Peripheral and central effects of intracerebroventricular microinjection of Hottentotta gentili (Pallary, 1924) (Scorpiones, Buthidae) venom

Accepted Manuscript Peripheral and central effects of intracerebroventricular microinjection of Hottentotta gentili (Pallary, 1924) (Scorpiones, Buthidae) venom Moulay Abdelmonaim El Hidan, Oulaid Touloun, Omar El Hiba, Jawad Laadraoui, Hind Ferehan, Ali Boumezzough PII:

S0041-0101(15)30157-4

DOI:

10.1016/j.toxicon.2015.12.010

Reference:

TOXCON 5270

To appear in:

Toxicon

Received Date: 22 September 2015 Revised Date:

9 December 2015

Accepted Date: 16 December 2015

Please cite this article as: El Hidan, M.A., Touloun, O., El Hiba, O., Laadraoui, J., Ferehan, H., Boumezzough, A., Peripheral and central effects of intracerebroventricular microinjection of Hottentotta gentili (Pallary, 1924) (Scorpiones, Buthidae) venom, Toxicon (2016), doi: 10.1016/ j.toxicon.2015.12.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Peripheral and central effects of intracerebroventricular microinjection of

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Hottentotta gentili (Pallary, 1924) (Scorpiones, Buthidae) venom Moulay Abdelmonaim El Hidan1*, Oulaid Touloun1,2, Omar El Hiba3, Jawad Laadraoui4,

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Hind Ferehan4,Ali Boumezzough1*

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Faculty of Sciences Semlalia, Cadi Ayyad University, BP 2390-40080 Marrakesh, Morocco

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Polydisciplinary Faculty, University Sultan Moulay Slimane, Beni Mellal, Morocco.

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Laboratory of Ecology and Environment L2E, (URAC 32, CNRST, ERACNERS 06)

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Polyvalent Laboratory of Research & Development LPVRD, Department of Biology,

Neurosciences, Pharmacology and Environment Unit, Faculty of Sciences Semlalia, Cadi

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Ayyad University, Marrakesh, Morocco

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of sciences Semlalia Cadi Ayyad University. Marrakesh, Morocco

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-Moulay Abdelmonaim El Hidan -Tel: +212 616558116 - Fax: +212 524434649 –

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E-mail address: [email protected]

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-Ali Boumezzough, -Tel: +212 661498880 - Fax: +212 524447412 –

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address E-mail: [email protected] / [email protected]

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Corresponding authors:

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Laboratory of Pharmacology, Neurobiology and Behavior. Department of Biology, Faculty

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ACCEPTED MANUSCRIPT Abstract

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Central effects of scorpion venom toxins have been neglected, due both to the common

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belief that scorpion venoms act by targeting peripheral organs and also to the

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misunderstanding that these peptides do not cross the brain-blood barrier (BBB).

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Determining whether scorpion neurotoxicity is restricted to peripheral actions or whether a

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central mechanism may be partly responsible for systemic manifestations could be crucial in

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clinical therapy trends. The present study therefore aims to assess histopathological

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damages in some organs (heart, kidney, liver, and lungs) and the related biochemical

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impairments, together with a neurobehavioral investigation following an

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intracerebroventricular (i.c.v) micro-injection of Hottentotta gentili (Scorpiones, Buthidae)

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venom (0.47 µg /kg). I.c.v. injection of venom produced focal fragmentation of myocardial

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fibers, while lungs showed rupture of the alveolar structure. Concurrently, there was a

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significant rise in the serum enzymes levels of ASAT, ALAT, CPK and LDH.Meanwhile,

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we observed behavioral alterations such as a hypoactivity, and in addition the venom seems

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to have a marked anxiogenic-like effect. The present investigation has brought new

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experimental evidence of a peripheral impact of central administration of H. gentili venom,

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such impact was manifested by physiological and behavioral disturbances, the last of these

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appearing to reflect profound neuro-modulatory action of H. gentili venom.

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Key words: anxiety; biochemical disturbances; histopathology; Hottentotta gentili;

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intracerebroventricular injection; locomotion; scorpion venom.

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1. Introdcution Scorpion envenomation constitutes one of the most important health problems in many

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countries, including North-Africa, the Middle East and South America (Al-Sadoon and

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Jarrar, 2003; de Roodt et al., 2003; Patil, 2009).

