THE SCIENTIFIC JOURNAL OF THE VETERINARY FACULTY UNIVERSITY OF LJUBLJANA SLOVENIAN VETERINARY RESEARCH SLOVENSKI VETERINARSKI ZBORNIK Slov Vet Res • Ljubljana • 2022 • Volume 59 • Number 4 • 169–222459 Volume S lo v Ve t R es 2 0 2 2 ; 5 9 (4 ): 16 9 –2 2 2 SLOVENIAN VETERINARY RESEARCH SLOVENSKI VETERINARSKI ZBORNIK Slov Vet Res 2022; 59 (4) Review Article Rajčević U, Smole A. Preclinical mouse models in adoptive cell therapies of cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 Original Research Articles Parlak K, Naseri A, Yalcin M, Akyol E T, Ok M, Arican M. Evaluation of trauma scoring and endothelial glycocalyx injury in cats with head trauma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .185 El-Bahr SM, Al-Sultan S, Hamouda AF, Atwa SAE, Abo-Kora SY, Amin AA, Shousha S, Alhojaily S, Alnehas A, Elzogby RR. Pectin improves hemato-biochemical parameter, histopathology, oxidative stress biomarkers, cytokines and expression of hepcidin gene in lead induced toxicity in rats . . . . . . . . . . . . . . . . . . 195 Case Report Wu B, Wang J, Cai T, Wang C, Li D, Deng L, Peng X. Pathological findings in an old female giant panda – a case report . . . . . . . . . . 211 Author Index Volume 59, 2022 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219 THE SCIENTIFIC JOURNAL OF THE VETERINARY FACULTY UNIVERSITY OF LJUBLJANA SLOVENIAN VETERINARY RESEARCH SLOVENSKI VETERINARSKI ZBORNIK 459 Volume Slov Vet Res • Ljubljana • 2022 • Volume 59 • Number 4 • 169–222 The Scientific Journal of the Veterinary Faculty University of Ljubljana SLOVENIAN VETERINARY RESEARCH SLOVENSKI VETERINARSKI ZBORNIK Previously: RESEARCH REPORTS OF THE VETERINARY FACULTY UNIVERSITY OF LJUBLJANA Prej: ZBORNIK VETERINARSKE FAKULTETE UNIVERZA V LJUBLJANI 4 issues per year / Izhaja štirikrat letno Volume 59, Number 4 / Letnik 59, Številka 4 Editor in Chief / Glavna in odgovorna urednica: Klementina Fon Tacer Co-Editors / Sourednici: Valentina Kubale Dvojmoč, Sara Galac Technical Editor / Tehnični urednik: Matjaž Uršič Assistant to Editor / Pomočnica urednice: Metka Voga Published by / Založila: University of Ljubljana Press / Založba Univerze v Ljubljani For the Publisher / Za založbo: Gregor Majdič, the Rector of the University of Ljubljana / rektor Univerze v Ljubljani Issued by / Izdala: Veterinary Faculty University of Ljubljana / Veterinarska fakulteta Univerze v Ljubljani For the Issuer / Za izdajatelja: Breda Jakovac Strajn, the Dean of the Veterinary Faculty / dekanja Veterinarske fakultete Editorial Board / Uredniški odbor: Vesna Cerkvenik Flajs, Robert Frangež, Polona Juntes, Tina Kotnik, Alenka Nemec Svete, Matjaž Ocepek, Joško Račnik , Jože Starič, Nataša Šterbenc, Marina Štukelj, Tanja Švara, Ivan Toplak, Modest Vengušt, Milka Vrecl Fazarinc, Veterinary Faculty / Veterinarska fakulteta, Tanja Kunej, Jernej Ogorevc, Tatjana Pirman, Janez Salobir, Biotechnical Faculty / Biotehniška fakulteta, Nataša Debeljak, Martina Perše, Faculty of Medicine / Medicinska fakulteta, University of Ljubljana / Univerza v Ljubljani; Andraž Stožer, Faculty of Medicine University of Maribor / Medicinska fakulteta Univerze v Mariboru; Cugmas Blaž, Institute of Atomic Physics and Spectroscopy University of Latvia / Inštitut za atomsko fiziko in spektroskopijo Univerze v Latviji Editorial Advisers / Svetovalca uredniškega odbora: Gita Grecs-Smole for Bibliography (bibliotekarka), Luka Milčinski for Electronic media (za elektronske medije) Reviewing Editorial Board / Ocenjevalni uredniški odbor: Breda Jakovac Strajn, Gregor Majdič, Ožbalt Podpečan, Gabrijela Tavčar Kalcher, Nataša Tozon, Jelka Zabavnik Piano, Veterinary Faculty University of Ljubljana / Veterinarska fakulteta Univerze v Ljubljani; Alexandra Calle, John Gibbons, Laszlo Hunyadi, Howard Rodriguez-Mori, Texas Tech University, School of Veterinary Medicine / Šola za veterinarsko medicino Univerze Texas Tech; Jovan Bojkovski, Faculty of Veterinary Medicine, University of Belgrade / Fakulteta za veterinarsko medicino Univerze v Beogradu; Antonio Cruz, Justus Liebig University of Giessen / Univerza Justus Liebig v Giessnu; Gerry M. Dorrestein, Dutch Research Institute for Birds and Special Animals / Nizozemski raziskovalni inštitut za ptice in eksotične živali; Zehra Hajrulai-Musliu, Faculty of Veterinary Medicine, University Ss. Cyril and Methodius, Skopje / Fakulteta za veterinarsko medicino Univerze Ss. Cirila in Metoda v Skopju; Wolfgang Henninger, Diagnostic Centre for Small Animals, Vienna / Diagnostični center za male živali, Dunaj; Aida Kavazovic, Faculty of Veterinary Medicine University of Sarajevo / Fakulteta za veterinarsko medicino Univerze v Sarajevu; Nevenka Kožuh Eržen, Krka d.d, Novo mesto; Eniko Kubinyi, Faculty of Sciences, Eövös Loránd University Budapest / Fakulteta za znanosti Univerze Eövös Loránd v Budimpešti; Louis Lefaucheur, French National Institute for Agriculture, Food, and Environment (INRAE) / Francoski nacionalni inštitut za kmetijstvo, prehrano in okolje; Peter O'Shaughnessy, University of Glasgow / Univerza v Glasgowu; Peter Popelka, University of Veterinary Medicine and Pharmacy in Košice / Univerza za veterinarsko medicino in farmacijo v Košicah; Uroš Rajčević, Novartis, Lek Pharmaceuticals d.d., Ljubljana; Dethlef Rath, Federal Research Institute for Animal Health, Friedrich-Loeffler-Institut, Germany / Zvezni raziskovalni inštitut za zdravje živali, Inštitut Friedrich-Loeffler, Nemčija; Alex Seguino, University of Edinburgh / Univerza v Edinburgu; Ivan-Conrado Šoštarić-Zuckermann, Faculty of Veterinary Medicine University of Zagreb / Fakulteta za veterinarsko medicino Univerze v Zagrebu; Henry Staempfli, Ontario Veterinary College, Canada / Veterinarska visoka šola Ontario, Kanada; Frank J. M.Verstraete, University of California Davis / Univerza v Kaliforniji, Davis; Thomas Wittek, University of Veterinary Medicine Vienna / Univerza za veterinarsko medicino na Dunaju Address: Veterinary Faculty, Gerbičeva 60, 1000 Ljubljana, Slovenia Naslov: Veterinarska fakulteta, Gerbičeva 60, 1000 Ljubljana, Slovenija Tel.: +386 (0)1 47 79 100, Fax: +386 (0)1 28 32 243 E-mail: slovetres@vf.uni-lj.si Sponsored by the Slovenian Research Agency Sofinancira: Javna agencija za raziskovalno dejavnost Republike Slovenije ISSN 1580-4003 Printed by/tisk: DZS, d.d., Ljubljana, December 2022 Number of copies printed / Naklada: 220 Indexed in/indeksirano v: Agris, Biomedicina Slovenica, CAB Abstracts, IVSI Urlich’s International Periodicals Directory, Science Citation Index Expanded, Journal Citation Reports – Science Edition https://www.slovetres.si/ This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License / To delo je ponujeno pod licenco Creative Commons Priznanje avtorstva-Deljenje pod enakimi pogoji 4.0 Mednarodna licenca SLOVENIAN VETERINARY RESEARCH SLOVENSKI VETERINARSKI ZBORNIK Slov Vet Res 2022; 59 (4) Review Article Rajčević U, Smole A. Preclinical mouse models in adoptive cell therapies of cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 Original Research Articles Parlak K, Naseri A, Yalcin M, Akyol E T, Ok M, Arican M. Evaluation of trauma scoring and endothelial glycocalyx injury in cats with head trauma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .185 El-Bahr SM, Al-Sultan S, Hamouda AF, Atwa SAE, Abo-Kora SY, Amin AA, Shousha S, Alhojaily S, Alnehas A, Elzogby RR. Pectin improves hemato-biochemical parameter, histopathology, oxidative stress biomarkers, cytokines and expression of hepcidin gene in lead induced toxicity in rats . . . . . . . . . . . . . . . . . . 