Histopathological Alterations Induced by Naja naja Crude Venom on Renal, Pulmonary and Intestinal Tissues of Mice Model
Abstract and Figures
Aim: Snake bite causes a significant number of mortality and morbidity throughout the world. So, the current study was carried out to estimate the extinct of damage caused by intraperitoneal introduction of cobra venom on kidney, lung and intestinal tissues of mice model using histological technique. Place and Duration of Study: The entire study including the treatment along with preparing histological slide was conducted in protein science laboratory, Department of Genetic Engineering and Biotechnology, University of Rajshahi, Bangladesh between December 2013 to July 2014. Methods: Twenty five mature female albino mice were divided mainly into two groups as control and envenomated group. Lyophilized Naja naja venom was dissolved in 0.9% NaCl solution and injected intraperitoneally into the mice of the envenomated group at dosages equivalent to LD 50 120 (0.25 mg/kg). Whereas the animals from control group were not received any venomous component. Both groups of animal were sacrificed for histological study and visualized under light microscope. Results: Injection of cobra venom induced a range of histological changes in all envenomated mice comparing with their control. Results from the histopathological examination showed mainly inflammatory cellular infiltration, vacuolation in renal tubules, shrinking of glomeruli, raising space between the walls of Bowman's capsule in renal tissue and alveolar haemorrhage, inflammatory cellular infiltration and edema in pulmonary tissue. No significant histopathological alterations in intestinal tissue were observed without infiltration and mild hemorrhage. Conclusion: The findings from the current study revealed that, cobra venom at lethal dose causes multiple organ failure in experimental animal which could be considered among the factors that lead to death. By observing the site and the mode of action on tissue level, these findings may help to allay the severity of damage by discovering novel anti venom drug.
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... These are typical symptoms of neurotoxicity as neurotoxins in cobra venom are known to affect the central nervous system by blocking transmission of nerve signals to muscles. Consequently, this action halts nerve transmission to the heart and lungs resulting to death due to complete failure of respiratory function ( Al-Mamun et al., 2015 ). Such observations have been reported in mice envenomed with N. nigricollis venom ( Abubakar et al., 2006 ). ...
... Cobra venoms are known to cause histological changes due to the presence of phospholipases ( Al-Mamun et al., 2015 ;Lougin et al., 2010 ). Phospholipase A 2 enzymes (PLA 2 ) is a major enzymes in cobra venom which plays a key role for tissue injuries by disrupting cell membrane permeability through disorganization of lipid bilayer on plasma membrane leading to pore formation with subsequent influx of Na + and water resulting in rapid degeneration of tissues ( Segelke et al., 1998 ). ...
... Renal injury as observed in this study has been documented in mice envenomed with cobra venom ( Al-Mamun et al., 2015 ). Action of the venom may be attributed to circulation of toxins through blood flow since blood is filtered in higher organism by the kidney. ...
Background
: Envenoming by Naja katiensis is known to constitute serious medical challenge. Moringa oleifera is a medicinal plant which have been reported as antivenom agent against pathologies caused by different snakes species. However, the ameliorative effects of extract and fractions of M. oleifera against toxicities induced by N. katiensis venom remain unreported.
Purpose
: This study therefore investigated the ameliorative effects of n-hexane and ethyl acetate fractions obtained from ethanol extract of Moringa oleifera leaves against N. katiensis venom-induced physiological and histological pathologies.
Methods
: Histopathology and antiheamorrhagic activity of M. oleifera extract and fractions were studied using albino male Wistar rats while bovine citrated erythrocytes were used to determine the antiheamolytic and anticoagulant effects. High Performance Liquid Chromatography was performed to chemically characterize the most active fraction of M. oleifera found to be most effective against venom-induced pathologies.
Results
: N. katiensis venom induced severe cellular alterations in the brain, liver and kidney tissues of envenomed untreated rats. However, treatments with the fractions obtained from M. oleifera ethanol extract ameliorated tissue injuries in these organs and the most effective was ethyl acetate fraction. M. oleifera extract and fractions showed a dose dependent inhibitory effects on anticoagulant, hemolytic, and heamorrhagic activities of N. katiensis venom. The HPLC analysis of ethyl acetate fraction of M. oleifera leaf led to the identification of two major bioactive constituents as quercetin and kaempferol.
Conclusion
: This study proved that the ethyl acetate fraction of M. oleifera could serve as a potential source of effective compounds against N. katiensis venom-induced pathologies.