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Depending on the species and the venom dose injected, the body region of venom

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inoculation, and on the patient’s age, the clinical symptoms of scorpion venom can vary

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from mild to moderate local pain and paresthesia (90% of poisoning accidents) to serious

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systemic dysfunction such as cardio-vascular alterations, gastrointestinal dysfunction, lung

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edema, pancreatitis, convulsions, neurological lesions, coma, and death (Amaral et al.,

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1993; Osnaya-Romero et al., 2001). The most serious poisoning cases are observed in

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children and in senior citizens (Guidine et al., 2008). Currently, 25,000 scorpion stings are

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recorded per year in Morocco and 90% of fatal cases are younger than 10 years old

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(Abourazzak et al., 2009).

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Among all components of scorpion venoms, toxins that affect ion-channels are the most

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important venom components responsible for human intoxication (Quintero-Hernández et

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al., 2013). The scorpion α-toxins are the most important neurotoxins, consisting of 61 to 76

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polypeptides that act on a specific site on the voltage-gated sodium channel. These toxins

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inhibit the inactivation of the channel, inducing a prolonged depolarization and neuronal

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excitation. There are other toxins with less important effects on human. These toxins bind

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on potassium and calcium channels (Quintero-Hernández et al., 2013).

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Scorpion venom action on the central nervous system (CNS) was previously neglected due

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to the common perception that peptides constituting scorpion venom didn’t cross the brain-

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blood barrier (BBB). However recent studies have emphasized that scorpion venom could

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act on CNS and even cross the BBB. In fact, Clot-Faybesse et al. (2000) and Nunan et al.

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(2003) report finding scorpion toxins in the CNS after systemic injection in newborn mice

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ACCEPTED MANUSCRIPT without a fully developed BBB. Additionally, Nunan et al. (2003) had observed the

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presence of tityostoxin (TsTX) toxin in the brain of young rats after subcutaneous injection.

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These authors moreover reported that the distribution of TsTX in the brain of young rats

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was threefold higher than that of adult rats, indicating a higher BBB permeability.

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Moreover, Mesquita et al. (2002), had reported that intra-muscular (i.m.) injections of

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Phenobarbital, a GABAergic agonist, were able to block the lung edema induced by

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intracerebroventricular ( i.c.v.) injections of TsTX, suggesting an important role of the

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central nervous system (CNS) in the mechanism of action for this fraction of the T.

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serrulatus scorpion venom. Nencioni et al. (2009) also demonstrate central changes in

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levels of homovanillic acid, a metabolite of the dopamine, after intraperitoneal injection of

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venom of both T. serrulatus and T. bahiensis.

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Hottentotta gentili is one of the most dangerous scorpions in Morocco, causing severe

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envenoming and even death. It was previously observed in our laboratory that subcutaneous

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injection of this scorpion species induced behavioral alterations characterized by

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hypersalvation, respiratory difficulty, squeaking, mouth rubbing, mastication, wild-runing,

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jumping, trembling, humpback and wet dog shakes. Also when neurobehavioral

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disturbances were evaluated based on the locomotor activity and other cognitive approaches

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such as depression and anxiety; we found that H. gentili venom causes time dependant

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neurobehavioral changes. In fact, H. gentili venom induces a hypoactivity state, and also

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elicits depression and anxiogenic effects mainly after the three first hours post

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envenomation (El Hidan et al., 2015a).

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Based on the above data and in view of the very small number of investigations on venom-

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elicited effects following either central or peripheral injection of scorpion venoms,

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especially with venom from Old World Scorpions common in North-Africa and the Middle

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East, the present study was designed to determine whether venom from the Old World H.

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ACCEPTED MANUSCRIPT gentili scorpion centrally microinjected could reproduce the same effects observed after

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subcutaneous injection.

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We performed a study to investigate, in experimental Sprague-Dawley rats, the possible

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histopathological damages in some organs (heart, kidney, liver, and lungs) and the

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subsequent biochemical impairments, together with a neurobehavioral investigation

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following a i.c.v micro-injection of a sublethal dose (0.47 µg /kg) of H. gentili venom. 2. Material and methods

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2.1. Animals

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Twelve Sprague-Dawley Male rats (200-250g) were used for determination of

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histopathological, biochemical and behavioral changes after intracerebroventricular (i.c.v)

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injection of H. gentili venom. Animals were kept at a constant room temperature (25 °C),

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with a 12 h dark–light cycle and free access to food. All animals were treated according to

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the European decree relating to the ethical evaluation and authorization of projects using

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animals for experimental procedures, 1st February 2013, NOR: AGRG1238767A. Thus, all

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efforts were made to minimize the number and suffering of animals used.