195 Case Report Wu B, Wang J, Cai T, Wang C, Li D, Deng L, Peng X. Pathological findings in an old female giant panda – a case report . . . . . . . . . . 211 Author Index Volume 59, 2022 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219 Received: 29 June 2022 Accepted for publication: 16 September 2022 Slov Vet Res 2022; 59 (4): 173–84 DOI 10.26873/SVR-1513-2022 UDC 606:616-056.7:616-006.4:602.68:606:61:615.37:57.084 Review Article Introduction Adoptive cell therapy (ACT) is a next-generation approach to treating cancer based on immune cells engineered to specifically recognize and effectively eliminate cancer cells. T-cell therapy using chimeric antigen receptors (CARs) has emerged as a leading approach in ACT (1). CARs are designed receptor molecules that merge specificity of monoclonal antibodies with the signalling capacity and effector functions of the T cell receptor (TCR) in T cells (2). The initial CAR designs, referred to as first-generation CARs, include an extracellular antigen-binding domain, usually in the form of a single-chain variable fragment (scFv) derived PRECLINICAL MOUSE MODELS IN ADOPTIVE CELL THERAPIES OF CANCER Uroš Rajčević1*, Anže Smole2* 1Lek, d.d., Technical Research and Development, Kolodvorska cesta 27, 1234 Mengeš, Slovenia, 2National Institute of Biology, Department of Genetic Toxicology and Cancer Biology, Immunology and Cellular Immunotherapy (ICI) Group, 1000 Ljubljana, Slovenia *Corresponding author, E-mail: uros.rajcevic@novartis.com, anze.smole@nib.si Abstract: Engineered T cell-based therapies are an advanced approach for cancer immunotherapy using genetically modi- fied T cells. To date, CD19 and BCMA targeting Chimeric Antigen Receptor (CAR) T cells have been approved for the treatment of certain hematologic malignancies. The success of CAR-T cells is offset by limited efficacy, particularly in solid tumors, and safety risks. Preclinical in vivo research, which is highly dependent on reliable mouse models, has been a cornerstone of the suc- cess story of adoptive cell therapies and continues to provide invaluable information for the development of the next generation of cellular immunotherapies. In this review we describe four of the most common preclinical mouse models: xenograft models, syngeneic models, immunocompetent transgenic models and humanized mouse models. All of these have advantages and disadvantages and no mouse model can fully recapitulate the human situation because of inherent differences and treatment complexity. Reports suggest that using a combination of mouse models in preclinical in vivo research prior to translating the treat- ment to humans in clinical trials can help incrementally improve the quality, safety, and efficacy of the treatment and provide more comprehensive information than a single model. Key words: mouse model; xenograft; syngeneic; transgenic; humanized; CAR-T; adoptive cell therapy from an antibody, linked to intracellular signalling domains, most often derived from the components of the TCR complex, such as the CD3 zeta chain (3, 4). This molecule is capable of recognizing antigens independently of HLA (human leukocyte antigens) presentation but does not support long- term T cell persistence and effector responses due to its limited signalling capacity (5). The second- generation CAR design incorporates additional co-stimulatory domains such as CD28 and 4-1BB (TNFRSF9) that enhance expansion, effector functions and persistence of CAR-T cells (2). The second-generation CAR-T design was the basis for successful clinical trials in relapsed or refractory paediatric and adult blood malignancies (6-10) that have led the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) to approve CD19-targeting CAR-T cells in 2017 U. Rajčević, A. Smole174 and 2018, respectively and BCMA-targeting CAR-T cells in 2021 (11). However, adoptive cancer immunotherapy is associated with safety risks such as cytokine release syndrome (CRS) and neurologic toxicities (12), which have led to life- threatening complications (13). In addition, the efficacy of cellular immunotherapy in solid tumor as well as hematologic malignancies is limited, due in part to the immunosuppressive tumor microenvironments, intrinsic T-cell dysfunction (14) and lack of unique surface antigens (15). Various approaches have been pursued to increase the efficacy of adoptive immunotherapy in cancer, and preclinical mouse models are currently irreplaceable to validate their efficacy and safety. Excellent reviews present the use of preclinical models for adoptive cell therapies (16-18). Here, we focus on how preclinical mouse models have supported recent advances in the development of next-generation cellular immunotherapies. Human xenograft models A xenograft is a cell, tissue, or organ transplant from a donor that is of a different species then the recipient. In the use of mouse models for the study of human diseases, the most common type of xenograft is the transfer of human tissue, including cells and biopsies, to a mouse recipient. The recipient must be immunocompromised to avoid rejecting foreign human cells. Cells are applied either non-original place (ectopic model) or in the organ from which the tissue is derived (orthotopic model) (reviewed in (19)). In this way, researchers can translate in vitro findings into preclinical in vivo stage to evaluate the efficacy and safety of cellular immunotherapy in a given disease. Patient-derived cancer xenografts (PDX) are a simulation of a specific patient’s tumor in an animal model. The various types of immunocompromised mice used in tumor xenograft models lack part or most of the immune system so they cannot reflect the immune system of humans. Their immune response to tumors is simplified and does not capture the full complexity of the response. In the past, different types of immunocompromised mice were developed to mimic the human immune system to varying degrees. The development of immunocompromised mice dates back to athymic mice reported as early as 1966 (20), non-obese diabetic severe combined immune-deficient mice (NOD SCID) and improved NOD SCID mice with Interleukin 2 Receptor Subunit Gamma (IL2Rg) deficiency (16, 21, 22). Since then, the use of NSG (NOD SCID IL2Rg-) (reviewed in (23)) in CAR-T has been reported by several authors including (24). The NSG mouse was developed by backcrossing the Il2rg−/−mouse resulting from a complete null mutation onto NOD/ShiLtSz-Prkdc SCID mouse (25). The NOG mouse was developed by backcrossing the Il2rg−/− mouse resultin from a truncated intracellular signaling domain onto NOD/ShiLtSz-Prkdc SCID mouse (26). In NOG mice, the Il2rg mutant gene is expressed and produces a protein that binds