... These inductions of toxic effects by the above biological active components generally attribute towards generating visible pathological anomalies on cardiac, nervous, renal, pulmonary, hepatic, and muscle tissues. Pathological alterations induced by snakebite usually depend upon two basic factors: the species of snake responsible for the bite and the biochemical composition of the specific venom of that species [12][13][14]. Many scientists are using different methodologies and techniques to address this biochemical and pharmacological properties of snakes' venoms. ...
... Numerous studies indicate that cobra venom can induce profuse pathological anomalies in vital tissue structures [12,13,18,51,53]. Histopathological tissue sections of gastrocnemius muscle, heart, kidney, liver, and lungs of mice injected with venom from all five regions manifested severe pathological anomalies including tissue necrosis associated with haemorrhage and congestion, while control mice group resulted in their normal tissue structures. ...
... Several studies with similar histopathology in mice tissues suggest that the potent toxic effect of nonenzymatic fraction of cytotoxins and phospholipase A 2 (PLA 2 ) enzyme leads to these extensive necrotic observations in venom affected tissues by the formation of pores and disturbing the phospholipid structure of lipid bilayer [12,16,54,55]. Other research reports state similar haemorrhages and congestion in several tissue types as haemostatic disturbances produced by N. naja venom components of metalloproteinases and PLA 2 [13,35,56]. Therefore, we can conclude that venom of Sri Lankan N. naja contains functionally similar components proceeding to damage the vessel endothelium and extracellular matrix causing blood leakage into tissues. ...
Several countries residing envenomation due to Naja naja had revealed a disparity in the venom composition according to their geographic location and Sri Lankan cobra still lacks the evidence to support this. Therefore, the current study was focused on addressing relationship between the histopathological changes according to geographic variation of Sri Lankan N. naja venom. The histopathological changes in vital organs and muscle tissues following intramuscular administration of venom of N. naja were studied using BALB/c mice. The median lethal dose of venom of N. naja in the present study was determined to be 0.55, 0.66, 0.68, 0.62, and 0.7 mg/kg for North (NRP), Central (CRP), Western, Southern, and Sabaragamuwa Regional Population venoms, respectively. Histopathological changes were observed in different levels in vital organs and muscle tissues of mice. NRP accompanied significantly higher infiltration of inflammatory and necrotic cells into skeletal muscle and CRP venom demonstrated high level of cardiotoxic effects comparing to other regions. This study revealed a certain extent of variations in the pathological effects of N. naja venom samples according to their geographical distribution.
... Biochemically, cobra venoms are mixtures of proteins, peptides, enzymes, cytotoxins, coagulants and neurotoxins exhibiting toxic actions in living systems [5]. The neurotoxins in cobra venom blocks the transmission of nerve impulses to the muscles consequently affecting the nervous system, heart and lungs thereby leading to organ malfunctioning and ultimately death as a result of complete failure of respiratory function [6]. ...
... Access of toxins to the nervous tissues enhances the possibility of a direct neurotoxic effect as neurotoxicity ultimately depends on the ability of the substance to bind to neural tissue targets and interfere with functional or structural integrity [51]. The assessment of the venom toxicities on the brain of envenomed rats was based on mechanism of action exhibited by N. nigricollis venom as neurotoxins in cobra venom are known to attack the central nervous system [6], disrupt its functions, facilitates nervous dysfunction and shutdown resulting to tetany and ultimately death. The mammalian nervous system has functional design features that predispose it to chemical perturbation. ...
Reproductive and neurological anomalies are often characterized by malfunctioning of reproductive and nervous organs sometimes attributed to systemic toxins. However, limited information is available on the impact of snake venom toxins on male reproductive and nervous system. This study investigated the toxicological effects of Naja nigricollis venom on male reproductive and neural functions in rat model. Twenty male Wistar rats weighing between 195 and 230 g were divided randomly into two groups of ten rats each. Group 1 served as normal control while rats in group 2 were envenomed with a single intraperitoneal injection of 0.25 mg/kg−1 (LD12.5) of N. nigricollis venom on first and twenty fifth day within the period of fifty days experiment. The venom significantly decreased sperm counts, motile cells and volume combined with increased sperm abnormalities. The venom induced hormonal imbalances in the envenomed group as levels of testosterone, luteinizing and follicle stimulating hormones depreciated compared to the control. Oxidative stress biomarkers: malondialdehyde significantly increased parallels with depletion of glutathione level and catalase activities in testis, epididymis and brain of envenomed rats. Furthermore, N. nigricollis venom up-regulated tumor necrosis factor-alpha (TNF-α) and interleukin1-beta (IL-1β) production in testis, epididymis and brain of envenomed rats compared to the control. Also, various histological alterations were noticed in tissues of testis, epididymis and brain of envenomed rats. Findings indicated that N. nigricollis venom is capable of inducing reproductive and neurological dysfunction in envenomed victims.