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2.2. Scorpions

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Scorpions were collected from Zagora province in the South-Eastern region of Morocco.

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They were housed in well ventilated wooden cages with free access to food and water. The

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species was determined according to the identification key as described by Kovařík (2007).

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Figure 1: Hottentotta gentili from Zagora.

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2.2. Methods

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2.2.1. Venom extraction

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Venom was obtained from mature H. gentili (Figure 1) scorpions by electrical stimulation

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of the telson as described by Ozkan et al. (2007). The venom was dissolved in double

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distilled water and centrifuged at 15,000g for 15 min. The protein content of supernatant

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was determined according to the method of Bradford (1976). Until use, the sample was

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stored at -20 °C.

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2.2.2. Surgery

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The animals were anesthetized with chloral hydrate (6% ip,) then they were submitted to

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stereotaxic surgery for guide-canula implantation in the right lateral ventricle (AP -0.8; LL -

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1.5; DV -2.5), coordinates derived from the Paxinos and Watson (1998) stereotaxic atlas. A

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stainless steel guide cannula, fastened to the skull bone with dental acrylat cement was used.

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After surgery, animals were housed individually and allowed to recover for 5 to 7 days.

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ACCEPTED MANUSCRIPT Animals were divided into two groups control groups (6 animals) injected with sterilized

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saline solution (0.9%) and treated groups (6 animals) injected with 0.47 µg/kg of H. gentili

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venom.

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The i.c.v microinjections were made as described by Mesquita et al. (2003), briefly a 5.0- µl

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Hamilton syringe was connected to the injector cannula through a polyethylene tube filled

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with distilled water, used to drive toxin into ventricular space. A small 1.0-µl bubble was

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inserted just before loading the injection needle with 4.0-µl H. gentili venom or Nacl (0.9%)

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solution, fourfold the amount actually injected. The injection setup, therefore, was designed

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to have no dead volume space, in order to confirm H. gentili venom injection, and to

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separate toxin from the distilled water. The i.c.v injections had always a volume of 1.0-µl.

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2.2.3. Histological study

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Rats were sacrificed by decapitation 6 hours post-injection and their vital organs such as:

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lungs, heart, liver and kidneys were dissected and fixed in 10% fomaline solution. After 48

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hours, the organs were dehydrated in a graded ethanol series and embedded in paraffin wax.

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Sections of 10 µm thickness were stained with hematoxylin–eosin (HE) for pathological

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studies as described by Kiernan (1999).

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In addition, brains were also dissected. After fixation, dehydration, embedded and sliced to

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a 20µm thickness, slices were stained with hematoxylin, then observed in an optical

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microscope for visualization of the tissue wound induced by the cannula in order to

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confirm the ventricular injection.

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2.2.4. Biochemical analysis

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Biochemical analysis was performed on the serum of rats described in the previous section

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(Histological study). The serum was obtained from the centrifugation of the blood samples

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collected from jugular vein of each animal. Aspartate aminotransferase (ASAT), Alanine 7

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transaminase (ALAT), Lactate Dehydrogenase (LDH) and Creatine phosphokinase (CPK)

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levels were measured in sera of both the control and test group, using CHRONOLAB kits

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applied to BA-88A Semi-Auto Chemistry Analyzer (Mindray-China).

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2.2.5. Behavioral study

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Each rat was examined in the open-field, elevated plus-maze and hot plate tests 1 hour post

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injection. Open Field test

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The open-field apparatus was similar to that described by Broadhurst (1960). Adapted for

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rats, each animal was placed in the middle of the field. Over a 5-minute observation period,

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multiple parameters were recorded such as locomotor activity (numbers of crossed boxes),

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rearing time (the duration the animal stood on its hind legs), grooming time (the time the

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animal licked, scratched. or cleaned any body part), time spent in the central areas of the

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field. All experiments were performed between 12:00 AM and 14.00 PM to obviate possible

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variations caused by circadian rhythm. Elevated plus maze

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The maze is made with black wood and elevated 50 cm above the floor. It consists of two