cytokines but does not signal. Conversely, Il2rg gene expression does not occur in NSG mice (27, 28). The use of the xenograft models gives us opportunity to assess the effect of the human CAR-T cells on human tumors but there are no interactions with other immune cells or healthy tissues. Nevertheless, xenograft models are often used as the initial preclinical mouse model in proof-of-concept studies in the development of next-generation cellular immunotherapies. They also allow functional validation of engineered human T cells including tumor targeting, anti- tumor activity, secretion of cytokines, tonic signaling, and intrinsic dysfunction such as T cell exhaustion. Here are selected recent examples that represent only a snapshot of numerous studies in this highly active and explored field of research. As described in the introduction, the development of second-generation CAR-T cells that contain costimulatory domains in addition to CD3 zeta has been critical for clinical efficacy. Currently, so-called third-generation CAR-T cells, which integrate multiple costimulatory domains into the same CAR molecule, are being tested for efficacy and safety (29). Xenograft models have been useful in the development of second and third generation CAR-T cells (30). Route of administration is important in solid tumors (31). Xenograft models revealed the main differences between the two domains, CD28 and 4-1BB (TNFRSF9). Exhaustion was ameliorated by 4-1BB (TNFRSF9) and exacerbated by CD28 (32). CD28 promoted faster tumor regression, while 4-1BB (TNFRSF9) promoted multiple cytokine secretion (33). CAR-T cells, which can be remotely controlled by the addition of small-molecule were tested Preclinical mouse models in adoptive cell therapies of cancer 175 in immunodeficient NSG mice. The authors demonstrated that the use of a split receptor, in which antigen recognition and intracellular signaling domains assemble into a functional unit only after the addition of a heterodimerizing small molecule, allows remote control of the activity of the engineered CAR-T cells. Such regulation provides additional control of the T cell activity, with the rationale of improving safety (34). Another landmark study at the interface between cellular immunotherapy and synthetic biology is the development of designer T cells equipped with tailored therapeutic response programs through the use of synthetic Notch receptors (synNotch). Using NSG mice with subcutaneously implanted CD19 negative or CD19 positive target cells, authors demonstrate that their system functions as designed in vivo. Specifically, they demonstrated in vivo expression of cytokines and bi-specific tumor-targeting antibodies by the SynNotch T Cells (35). Distinct approach that allows additional control over the injected CAR-T cells to increase safety, but also to allow multiple antigen targeting to mitigate potential antigen escape in CAR-T cell therapy, is the prototype universal immune receptor called SpyCatcher. This universal immune receptor enables covalent binding of targeting ligands to the T cell surface using SpyCatcher-SpyTag chemistry. The SpyCatcher immune receptor redirects primary human T cells by addition of SpyTag-labeled targeting ligands in vivo in a solid tumor xenograft model. (36). As mentioned in the introduction, intrinsic dysfunction of T cells is one of the important limiting factors in cellular immunotherapies. One such example is T cell exhaustion, which leads to defects in T cell functionalities. In an attempt to counteract exhaustion, CAR-T cells were engineered to overexpress the transcription factor c-Jun. In this study, human xenograft models in immunocompromised NOD-SCID-Il2rg−/− (NSG) mice were used as models to demonstrate enhanced expansion, improved function, limited terminal differentiation and enhanced anti-tumor activity (37). Current clinically used CAR-T cells use lentiviral or retroviral vectors to introduce CARs into primary T cells. While this is effective, it poses certain problems related to random integration. With the advent of genome editing approaches most notably CRISPR/Cas systems, the CARs (38) or TCRs (39, 40) were introduced into the endogenous TCR genomic locus. These protocols utilize CRISPR/Cas9 and the homology-directed repair (HDR) pathway with either viral (38) or non-viral (39, 40. 41) donor template delivery. Both landmark approaches were validated in NOD/SCID/ IL2gr-null (NSG) xenograft models. In these studies, xenograft models were sufficient to provide evidence of the concept of these novel platforms, specific targeting and tumor control, as well as improved functionalities of human T cells engineered with designed immune receptors. Tasian reports that CAR-T's orchestration of off-target toxicities may only be found in early clinical trials (41). Mouse xenografts are useful in screening for basic CAR-T efficacy and for answering specific human biology questions. Additional studies in immune competent hosts are required to evaluate CAR safety. The hostile tumor microenvironment (TME) includes Tregs and MDSCs, but it’s largely ignored in preclinical immunocompromised models (42). Tregs may partially explain worse CAR-T clinical trial results in solid tumors, and their inclusion in xenograft models may provide more accurate results. In xenograft models it is difficult to distinguish between xenogeneic rejection, graft versus host disease (GVHD), allogenic response of human CAR-T cells to the tumor and actual CAR-T therapeutic efficacy. Therefore, appropriate and rigorous controls in experimental design are very important to obtain reliable results and draw correct conclusions. One such control that aids in differentiating the above-mentioned effects from the on-target responses of designed cellular immunotherapies are T cells engineered with a non-targeting immune receptor, such as CAR directed against target that is not expressed on tumor cells. In the absence of the host immune system, it is not possible to test the TME, tumor metastatic potential, or host response to CAR-T. Taken together, xenograft models provide key insights into the function of human CAR-T cells against human tumors in vivo, which has been a basis for clinical success. This allowed the study of basic properties of human CAR-T cells such as anti-tumor activity, secretion of cytokines, expansion and persistence in vivo. However, in the absence of an interacting immune system, these models do not allow for comprehensive evaluation of immune-mediated mechanisms as well as on- target off-tumor toxicities (Figure 1). U. Rajčević, A. Smole176 To gain deeper insight into mechanism of action, interactions with the endogenous immune system and the role of endogenous immune response, xenograft models need to be complemented with syngeneic mouse models. Syngeneic mouse models Syngeneic models refer to genetically identical or sufficiently identical and immunologically compatible individuals to allow transplantation. The main feature of syngeneic mouse models is their immunocompetence, including full mouse immunity and comprehensive stroma. Important factor in their