e17 (Acosta et al., 2003; Peichoto et al., 2004, 2005, 2006; Lopes, 2008) groups (n ¼ 10/group): Group 1 e mice injected with phosphate- cause edema, hemorrhage and myonecrosis in experimental ani- buffered 150 mM saline (PBS) solution (control group) in the left mals, while in humans the primary manifestations are local effects gastrocnemius muscle and Groups 2e4 e mice injected with 10 mg, such as pain, edema, erythema and ecchymosis (Ribeiro et al., 1999; 30 mg and 60 mgofP. olfersii venom, respectively, in a volume of 50 Medeiros et al., 2010). A few components have been isolated from ml/gastrocnemius muscle. Six hours after saline or venom injection, these venoms, including a myotoxin (Prado-Franceschi et al., 1998) the mice were anesthetized with a mixture of xylazine hydro- and five fibrinogenolytic proteases (four metalloproteinases and chloride (Xilazin™ 2% injectable solution; 10 mg/kg, i.p.) plus ke- one serine protease, with two of these enzymes also being hem- tamine hydrochloride (Cetamin™ 10% injectable solution; 100 mg/ orrhagic) (Assakura et al., 1992) from P. olfersii, and a metal- kg, i.p.). Once satisfactory anesthesia had been reached, blood was loproteinase (patagonfibrase) (Peichoto et al., 2007, 2010, 2011) and collected via the inferior vena cava in a 1 ml syringe containing cysteine-rich secretory protein (CRISP; patagonin) (Peichoto et al., 0.1 ml of EDTA. Ten microliters of blood were used for a complete 2009) from P. patagoniensis. The identification of these isolated blood count and the remainder was centrifuged (900 g, 10 min, components agrees with proteomic and transcriptomic analyses 4 C) and the plasma then collected and stored at À80 C for sub- indicating the presence of metalloproteinases, serine proteases, sequent quantification of inflammatory mediators. After blood CRISPs and other components in these venoms (Ching et al., 2006; collection, the left gastrocnemius muscle was removed from the Peichoto et al., 2012). exsanguinated mice and three samples of each muscle were placed Philodryas olfersii venom degrades fibrinogen in vitro and in vivo in separate polypropylene microtubes and stored at À80 C. via the action of metalloproteinases and serine proteinases (Assakura et al., 1994), but is devoid of thrombin-like activity; this 2.3. Hematological analysis degradation delays the clotting of fibrinogen by thrombin (Assakura et al., 1992). The venom also has fibrinolytic activity, but Hematological analyses were done in an automated hemato- is devoid of platelet-aggregating or inhibitory effects (Assakura logical analyzer (Sysmex 800i, Roche, Germany) using blood sam- et al., 1992). In contrast to these effects on hemostasis, the effect ples collected from the inferior vena cava. The parameters of P. olfersii venom on general hematological parameters is un- measured included red blood cell count (RBC), hemoglobin, he- known. In addition, compared to P. patagoniensis (Peichoto et al., matocrit, mean corpuscular volume (MCV), mean corpuscular he- 2004; Lopes, 2008), the edematogenic response to P. olfersii moglobin (HCM), mean corpuscular hemoglobin concentration venom (Assakura et al., 1992; Acosta et al., 2003) has not been (CHMC), white blood cell (WBC) count, neutrophils, lymphocytes, investigated in detail, particularly with regard to the profile of in- monocytes and platelets. flammatory cells involved and the possible changes in the con- centrations of cytokines in the general circulation. 2.4. Cytokine quantification In this work, we therefore examined the profile of the cellular infiltrate associated with the inflammatory response after the Plasma cytokine levels were quantified by ELISA using com- intramuscular injection of P. olfersii venom in mice. We also mercial kits obtained from Biolegend (San Diego, CA, USA) and R&D quantified a variety of cytokines (IL-1b, IL-6, IL-10, IL-13, IL-17, Systems (Minneapolis, MN, USA). The cytokines investigated were TNFa, IFNg, MIP-2 and KC) and the growth factor IGF-1 known to be IL-1b, IL-6, IL-10, IL-13, IL-17,MIP-2,KC, TNF-a and IFN-g and the involved in the development and modulation of inflammation and