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open arms 10 cm wide and 50 cm in length connected perpendicular to two closed arms of

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equal dimensions with a 10 cm square center region. The closed arms have black walls 30

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cm in height. Each animal was placed in the center of the elevated plus maze facing toward

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a closed arm and allowed to explore for 5 min as described by Braun et al. (2011). Later, the

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videos recorded were analyzed and for each animal of each group, the percentage of time

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spent in the open arms is calculated as: (time spent in the open arms / total time spent in

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open and closed arms) x 100, with the percentage of open arms entries calculated as:

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(Number of open arms entries / number of total entries in the closed and open arms) x

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100, as indicators of anxiety. Between trials, the maze was cleaned with 70% ethanol.

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Thermal hyperalgesia (hot plate test)

In this test, rats were individually placed on a hot plate (Eddy’s hot plate) with the

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temperature adjusted to 55 ± 1°C. The latency to the first sign of paw licking or jump

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response to avoid the heat was taken as an index of the pain threshold; the cut off time was

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10 s in order to avoid damage to the paw (Tiwari et al., 2011). 2.2.6. Statistical analysis

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Data are reported as mean ± SEM, and were subjected to a one-way analysis of variance

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(ANOVA). Post hoc differences between group means were tested using the Tukey test.

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Values of p lower than 0.05 were considered significant. Statistical analyses were then

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performed using the computer software SPSS 10.0 for Windows.

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3. Results

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3.1. Histopathology analysis

The histopathological analysis of certain rat organs after i.c.v microinjection of H. gentili

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venom (0.47 µg/kg) revealed remarkable alterations in heart and lung tissues. Heart tissue

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showed focal fragmentation of myocardial fibers, some with cytoplasmic condensation

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which characterize myocardial infarct (Figure2a’, Table 1). Lungs showed thickening of the

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alveolar septa and rupture of the alveolar structure due to edema and hemorrhage

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(Figure2b’, Table 1). In addition, kidneys of some envenomed rats showed significant

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changes of the glomerular manifested by congestion and swelling (Figure2c’, Table 1).

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Meanwhile the livers do not exhibit any noticeable alteration of tissue.

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Heart Haemorrhage 83 0

Haemorrahge 100 0

Lung Thickning of alveoral septa 100 0

congestion 33 0

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Table 1: Percentages of animals showing histopathological changes in selected organs of male adult rats injected with H. gentili venom by i.c.v routes.

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Figure 2: Sections of heart and lungs from rats after 6 hours of i.c.v microinjection of H.

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gentilli venom. Light microscopic analysis of heart (a), lungs (b) and kidney (c) of tissue

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specimen in healthy rats as control. (a’) Heart muscle showed massive deleterious

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Kidney Glomerular Disorganization 50 0

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degeneration, (b’) lungs showed massive hemorrhages, (c’) kidneys showed congestion and

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glomerular disorganization.

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3.2. Biochemical analysis I.c.v injection of H. gentili venom caused a significant increment in the level of ASAT

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(p<0.05), CPK (p<0.05), ALAT (p<0.05) and LDH (p<0.05) activities (Figure 3).

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Figure 3: Histograms showing the enzymatic activities in sera of envenomed rats vs.

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control rats. ALAT, ASAT, CPK and LDH activities levels are significantly enhanced in the

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treated rats compared to control. Data are reported as mean ± SEM. Data were subjected to

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the Student t-test. A value of P < 0.05 was considered to indicate statistical significance

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between control and treated groups.

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3.3. In vivo effects of i.c.v injection of venom

After venom administration, rats showed several intoxication symptoms. They exhibited the

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following symptoms: squeaking, mouth rubbing, mastication, jumping, trembling, wild-

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running, humpback and wet dog shakes.

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Symptoms

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Squeaking

mouth rubbing mastication

wild-running Jumping trembling wet dog shakes

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Envenomed (%) 83

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Table 2: Percentage of animals that showed behavioral alterations after i.c.v administration

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of H. gentili venom (0.47 µg/kg) vs. control group. 3.4. Neuro-behavioral studies

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To further assess the effect of i.c.v injection of the H. gentili venom on the central nervous

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system, we were focused on the assessment of the possible neurobehavioral impairments

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occurring in the envenomed rats. Our finding reports marked effects of H. gentili venom on

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all behavioral parameters (Figure 4). In fact, analysis of locomotor activity revealed a

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general and significant loss of locomotor activity after i.c.v injection (p<0.05), decreased

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rearing time (p<0.05), and time at the center square (p<0.05), in comparison with the

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control. In addition, our data reports a significant increase in immobility time (p<0.05) post

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injection.