comprehensive use is their relative simplicity compared with other immunocompetent models. The field of engineered cellular immunotherapies is moving towards increasing the efficacy and improving safety. Often, designed T cells rely upon recruiting endogenous immune response or counteracting immunosuppressive TME. Elucidation of the effects and mechanisms cannot be performed in a comprehensive manner in immunocompromised mice because the interacting immune system is lacking. Here, we list selected reports of upgraded CAR-T cells whose functions have been studied in various syngeneic mouse models. In preclinical studies of adoptive cell therapy in a syngeneic model, the CAR-T, tumors, and target antigens are all mouse derived. Thus, the model allows observation of the CAR-T cells in the context of a functional immune system (16). This model can reveal-on-target off-tumor toxicity (43). Its major drawback is that mouse biology does not fully recapitulate human biology. Mouse syngeneic models have largely been unable to mimic CRS. Mouse CAR-T have shorter persistence and are more susceptible to activation–induced cell death compared to human. Syngeneic models do not provide much insight into the mechanisms of human CAR-T cells (44). Initial syngeneic models of CAR-T demonstrated the superior efficiency of CAR-T over monoclonal antibodies. Preconditioning of the patient by irradiation or chemoablation was crucial for the efficacy of CAR-T therapy (45, 46). In this way, B-cell aplasia was shown as toxicity – mice were clear of lymphoma, but B cells were also absent. Cheadle (47) showed that first generation CAR-T were efficient in removing lymphoma, but not persistent. Second generation CAR-T cells induced B-cell aplasia and chronic toxicity accompanied by CD11b+Gr1+ myeloid derived suppressor cells; elevated TNFα and IFNγ point toward CRS–BALB/c (48), but not in C3H/HeJ or C57BL/6J – side effects vary between strains. Better and more accurate preclinical models are needed for CAR-T in solid tumors. There the combination with checkpoint blockade regimens was shown to be successful (49). Syngeneic model also demonstrated potential toxicities and strain- specific effects (50). Chinnasamy (51) emphasized that mouse strains must be carefully selected or tested on multiple strains to ensure that toxicity is accurately modeled. Widely varying results in studies using the same tumor associated antigen (TAA) but different mouse strains as seen in anti-CD19, anti-NKG2D and anti-VEGF studies caution against using a single mouse strain to determine safety before moving to clinical trials (16). One approach to generate CAR-T cells with improved functionalities is to overexpress an additional accessory molecule along with the immune receptor. Constitutive expression of IL- 12 in CAR-T cells was shown to augment CAR-T cell functions in a syngeneic model. Additional modification of infused hCD19-targeted CAR-T cells to secrete IL-12 allowed for efficient eradication of systemic EL4 (hCD19) tumors, as well as induction of B-cell aplasias, in the absence of prior cyclophosphamide conditioning. This outcome was dependent on both CD4 and CD8 T-cell subsets and required continued in vivo autocrine stimulation of IL-12 as well as modified T cell–IFNγ secretion, which in turn resulted in resistance to Treg-mediated suppression (52). In addition to IL-12, IL-18 emerged as a promising candidate (53-56). In one of these studies (54) syngeneic model of pancreatic and lung tumors revealed that release of IL-18 modulated the immune cell landscape in the tumor. Increased numbers of CD206- M1 macrophages and NKG2D+ NK cells were observed, while Tregs, suppressive CD103+ DCs, and M2 macrophages decreased. These observations were possible because an intact interaction immune system was present. CAR-T cells were also engineered to secrete a combination of IL-7+CCL19 with the rationale that these factors contribute to the maintenance of T-cell zones in lymphoid organs. Upgraded CAR-T cells eradicated established solid tumors Preclinical mouse models in adoptive cell therapies of cancer 177 and prolonged survival compared to conventional CAR-T cells. The syngeneic model showed increased infiltration of dendritic cells and T cells into tumor tissues. Depletion of recipient T cells reduced the therapeutic effects of upgraded CAR-T cell treatment, demonstrating that endogenous immune responses were induced (57). In another study, CAR-T cells were engineered to secrete single-chain variable fragments (scFv) that block PD-1 (PDCD1). Clinically relevant syngeneic model with PD-L1 (CD274) positive tumor targets made it possible to demonstrate that secreted scFv acted on both CAR-T cells and bystander tumor-specific T cells to improve anti- tumor activity (58). CAR-T cells constitutively expressing the immune-stimulatory molecule CD40 ligand (CD40L) demonstrated improved anti-tumor activity. In relevant syngeneic models, the authors investigated the underlying mechanisms and found that CD40L+ CAR-T cells were able to counteract tumor antigen escape variants via CD40/CD40L-mediated cytotoxicity and induction of an endogenous immune response. After adoptive cell transfer, upgraded CAR-T cells licensed antigen-presenting cells and recruited endogenous tumor-recognizing T cells (59). Another study demonstrating the importance of syngeneic mouse models explored how depletion of immunosuppressive M2 tumor- associated macrophages (TAMs) may improve the efficacy of CAR-T cells. The authors found that a folate receptor β (FRβ) positive subset of TAMs exhibited an immunosuppressive M2- like profile. When CAR-T cells were engineered to eliminate these FRβ+ TAMs, an enrichment of proinflammatory monocytes, a recruitment of endogenous tumor-specific CD8+ T cells, delayed tumor growth, and prolonged survival were observed (60). Syngeneic models are useful for evaluating immunotherapies, e.g. in combination studies, particularly using checkpoint inhibitors. Syngeneic model panels can be extensively characterized (e.g. RNA sequencing of cell lines and tumors, immunophenotyping, biomarker identification), and these data can be combined with in vivo efficacy benchmarking profiling results from common checkpoint inhibitors (anti-PD-1, PD-L1, CTLA-4). Syngeneic models are therefore an indispensable for evaluating the safety and efficacy of cellular immunotherapy, as they allow the study of adoptively transferred cells in the context of an interacting immune system. This allows for a rigorous assessment of immune-mediated mechanisms involved in successful therapy as well as toxicities. Potential drawbacks of syngeneic models include difficulties in generating cellular products and limited persistence and efficacy after infusion (Figure 1). Immunocompetent transgenic mice Transgenic animals have been around for sev- eral decades (61). Immunocompetent transgenic mice tolerant to human tumor associated anti- gens (TAAs) have been described in hematologic and solid tumors and for evaluating the safety and efficacy of antitumor immunotherapies (62- 67). Although most anti CD19 CAR-Ts are studied