... Biochemically, cobra venoms are mixtures of proteins, peptides, enzymes, cytotoxins, coagulants and neurotoxins exhibiting toxic actions in living systems [5]. The neurotoxins in cobra venom blocks the transmission of nerve impulses to the muscles consequently affecting the nervous system, heart and lungs thereby leading to organ malfunctioning and ultimately death as a result of complete failure of respiratory function [6]. ...
... Access of toxins to the nervous tissues enhances the possibility of a direct neurotoxic effect as neurotoxicity ultimately depends on the ability of the substance to bind to neural tissue targets and interfere with functional or structural integrity [51]. The assessment of the venom toxicities on the brain of envenomed rats was based on mechanism of action exhibited by N. nigricollis venom as neurotoxins in cobra venom are known to attack the central nervous system [6], disrupt its functions, facilitates nervous dysfunction and shutdown resulting to tetany and ultimately death. The mammalian nervous system has functional design features that predispose it to chemical perturbation. ...
Reproductive and neurological anomalies are often characterized by malfunctioning of reproductive and nervous organs sometimes attributed to systemic toxins. However, limited information is available on the impact of snake venom toxins on male reproductive and nervous system. This study investigated the toxicological effects of Naja nigricollis venom on male reproductive and neural functions of envenomed rats. Twenty male Wistar rats weighing between 195 and 230 g were divided randomly into two groups of ten rats each. Group 1 served as normal control while rats in group 2 were envenomed with a single intraperitoneal injection of 0.25 mg/kg⁻¹ (LD12.5) of N. nigricollis venom on first and twenty fifth day within the period of fifty days experiment. The venom significantly decreased sperm counts, motile cells and volume combined with increased sperm abnormalities. The venom induced hormonal imbalances in the envenomed group as levels of testosterone, luteinizing and follicle stimulating hormones depreciated compared to the control. Oxidative stress biomarkers: malondialdehyde significantly increased parallels with depletion of glutathione level and catalase activities in testis, epididymis and brain of envenomed rats. Furthermore, N. nigricollis venom up-regulated tumor necrosis factor-alpha (TNF-α) and interleukin1-beta (IL-1β) production in testis, epididymis and brain of envenomed rats compared to the control. Also, various histological alterations were noticed in tissues of testis, epididymis and brain of envenomed rats. These findings indicated that N. nigricollis venom is capable of inducing reproductive and neurological dysfunction in envenomed victims.
... Similarly, the venom of N. naja from Rajshahi, Bangladesh has a low i.p. LD 50 of 0.25 µg/g [107], although its venom proteomics has not been studied to reveal the quantitative abundance of alpha-neurotoxins in the venom. On the other hand, the venom of NN-SL tested in the present study (representing the southernmost dispersal of N. naja) has an i.v. ...
... Comparison of the relative expression levels of alpha-neurotoxins and cytotoxins reported in the venom proteomes and/or venom-gland transcriptomes of Naja naja for snake populations from different localities in South Asia, including Pakistan, India, Sri Lanka (current work) and Bangladesh. Values of intravenous median lethal doses of the venoms were included where applicable, based on available references[6,8,44,107] except for locality #8 (Tamil Nadu, India, unpublished data from authors). Details of toxin abundances sorted were provided in Supplementary File S3. ...