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Figure 4: Open-field behavior of rat i.c.v microinjected with 0.47 µg/kg of Hottentotta

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gentili venom. The 5 min observation started after 1 hour of microinjection. Venom

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injection causes behavioral changes in rats. Values shown for locomotion are numbers of

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floor units entered (fig. a). Central activity (fig. d). Values shown for rearing is the duration

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ACCEPTED MANUSCRIPT the animal stood on its hind legs (fig. b). Grooming time (fig. c) and immobility time (fig.

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e).

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Regarding the elevated plus-maze test, i.c.v injections have shown a significant decrease in

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the percentage of open arms entries and time spent on the open arms (p<0.05) (Figure 5).

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Additionally there was no significant difference in pain sensitivity between the control and

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envenomed group (Figure 6).

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Figure 5: The Elevated Plus-Maze behavior of rat i.c.v microinjected with 0.47 µg/kg of

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Hottentotta gentili venom. The 5 min observation started after 1 hour of microinjection. The

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categories (a) and (b) respectively denote: (a), the percentage of entries into open

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arms/entries into all arms, (b), the percentage of time spent in open arms/time spent in all

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arms. The microinjected rats with H. gentilli venom showed decrease in the percentage of

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time spent in open arms and in the percentage of entries into open arms/entries.

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Figure 6: Effect of Hottentotta gentili venom i.c.v microinjected on reaction time in hot

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plate test. The 10 seconds observation started after 1 hour of microinjection. There is no

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significant difference between the control and envenomed groups.

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4. Discussion

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In severe scorpion envenoming effects such as agitation, brain infarcts and convulsions are

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commonly observed (Fernandez-Bouzas et al., 2000), indicating a clear neurological

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involvement in scorpion envenomation. However, central effects of scorpion venom toxins

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have been neglected, due to the common belief that scorpion venoms act by targeting

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peripheral organs and also the misconception that these peptides do not cross the BBB

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(Revelo et al., 1996). Determination of whether scorpion neurotoxicity is restricted to

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peripheral actions, or whether a central mechanism may be partly responsible for systemic

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manifestations could be crucial in the trends of clinical therapy (Guidine et al., 2009).

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Our results clearly demonstrate that low doses of centrally injected H. gentili venom induce

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similar systemic manifestations as those observed in experiments using subcutaneous

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injections of much higher doses of scorpion toxins (El Hidan et al., 2015a). In fact, i.c.v

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administration of venom produce heart necrosis, lungs hemorrhage and in some cases

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ACCEPTED MANUSCRIPT congestion and disorganization in glomeruli, alterations that were also observed after

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subcutaneous injection of H. gentili venom especially after 12 h post-envenomation.

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Such similarities between the peripheral outcomes following the systemic and the central

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administrations of the venom could be related to a possible communication between those

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compartments (brain and other organs). Hence, support of this view is provided by the

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Drumond et al. (1995) who postulated that peripheral effects of central injection could be

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due to retrograde circulation of toxin, from ventricular to venous systems through

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absorption at the level of the arachnoidal granulations, favoring direct peripheral action for

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centrally injected toxins. These views have been challenged and refuted. Mesquita et al.

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(2003) showed that intravenous inoculation of the same amount of toxins injected centrally

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did not reproduce any of the peripheral effects of i.c.v injections. It is quite unlikely that

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retrograde toxin circulation would have any significant role on the systemic manifestations

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observed during i.c.v injections in adult animals (Mesquita et al., 2003).