in syngeneic or xenograft models, immunocompe- tent transgenic mouse models can also be used to better determine CAR-T safety. In transgenic mice, human TAA (murine TAA knockout and human TAA knock in) are expressed in mice to highlight the on-target off-tumor effect in healthy tissues. The mice are bred to have TAA expres- sion similar to humans (68). Mice have their own T cells and an intact immune system (like in syngeneic models), but allow the use of human TAA-specific CAR-Ts (like xenografts) (16). A study in C57BL/6J with mouse CD19 knockout and hu- man CD19 knockng restricted to B cells showed no toxicities other than B-cell aplasia (52). CARs targeting different antigens (CD19, CEA, HER2) have been tested in the clinic. The positive effect of preconditioning on adoptively transferred cells was confirmed in transgenic and not xenograft models (16, 69, 70). Engrafted tumors do not recapitulate many of the properties of naturally occurring tumors. A transgenic model in which tumors develop spontaneously can better mimic clinical progression and predict off-tumor toxici- ties (16). Transgenic CEA mice were developed by Zimmerman and colleagues (71) and have been frequently used to test CEA-targeting- CAR-T-cell therapy. In this model with high CEA expression, the toxicity associated with CAR-T cells appears to be limited to high-affinity CAR-T cells (65, 67). One of transgenic mouse models with CEA lev- els equivalent to those in humans (72) has also shown severe side effects associated with CAR-T- cell therapy (73, 74). U. Rajčević, A. Smole178 Figure 1: Schematic representation of preclinical mouse models for adoptive cell therapies Figure created with BioRender.com Humanized models Currently, NOD/SCID/Il2rg−/− (NSG) or BALB/c/RAG2−/−/Il2rg−/− (BRG) mice are the standard recipients in the generation of humanized mice because they are deficient in mouse T cells, B cells, and NK cells (75-77). Transfer of human CD34+ hematopoietic stem and progenitor cells (HSPCs) into newborn NSG or BRG mice results in long-term engraftment of CD34+ cells and reconstitution of multilineage human immune cells (77-79). The comprehensive transfer protocol involves transfer of human CD34+ cells into NSG recipient mice and implantation of human fetal thymus and liver tissue under the kidney capsule of these mice (77, 80, 81). The resulting humanized mice are called bone marrow-liver-thymus (BLT) mice. They support the long-term engraftment and systemic reconstitution of a nearly complete human immune system, including multilineage human adaptive and innate immune cells consisting of T cells, B cells, NK cells, dendritic cells, and macrophages (77, 80, 81). Importantly, human immune cells developed in BLT mice, particularly T cells, are functional and have shown productive responses to skin xenografts and various viral/ bacterial infections (77, 80-82). The next generation of humanized mouse models including NSG-SGM3 (or NSGS), NSGW41, NOG-EXL and MISTRG, support human myelopoiesis at varying degrees and through different strategies (28). Humanized mouse models may bridge the gap between the syngeneic and xenograft models, because they are tolerant to human cells and exhibit aspects of a functional human immune system (16). Until recently, CRS, which typically develops within the first few days after infusion of CD19 CAR-T cells, and neurotoxicity, the two major toxicities associated with clinically used CD19 CAR-T cells in humans, could not be reproduced in preclinical mouse models. This changed with Preclinical mouse models in adoptive cell therapies of cancer 179 the development of a novel xenograft model in humanized mice that faithfully recapitulated both major toxicities (83). This model was developed by engrafting human cord blood hematopoietic stem and progenitor cells (HSPCs) into sub lethally irradiated newborn triple transgenic NSG (SGM3) mice. These mice express human stem cell factor, granulocyte-macrophage colony-stimulating factor (CSF2), and IL-3 to support the engraftment. Successful reconstitution of hematopoiesis was demonstrated, which included human B cells, monocytes, and T cells as well as cells from other lineages. Interestingly, the timing of HSC injection shortly after birth was important for successful human T lymphopoiesis. Circulating T cells exhibited a physiological CD4/CD8 ratio and differentiated into all major T cell differentiation subsets. T cells from these mice were then used to generate second-generation CAR-T cells targeting either CD19 or CD44v6. These CAR-T cells were injected into adult SGM3 mice that had previously been engrafted with ALL-CM leukemia cells. CAR-T cells cleared leukemia, which was associated with CRS characterized by weight loss, fever, and elevated systemic levels of human inflammatory cytokines including IL-6, resembling CRS in humans receiving CD19-targeting CAR-T cells. The authors used this model to show that monocytes are the major sources of IL-1 and IL-6 during CRS and that the syndrome could be prevented by depleting monocytes or by blocking the IL-6 receptor with tocilizumab (IL-6 receptor-blocking antibody). This model also recapitulated the lethal neurotoxicity, characterized by inflammation of the meninges. Interestingly, authors demonstrated that anakinra (IL-1 receptor antagonist) but not tocilizumab ameliorated neurotoxicity. In the follow-up study by the same group, this model was further developed to investigate the efficacy and safety of CAR-T cells derived from preselected T cell subsets. When preselected naive/stem memory T cells were used to generate the CAR-T cellular product, superior anti-tumor activity, expansion and functional phenotype were observed compared to unselected bulk T cells. Surprisingly, this was accompanied by limited incidence of severe CRS and neurotoxicity. Overall, this model reveled improved efficacy and safety when preselected T cells are used to generate CAR-T cell products (84). Therefore, in contrast to all other in vivo models using mice, humanized mouse models were able to reveal and investigate critical toxicities associated with CAR-T cell therapy (Figure 1). In addition to humanized mouse models for T cell-based therapies, models have also been developed that allow adoptive transfer of engineered B cells. In this example, B cells were first isolated from the spleens of humanized donor mice, then genetically engineered and infused into “autologous” humanized recipient mice. Because the recipient mice were humanized with the same source of CD34+ cells as the humanized donor mice, such approach rendered enigneered human B cells tolerant to the host (85). Conclusions Cellular immunotherapy with CAR-T cells is a paradigm shifting approach for the treatment of certain blood cancers. However, limited efficacy and safety risks are the major barriers to advance the field and plethora of academic research groups, biotech and pharmaceutical companies are developing innovative next-generation cellular immunotherapies. After a novel approach has been validated in vitro, the next steps are experiments in preclinical mouse models. Typically, the