Inadequate effectiveness of Indian antivenoms in treating envenomation caused by the Spectacled Cobra/Indian Cobra (Naja naja) in Sri Lanka has been attributed to geographical variations in the venom composition. This study investigated the de novo venom-gland transcriptomics and venom proteomics of the Sri Lankan N. naja (NN-SL) to elucidate its toxin gene diversity and venom variability. The neutralization efficacy of a commonly used Indian antivenom product in Sri Lanka was examined against the lethality induced by NN-SL venom in mice. The transcriptomic study revealed high expression of 22 toxin genes families in NN-SL, constituting 46.55% of total transcript abundance. Three-finger toxins (3FTX) were the most diversely and abundantly expressed (87.54% of toxin gene expression), consistent with the dominance of 3FTX in the venom proteome (72.19% of total venom proteins). The 3FTX were predominantly S-type cytotoxins/cardiotoxins (CTX) and α-neurotoxins of long-chain or short-chain subtypes (α-NTX). CTX and α-NTX are implicated in local tissue necrosis and fatal neuromuscular paralysis, respectively, in envenomation caused by NN-SL. Intra-species variations in the toxin gene sequences and expression levels were apparent between NN-SL and other geographical specimens of N. naja, suggesting potential antigenic diversity that impacts antivenom effectiveness. This was demonstrated by limited potency (0.74 mg venom/ml antivenom) of the Indian polyvalent antivenom (VPAV) in neutralizing the NN-SL venom. A pan-regional antivenom with improved efficacy to treat N. naja envenomation is needed.
... N. nigricollis is a cobra (Naja spp.) with a high abundance of cytotoxins, cardiotoxins and neurotoxins present in the venom which contribute to cell destruction, tissue necrosis and systemic neurotoxicity after envenomation (Bala et al., 2022;Chong et al., 2020;Gomez et al., 2023). Neurotoxins such as phospholipase A2 (PLA2) is well known presynaptic neurotoxic enzyme present in N. nigricollis venom (NnV) which distorts muscle contraction processes by blocking the binding of acetylcholine to its receptors and disrupting the transmission of nerve impulses to the muscles consequently leading to malfunctioning of major organs of the central nervous system (Ajisebiola et al., 2022;Al-Mamun et al., 2015). Furthermore, neurotoxic venoms have been reported to cause detrimental effect by inducing oxidative damage and inflammation in important organ of the nervous system resulting in neurological toxicities and other severe pathophysiological alterations after NnV envenomation in vivo (Adeyi et al., 2020;Ajisebiola et al., 2022). ...
Naja nigricollis venom (NnV) contains neurotoxins that influence neurological functions. Kaempferol is a bioactive compound present in edible plants with numerous pharmacological activities. This study investigated the ameliorative potential of kaempferol against NnV-induced neurotoxicity in rats. Fifty male Wistar rats were randomized into five groups (n = 10). Group 1 rats were the control while 1.0 mg/kg − 1 (LD 50) of NnV was injected intraperitoneally into rats in groups 2-5 to observed neurotoxicity. Group 2 was untreated post en-venomation, while groups 3-5 were treated with polyvalent antivenom, 4 and 8 mg/kg of kaempferol, respectively. The biochemical analysis, neurotoxicity, and pathomorphological defects were assessed in the brain of the envenomed treated rats. Envenomation with NnV elevated oxidative and inflammatory biomarkers, and induced neurotoxicity accompanied with neurobehavioral deficits, and severe pathohistological defects were seen in the brain of untreated envenomed rats. However, treatment with kaempferol significantly (p < 0.05) decreased malondialdehyde (MDA) levels and upregulated levels of reduce glutathione (GSH) antioxidant including su-peroxide dismutase (SOD) and glutathione peroxidase (GPX) antioxidant enzymes, while inflammatory bio-markers; nitric oxide (NO) levels and myeloperoxidase (MPO) activity significantly decreased in envenomed treated groups. Kaempferol upregulated dopamine concentration with significant suppression of acetylcholin-esterase (AchE) activity, and restored neurobehavioral and locomotor activities in envenomed treated rats. Also, severe pathomorphological alterations observed in the cortex of the brain were attenuated after kaempferol treatment. The underlaying ameliorative mechanisms of kaempferol are linked to its antioxidant activity, lipid peroxidation inhibition, anti-inflammatory activity, acetylcholinesterase suppression, and alleviation of dopa-mine system and neurobehavioral abilities. Abbreviations NnV Naja nigricollis venom
... The edema interstitial and congestion with blood vessel dilation have also been observed. Similarly, a study on Sri Lankan Naja naja venoms in mice showed vascular abnormalities such as capillary congestion, irregular capillary endothelium, arteritis, and a thickening of the walls and alveolar spaces alveolar, thus an infiltration of inflammatory cells into the interstitial tissue [36]. A. Al-Mamun also reported severe changes in the albino mice (26-30 g) injected with 1 2 LD 50 Naja naja venom at the pulmonary level such as significant inflammatory cell infiltration and interstitial edema [37]. The observation of the accumulation of leukocytes (neutrophils and macrophages) indicates the neurotoxic effect of Phospholipase A 2 on the lung tissue [36]. ...