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In our experiments, Microinjected rats with H. gentili venom have manifested symptoms

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accounting for cardio-vascular and respiratory systems perturbations, which are mainly

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observed in severe scorpion poisoning. In fact, i.c.v injection of H. gentili venom to rats

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showed several impacts on function and structure of the heart and lung tissues. Thus, we

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report a focal necrosis of the heart tissue. This is the first study to reports histopathological

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changes in heart tissue after i.c.v injection of H. gentili venom. Our results corroborate

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those found by Fatani et al. (2010) after i.c.v injection of venom from the Old World

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Leiurus quinquestriatus scorpion into conscious rabbits. Based on measuring cardiovascular

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parameters, several other studies have shown that centrally injected scorpion toxins induce

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an increment in heart rate and heart arrhythmia(Mesquita et al., 2003; Guidine et al., 2008;

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Guidine et al., 2009). Scorpion venom action on the cardiovascular system may be mediated

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by a stimulatory action on: the hypothalamus, the peripheral sympathetic nervous system or

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ACCEPTED MANUSCRIPT the central spinal and sympathetic preganglionic neurons. Otherwise, venom neurotoxins

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might modify the adrenal medullary secretory leading to cardiovascular impairments, with

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ultimate possible direct action on the heart (Henriques et al., 1968; Teixeira et al., 2001;

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Ismail, 1995). Our finding suggests a possible involvement of the CNS for cardiac

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alterations observed in severe cases of envenomation. Support of this view is provided by

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the study of Silva el al. (2015) on the role of dorsomedial hypotalamic ionotropic glutamate

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receptors in the hypertensive and tachycardic responses evoked by Tityustoxin (TsTX)

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intracerebroventricular injection which demonstrates that the injection of ionotropic

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glutamate receptors antagonists in dorsomedial hypotalamus abolished the pressor and the

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tachycardic response evoked by i.c.v injected TsTX, suggesting that the central circuit

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recruited by TsTX, whose activation results in an array of physiological and behavioral

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alterations, depend on the activation of dorsomedial hypotalamic ionotropic glutamate

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receptors.

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Hemorrhage and edema in pulmonary parenchyma is one of the most frequent

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complications in scorpionic accidents (Hering et al., 1993). According to literature, lung

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edema evoked by scorpion venom can be induced by two types of factors: a cardiogenic

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factor directly related to a cardiac dysfunction of the left ventricle, and a noncardiogenic

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one caused by the release of inflammatory mediators (Adi-Bessalem et al., 2008). Whereas,

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our results clearly demonstrate that centrally injected, venom alters also the pulmonary

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function by inducing hemorrhage and edema, suggesting an involvement of the CNS in the

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action of scorpion toxins on the pulmonary parenchyma. The microscopic aspect of the

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lungs (extensive hemorrhage) from animals i.c.v injected with H. gentili venom, suggest an

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increase in lung vascular permeability and a higher vascular fragility. Such impairments

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might be due to the left ventricular failure, which induce an enhancement of blood pressure

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in the lung vascular system, leading, to edema and to vascular rupture, explaining the

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ACCEPTED MANUSCRIPT hemorrhage (Mesquita et al., 2003). Moreover Santana et al. (1996), in the study of the

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effect of Tityus serrulatus crude venom (5 mg/100 g, s.c.) in anesthetized rats, reported

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finding of a unilateral lung edema without the presence of cardiac dysfunction, which would

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indicate a specific and direct lung target of the venom (Santana et al., 1996).

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At the biochemical level, i.c.v injection of H. gentili venom was strongly accompanied by

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an alteration of several parameters which in part reflect the abnormal functional aspects of

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some vital organs and supports the above observations. Thus, following i.c.v injection of H.

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gentili venom, an increment in levels of plasmatic LDH and CPK enzymes was noticeable

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as a consequence of myocardial and pulmonary damages. After extensive tissue destruction

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that supports the cardiac histopathological abnormalities described above. such enzymes are

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liberated into serum (Adi-Bessalem et al., 2008). Based on literature, blood biochemical

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parameter disorders were observed after scorpion envenomation, in humans as well as

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animals (Ben Nasr et al., 2009). In addition to heart and lungs, liver seem to be a vulnerable

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target of H. gentili venom. In fact, assessment of ALAT and ASAT showed a significant

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increment 6 h post-envenomation indicating a hepatotoxic potential of the centrally injected

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venom. However, our histopathological analysis has not revealed any obvious profound

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alteration of the liver tissue, which suggest that envenomation could act on the functional

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aspect but not the structure of the hepatocytes. Fatani et al. (2010) had shown that i.c.v

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injection of Leiurus quinquestriatus venom (2 µg/kg) produces marked diffuse fatty

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changes in hepatocytes and focal hemorrhagic areas and also an elevation in the level of

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ASAT and ALAT activities, however, the underlying mechanism by which i.c.v

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administration of the venom could affect the liver function is still extremely far from being

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completely understood.