initial experiments are conducted in human xenograft models, which are well suited for evaluating of novel genetic constructs and designs, immune receptor signaling, anti-tumor activity and intrinsic properties of human CAR-T cells, such as expansion and persistence, as well as certain dysfunctions, including T cell exhaustion. When more in-depth information about the immune mechanisms is required, which is the case when improved cellular immunotherapies aim to recruit endogenous immune responses and/or counteract an immunosuppressive tumor microenvironment, xenograft models are inadequate because they lack an interacting immune response. To answer such questions, syngeneic models are needed and have already provided valuable information for clinical translation. Since syngeneic models are associated with certain challenges including difficulties in the manufacturing of mouse cellular products, in vivo expansion, persistence, and efficacy, studies that systematically develop optimized protocols and procedures are very important (86). In human clinical trials using CAR-T cells, certain toxicities including CRS and neurotoxicity U. Rajčević, A. Smole180 were observed that were not predicted by any of the preclinical models available at the time. Recently, a humanized mouse model was successfully developed to specifically address this challenge and replicate the pathologies observed in humans in preclinical mouse models as well (83). This example highlights the importance of continued development of preclinical mouse models as the field of cellular immunotherapy rapidly grows. Preclinical mouse models continue to be a cornerstone in the development of the next generation of cellular immunotherapies. In this review recent advances and use of the four most common preclinical mouse models are presented. In addition, we presented the selected recent studies in which these models have been used to demonstrate innovative CAR-T cell approaches. The use of multiple models may provide a better understanding of a particular CAR-T therapy than a single model and we anticipate that increasingly sophisticated models will be developed, aided in part by recent advances in genome editing technologies, to comprehensively address the complexities of immunology and cellular immunotherapy. However, we must be aware of the limitations of preclinical mouse models, including the inherent differences between human and mouse biology and immunology. Careful design of properly controlled experiments is essential to generate reliable, high-quality data and draw the right conclusions required for clinical translation of cellular immunotherapies. Acknowledgements We thank K. Butina Ogorelec for reviewing and providing valuable feedback on the manuscript. A.S. received funding from the Slovenian Research Agency (ARRS) for Project J3-3084 and Program P1-0245, and from Research fund of the National institute of biology for Project 10ICIGEN (ICI). 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Predklinične raziskave in vivo, ki so močno odvisne od zanesljivih mišjih modelov, so bile kritični dejavnik zgodbe o uspehu adoptivnih celičnih terapij in še vedno zagotavljajo neprecenljive podatke za razvoj naslednje generacije celičnih imunoterapij. V preglednem članku povzemamo štiri najpogostejše predklinične mišje modele: ksenografte, singenetske modele, imunokompetentne transgenske modele in humanizirane mišje modele. Vsi opisani modeli imajo svoje prednosti in slabosti in noben mišji model ne more do popol- nosti preslikati situacije v človeškem pacientu zaradi medvrstnih razlik ter izjemne zapletenosti zdravljenja. Podatki iz literature kažejo na to, da lahko uporaba kombinacije mišjih modelov v predkliničnih in vivo raziskavah pred translacijo zdravljenja na ljudi v kliničnih poskusih pripomore k postopnemu izboljšanju kakovosti, varnosti in učinkovitosti zdravljenja in zagotovi bolj celostni nabor podatkov kot en sam model. Ključne besede: mišji model; ksenograft; singenetski; transgenski; humanizirani; CAR-T; adoptivna celična terapija Received: 9 August 2021 Accepted for publication: 19 December 2022 Slov Vet Res 2022; 59 (4): 185–93 DOI 10.26873/SVR-1368-2022 UDC 636.8.09:616.831:616-001.1:614.86:616-089.17:005.585 Original Research Article Introduction Head trauma is the most common injury in cats after extremity trauma (1). This type of trauma is commonly associated with motor vehicle accidents and high-rise syndrome (2, 3). Modified Glasgow Coma Scale (mGCS) and Animal Trauma Triage (ATT) are trauma-specific scoring systems used to classify trauma patients for prognostic purposes with quantification of injury severity (4–6). Trauma scores can facilitate the objective assessment of traumatized animals, and improve the outcome of treatments by predicting prognosis. The ATT score assesses six categories of body systems (perfusion, cardiac, respiratory, eye/muscle/ EVALUATION OF TRAUMA SCORING AND ENDOTHELIAL GLYCOCALYX INJURY IN CATS WITH HEAD TRAUMA Kurtulus Parlak1,*, Amir Naseri2, Mustafa Yalcin1, Eyup Tolga Akyol3, Mahmut Ok2, Mustafa Arican1 1Department of Surgery, 2Department of Internal Medicine, Faculty of Veterinary Medicine, University of Selcuk, 42130, Konya, 3Department of Surgery, Faculty of Veterinary Medicine, Balikesir University, 10145, Balikesir, Turkey *Corresponding author, E-mail: kparlak@selcuk.edu.tr Abstract: This study aim to evaluate the modified Glasgow Coma Scale (mGCS) and Animal Trauma Triage (ATT) scores, labo- ratory variables, and prognostic features of trauma-induced endothelial glycocalyx injury in cats with head trauma. Twenty-five cats with head trauma and 10 healthy cats were evaluated in this study. The enzyme-linked immunosorbent assay (ELISA) method was used to measure the levels of syndecan-1 and thrombomodulin in the serum of the 25 cats with head trauma (within 48 hours) and the 10 healthy cats. In addition, mGCS scores, ATT scores, laboratory values, syndecan-1 and thrombomodulin levels were compared between the cats that survived following treatment and the cats that did not survive despite treatment. Syndecan-1 and thrombomodulin levels were not statistically different between healthy cats and cats with head trauma. In the cats with head trauma, the mGCS scoring system was found to be more sensitive than the ATT scoring system. In conclusion, syndecan-1 and thrombomodulin levels did not yield statistically significant results in the cats with head trauma. Key words: animal trauma triage; cat; head trauma; modified Glasgow coma scale; endothelial glycocalyx integument, skeletal, and neurologic) and a scale of 0 to 3 for each category (0-slight or no injury, 3-severe injury), which contribute equally to the total score of 0 to 18 for prediction (5, 7). The mGCS was modified for veterinary use from the Glasgow Coma Scale, which has been described for humans with traumatic brain injury. This scale is based on the assessment of three categories, motor activity, brainstem reflexes, and level of consciousness. For each category, there is a scale from 1 to 6 (1-severely abnormal, 6-normal), with a lower