... The edema interstitial and congestion with blood vessel dilation have also been observed. Similarly, a study on Sri Lankan Naja naja venoms in mice showed vascular abnormalities such as capillary congestion, irregular capillary endothelium, arteritis, and a thickening of the walls and alveolar spaces alveolar, thus an infiltration of inflammatory cells into the interstitial tissue [36]. A. Al-Mamun also reported severe changes in the albino mice (26-30 g) injected with 1 2 LD 50 Naja naja venom at the pulmonary level such as significant inflammatory cell infiltration and interstitial edema [37]. The observation of the accumulation of leukocytes (neutrophils and macrophages) indicates the neurotoxic effect of Phospholipase A 2 on the lung tissue [36]. ...
In Morocco, eight species of venomous snakes belonging to the Viperidae and Elapidae families are responsible for severe envenomation cases. The species from the Elapidae family is only represented by the medically relevant cobra Naja haje, which is widely distributed in North Africa. However, there is little information on the systemic effects of Moroccan cobra venom on vital organs due to regional variations. It has been demonstrated that the venom of Naja haje from Egypt causes hemorrhage, while the venom of the Moroccan cobra is neurotoxic and devoid of systemic bleeding. This variability is known to significantly influence treatment efficacy against Naja haje cobra bites in the Middle East. In this study, we examined the pathophysiological mechanisms responsible for the lethality induced by Naja haje venom, as well as the evaluation of the neutralizing capacity of two antivenoms; the monospecific antivenom made for Naja haje only and the antivenom marketed in the Middle East and North Africa. We first determined the toxicity of Naja haje venom by LD50 test, then compared the neutralizing capacity of the two antivenoms studied by determining the ED50. We also performed histological analysis on Swiss mice envenomed and treated with these antivenoms to observe signs of cobra venom envenomation and the degree of reduction of induced systemic alterations. The results showed significant differences between both antivenoms in terms of neutralization. The monospecific antivenom was four times more effective than the marketed antivenom. These results were confirmed by a histological study, which showed that monospecific antivenoms neutralized severe signs of mortality, such as congestion of blood vessels in the heart and kidneys, pulmonary and renal edema, cytoplasmic vacuolization of hepatocytes in the liver, and infiltration of inflammatory cells in the brain and spleen. However, the polyvalent antivenom failed to protect all severe lesions induced by Naja haje venom in mice. These findings highlight the negative impact of geographic variation on the effectiveness of conventional antivenom therapy and confirm the need for a specific Naja haje antivenom for the effective treatment of cobra envenomation in Morocco.
... The liver is the body's primary detoxifying organ, could be damaged by a variety of toxic components found in venom. More severe tissue damages are caused by elapid and viper (Al-Mamun et al., 2015b;Dissanayake et al., 2018) venoms. ...
As a disaster-prone country with unique geographical features, snake biting is a major public health concern in Bangladesh. The primary reasons of mortality from snakebite include late presentation to the hospital, low efficacy of antivenom, and a lack of adequate management facilities. Because snake venom characteristics vary depending on geographical location, antivenom should be manufactured from snakes native to the region in which it would be administered. Bungarus caeruleus is a highly venomous snake contributing to the major snakebite issue in Bangladesh. Therefore, the neutralization efficacy of the antivenom against B. caeruleus venom was evaluated in the current study along with the characterization of venom. For biological characterization of venom, RP-HPLC and SDS page profiling, hemolytic activity, hemorrhagic activity, phospholipases A2 (PLA2) activity, edema inducing activity and histopathological observations were carried out following standard protocol. LD50 of the venom was calculated along with neutralization potency of Incepta antivenom through probit analysis. Results showed that venom possesses phospholipase A2 activity, hemolytic activity and edema inducing activity while hemorrhagic activity was absent in the skin of envenomed mice. Histopathological alterations including necrosis, congestion and infiltrations were observed in envenomed mice organs after hematoxylin and eosin staining. Neutralization study showed that Incepta polyvalent antivenom could neutralize (potency 0.53 mg/ml) the lethal effect in in vitro study on mice. Further investigation on snakebite epidemiology and clinical observations of the envenomed patients will help in combating the snakebite problem more efficiently.
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