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Scorpion venom contains small neurotoxins polypeptides consisting of low-molecular

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weight proteins with lethal and paralytic effects (Rodríguez De La Vega and Possani, 2004).

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ACCEPTED MANUSCRIPT These toxins act on ion channels, promoting an impairment that may result in an abnormal

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release of neurotransmitters; known to be involved in the control of several cognitive

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behaviors (El Hidan et al., 2015b). Therefore, in the present investigation, we assessed the

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possible neurobehavioral disturbances occurring in rats i.c.v injected with H. gentili venom.

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A behavioral approach has been focused on the locomotor activity and other cognitive

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impairment such as depression and anxiety. The i.c.v. injection of H. gentili scorpion venom

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induced behavioral alterations characterized by, squeaking, wild-running, jumping,

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trembling, humpback and wet dog shakes. These symptoms were also observed after

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subcutaneous injection of high doses ranged from 0.7 to 1 mg/kg of H. gentili venom (El

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Hidan et al., 2015a). Anterior data report the same finding after i.c.v. injection of Iurus

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dufoureius asiaticus venom (Ozkan et al., 2007). The mice showed excitability, rapidly

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walking as humpback, excessive salivation, weakness, paralysis, coma and consequent

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death. Moreover, behavioral changes such as immobility, wet dog shakes and wild running

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are also described in rats i.c.v. injected with fractions of Tityus serrulatus venom (Nencioni

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et al., 2000).

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As found after subcutaneous injection of H. gentili venom, the i.c.v injection induces the

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same behavioral alterations evident in decreased locomotion and rearing frequencies and

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increased time of immobility. Furthermore, i.c.v injection produces a significant decrease in

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percentage of entries and time into open arms. All these behavioral responses indicate a

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possible direct anxiogenic-like effect of H. gentili venom centrally injected. Our result

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corroborate those found by Bhattacharya (1995), who reports that Mesobuthus tamulus

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venom administered i.c.v. induced a reduction of exploratory activity and rears, and

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increased immobility in the open-field test. In addition, author had observed an increased

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number of entries and time spent on the closed arms of the elevated plus-maze. Recently,

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Yang et al. (2014) had discovered a new neurotoxin from the scorpion Androctonus bicolor

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ACCEPTED MANUSCRIPT with six disulfide bridges, referred as Androcin. Toxicological analysis using the

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recombinant Androcin peptide revealed that this neurotoxin is able to induce severe akinesia

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and anxiogenic-like symptoms in mice. Nevertheless, it is still indistinguishable whether

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such impairments were a direct consequence of the central action of the scorpion venom

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neurotoxins or were derived from the systemic changes induced by the venom injection.

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The first plausible explanation of the mechanism by which venom induces hypo-activity

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and anxiogenic-like effect could be linked to the ability of venom neurotoxins to produce

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neurotransmitters and neuropeptides disorders. However, an indirect action of the

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neurotoxins on the CNS mediated through alterations on the peripheral systems trigged after

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scorpion venom administration cannot be excluded.

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The present investigation has brought new experimental evidence of a peripheral impact of

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the central administration of H. gentili venom. Such impact was manifested by

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physiological and behavioral disturbances, the last of these appearing to reflect a profound

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neuro-modulatory action of H. gentili venom. The lack of established mechanisms of the

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central neuronal responsiveness to scorpion venom requires further studies to complete our

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understanding of the pathophysiological action of those kinds of venom.

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Ethical statement:

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The authors declare that this manuscript complies with the Elsevier Ethical Guidelines for

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Journal Publication.

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Conflict of interest: The authors declare that there are no conflicts of interest. Acknowledgements

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The Authors would like to thank Alison Judge for linguistic consultation. Thanks are also

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conveyed to Mr. Regragui Abderrazzak for technical assistance. References:

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Highlights: • ICV injection of H. gentili venom induces histological changes in heart and

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lungs. • ICV injection of H. gentili venom induces an increase in AST, ALT & LDH activities. • ICV H. gentili venom induces an anxiety-like behavior.

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Ethical statement:

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• The research meets all applicable standards with regard to the ethics of experimentation and research integrity, and the following is being certified/declared true. • As an expert scientist and along with co-authors of concerned field, the paper has been submitted with full responsibility, following due ethical procedure, and there is no duplicate publication, fraud, plagiarism, or concerns about animal or human experimentation.