total score indicating the more severe neurological deficits (4). Recently, resuscitation efforts in severe trauma patients (humans, cats, dogs, etc.) have focused not only on restoring lost blood volume, but also on improving the recovery of inflammatory and coagulation responses, vascular permeability, K. Parlak, A. Naseri, M. Yalcin, E. T. Akyol, M. Ok, M. Arican186 and endothelial dysfunction (8, 9). In humans, head trauma and hemorrhagic shock are the most common causes of death in patients with trauma (10). In particular, hemorrhagic shock leads to systemic degradation of the endothelial glycocalyx layer, and these changes are thought to lead to traumatic endotheliopathy (EoT), a syndrome associated with high mortality (11–13). Therefore, biomarkers such as syndecan-1 and thrombomodulin have emerged for the assessment of coagulation and endothelial integrity (endothelial glycocalyx). In particular, hemorrhagic shock, sepsis, multiorgan failure, endothelial dysfunction, and damaged vascular permeability have been associated with increased morbidity and mortality (14, 15). Overall, the purpose of this study is to investigate the mGCS and ATT scores as well as laboratory variables and markers of endothelial glycocalyx dysfunction for prognosis in cats with head trauma. Materials and methods Criteria for the selection of cases Cats with a history of head injury (within 48 hours) that met clinical and neurologic examination criteria (mGCS and ATT scores) were included in the study. Delayed (over 48 hours), treated cases, and cats with polytrauma were excluded from the study. Scoring methods Clinical examinations of the cats with head injuries were performed by the same observer (KP) using the mGCS and the ATT scoring systems before analgesic medication was administered. On the mGCS, consciousness, brainstem reflexes, and motor activities were rated in three scoring categories: 3 to 8, “grave”; 9 to 14, “guarded”; 15 to 18, “good.” As with the ATT assessment, body systems were rated in six categories (perfusion, cardiac, respiratory, eye/muscle/integument, skeletal, and neurologic) and scored on a scale of 0 to 3 in each category (4, 5). CT imaging A CT scan of the head was performed within 72 hours after trauma when the patient’s condition had stabilized. The patient was premedicated with medetomidine hydrochloride (Domitor®, Zoetis) (0.08 ml/kg b.w., intramuscularly), and anesthesia was induced with propofol (Propofol %1, Fresenius Kabi) (4 - 6 mg/kg b.w., intravenously) and maintained with isoflurane (Forane®, Abbott) in oxygen via intubation tube. For the CT 120 kV, 100 mA, and 2 mm cross- sectional thickness were selected and performed in helical cranial scanning mode. CT was used for head bone assessment only. Blood sample collection On admission, 2 ml of blood was collected by jugular venipuncture. Depending on the clinical condition of the cases, blood analyzes were repeated during the monitoring process. One part (0.5 ml) of the collected sample was immediately used for venous blood gas analysis, and the rest for complete blood count (CBC) and biochemistry (serum) analysis. Serums were stored at – 80 °C and thawed immediately before ELISA (enzyme- linked immunosorbent assay) analysis. Blood gases and complete blood count Venous blood gas analysis, which includes pH, partial pressure of carbon dioxide (pCO2), partial pressure of oxygen (pO2), venous oxygen saturation (sO2), lactate, sodium (Na), calcium (Ca), chloride (Cl), potassium (K), glucose, base excess (BE), and bicarbonate (HCO3) was performed using an automated blood gas analyzer (ABL 90 Flex, Radiometer, USA). Blood counts including total leukocytes, lymphocytes, monocytes, granulocytes, erythrocytes, hematocrit (HCT), hemoglobin (Hgb), and platelets were obtained using an automated cell counter (MS4e, Melet Schlosing Laboratories, France). Measuring syndecan-1 and thrombomodu- lin by the ELISA method Serum concentrations of syndecan-1 and thrombomodulin were measured according to the manufacturer’s protocol using the feline syndecan-1 commercial sandwich ELISA kit (catalog number: MBS1603385, USA) and the feline thrombomodulin commercial ELISA kit (catalog number: MBS1603383, USA) with an ELISA reader (Biotek 800TS, BioSPX, The Netherlands). Intra- Evaluation of trauma scoring and endothelial glycocalyx injury in cats with head trauma 187 assay coefficients, inter-assay coefficients, and minimum detectable concentrations were < 8%, < 10%, and 0.025 ng/ml for syndecan-1 cat, < 8%, < 10%, and 0.017 ng/ml for thrombomodulin. Treatment protocol The treatment protocol was applied to the cats with head trauma immediately after scoring and blood collection, and this protocol was repeated depending on the condition of the patients in the intensive care unit. The goals of the treatment protocol were based on the stimulation of circulation and oxygen delivery to vital organs. Fluid therapy with crystalloid solution 0.9% NaCl (60 ml/kg/hour, b.w., intravenously), osmotic diuretic mannitol (20% Mannitol, Polifarma) (2 g/kg b.w., intravenously, in 15 minutes), oxygen therapy (flow rate 100 ml/kg/min, intranasal or flow-by, oxygen cage), butorphanol (Butomidor®, Richter Pharma) (0.4 mg/kg b.w., subcutaneously) for analgesia, levetiracetam (Keppra®, UCB Pharma) (40 mg/kg b.w, intramuscularly) for anticonvulsant therapy, and nutrition (oral feeding or nasogastric tube feeding and esophageal feeding) were performed in cats with head trauma. Statistical methods To determine if the variables had a normal distribution, the Shapiro-Wilk test was used. In addition, parametric data were analyzed with the Student t-test (as mean ± standard deviation (SD)), nonparametric data were analyzed with the Mann- Whitney U test (as median (min/max)), and linear regression analysis was performed to determine the independent predictors of mortality. The prognostic value of serum endothelial biomarkers, mGCS, and hematologic variables was also evaluated using a receiver operating characteristic curve (ROC) to determine the prognostic cut-off values for best discrimination between survivors and nonsurvivors. Finally, the Kaplan-Meier survival curve from GCS was constructed. Overall, the statistical significance was P < 0.05, and data analysis was performed using SPSS statistical software. Results The animals in this study consisted of 25 cats with acute head trauma (24 mixed-breed and one British Shorthair; 14 males and 11 females; mean age, 13.4 (1-48) months) and 10 healthy cats (control group) (10 mixed-breed; 6 females and 4 males; mean age, 15.2 (8-28) months). Of the 25 cats with head trauma, 5 cats (20%) had trauma due to high-rise syndrome and 20 cats (80%) had trauma due to motor vehicle accidents. In addition, 8 cats (32%) had severe head trauma (mGCS score 3 - 8, “grave”), 10 cats (40%) had moderate head trauma (mGCS score 9 - 14, “guarded”), and 7 cats (28%) had mild head trauma (mGCS score 15 - 18, “good”). Compared with the mGCS scores, the ATT scores ranged from 5 - 14 (mean 9) in the cats with severe trauma, from 4 - 12 (mean 7.5) in the cats with moderate trauma, and 2 - 7 (mean 4.7) in the cats with mild trauma (Table 1). CT scan was performed in 15 / 25 cats with head trauma. The CT could not be performed in 8 cats because of their poor clinical condition and in 2 cats because their owners did not give permission. There were no abnormal findings in 2 / 15 cats. In addition, in 1 of the cats in which a CT scan was not performed, separation of the mandibular symphysis was clinically detected and treated. Regarding the CT findings of the other cats, separation of the symphysis mandible (n = 8), temporomandibular joint (TMJ) fracture (n = 5), os temporale fracture (n = 1), os zygomaticus fracture (n = 2), and separation of the palatal bone (n = 8) were observed (Table 1). There were no complications related to anesthesia during the CT scans. Finally, 12 of the 25 cats with head trauma were discharged after treatment (the average treatment time for discharged cats is 96 hours), whereas the remaining 13 cats died (no cat was euthanized). In this context, hematologic values, trauma scores (mGCS, ATT), and endothelial glycocalyx layer data (syndecan-1, thrombomodulin) were statistically compared between non-surviving and surviving cats (Figure 1) (Table 2). Linear regression analysis showed that mGCS, K+, HCO3, WBC, and Hb were independent risk factors for mortality in the head trauma group, whereas ROC analysis for the benefit of mGCS in discriminating between surviving and non- surviving cats yielded an area under the curve of 0.76 (p = 0.028, 95% CI = 0.569-0.950) (Fig- ure 2). Furthermore, the optimal cut-off point of 14.50 for the mGCS corresponded to a sensitiv- ity of 76% and a specificity of 70% for predicting K. Parlak, A. Naseri, M. Yalcin, E. T. Akyol, M. Ok, M. Arican188 Case TraumaType CT Results mGCS ATT Survivors/ Non-survivors Cat 1 HRS TMJ Fracture (Left)Os zygomaticus fracture 14 8 N Cat 2 MVA CT not applied 17 2 N Cat 3 MVA TMJ Fracture (Right) 16 7 N Cat 4 MVA CT not applied 5 9 Y Cat 5 MVA CT not applied 6 9 N Cat 6 MVA CT not applied 3 5 N Cat 7 MVA CT not applied 4 6 N Cat 8 MVA Separation of the symphysis mandible 18 7 Y Cat 9 MVA TMJ fracture (Left)Separation of the symphysis mandible 16 5 Y Cat 10 MVA Separation of the palatal bone 16 3 Y Cat 11 MVA TMJ Fracture (Right) Separation of the symphysis mandible Separation of the palatal bone 3 11 N Cat 12 MVA Os temporale fractureSeparation of the palatal bone 12 5 Y Cat 13 MVA Separation of the symphysis mandibleSeparation of the palatal bone 15 7 Y Cat 14 HRS Different structure not seen 14 9 N Cat 15 MVA Separation of the symphysis mandible 14 9 N Cat 16 MVA Separation of the symphysis mandibleSeparation of the palatal bone 10 6 Y Cat 17 MVA TMJ fracture (Right)Separation of the palatal bone 11 7 N Cat 18 MVA Os zygomaticus fracture Separation of the symphysis mandible Separation of the palatal bone 5 10 N Cat 19 HRS Different structure not seen 13 8 N Cat 20 MVA Separation of the palatal bone 9 4 Y Cat 21 MVA CT not applied 6 7 Y Cat 22 HRS CT not applied 18 2 Y Cat 23 MVA CT not applied 13 7 Y Cat 24 MVA CT not applied 13 7 Y Cat 25 HRS CT not applied 4 14 N Table 1: Trauma scores and findings on cases HRS: High-Rise Syndrome, TMJ: Temporomandibular Joint, MVA: Motor vehicle accidents, mGCS: Modified Glasgow Coma Scale, ATT: Animal Trauma Triage, Y: Survivors, N: Non-survivors mortality. Finally, a probability curve for the mGCS score showed a 72% probability of nonsurvival at a score of 6 and a 56% probability of non-survival at a score of 13, whereas a mentation score of 3 showed a 92% probability of nonsurvival (Figure 3). Discussion The primary causes of head injuries are traffic accidents, followed by high-rise syndrome (3). Of the 25 cats with head trauma in the present study, 80% were due to motor vehicle accidents, whereas 20% were due to high-rise syndrome, which is consistent with the results of previous studies. Some researchers have evaluated different scoring systems, such as the mGCS and ATT scoring systems, as prognostic indicators of trauma in cats and dogs (4, 7, 16). For instance, Lapsley et al (7) studied the mGCS and ATT scoring systems in 711 cats with trauma. Evaluation of trauma scoring and endothelial glycocalyx injury in cats with head trauma 189 However, when they limited the study to patients with head trauma, they found that there was no significant difference in differential capacity between the two scoring systems. In a related study, Sharma and Holowaychuk (16) performed prognostic assessments of 72 dogs Figure 1: Comparison of the syndecan-1 (left) and thrombomodulin (right) levels of healthy cats and cats with head trauma Figure 2: Receiver operating characteristic curve (ROC) analysis for the utility of the modified Glasgow Coma Scale to discriminate between surviving and nonsurviving cats yielded an area under the curve of 0.76 (p = 0.028, 95% CI = 0.569 - 0.950) Figure 3: A probability curve for the modified Glasgow Coma Scale score showed a 72% probability of nonsurvival at a score of 6 and a 56% probability of nonsurvival at a score of 13, whereas a mentation score of 3 showed a 92% probability of nonsurvival with head trauma and found that both scoring systems, particularly the mGCS, had prognostic value. In addition, Platt et al (4) reported that the mGCS can be evaluated as prognostic data in dogs with head trauma. In the present study, when the mGCS and ATT scores of the non-surviving and K. Parlak, A. Naseri, M. Yalcin, E. T. Akyol, M. Ok, M. Arican190 surviving cats were compared, it was found that the mGCS was statistically more prominent in head trauma in terms of prognosis. It was also suggested that the lack of statistical prognostic value of the ATT system was since this study focused only on cats with isolated head trauma and not on cats with polytrauma. It is important to note that the ATT scoring system generally assesses all body systems, including the musculoskeletal system, whereas the mGCS provides a more specific assessment of the central nervous system. In general, CT scans are used to diagnose hematomas, contusions, hernias, and cerebral ischemia, especially fractures in patients with head trauma (17, 18). It has also been used in the determination of craniomaxillofacial fractures in cats with head trauma. Recent studies report that mandibular fractures, particularly the separation of the mandibular symphysis and TMJ fractures, are the most common injuries diagnosed with CT Parameter Survivors(n: 12) Non-survivors (n: 13) P-value mGCS 14 (6 - 18) 6 (3 - 17) 0.026 ATT 6 (2 - 12) 8 (2 - 14) 0.110 Syndecan-1 (ng/ml) 2.49 ± 0.94 2.59 ± 1.34 0.827 Thrombomodulin (ng/ml) 1.14 ± 0.30 1.02 ± 0.29 0.319 pH 7.32 ± 0.49 7.26 ± 1.12 0.076 pCO2 (mmHg) 35.02 ± 5.90 35.56 ± 5.73 0.818 pO2 (mmHg) 36.54 ± 6.18 34.06 ± 4.01 0.255 sO2 (%) 47.62 ± 14.17 44.38 ± 8.64 0.503 cK+ (mmol/L) 3.72 ± 0.58 3.19 ± 0.68 0.047 cNa+ (mmol/L) 159.58 ± 3.08 159.53 ± 9.68 0.988 cCa+ (mmol/L) 0.87 ± 0.19 0.81 ± 0.22 0.452 cCl- (mmol/L) 120 (116 - 125) 121 (92 - 181) 0.205 cGlu (mg/dL) 209.58 ± 52.64 212.76 ± 66.50 0.895 cLac (mmol/L) 3.00 ± 1.75 2.13 ± 1.34 0.182 cBase (Ecf) (mmol/L) -7.70 ± 1.55 - 10.21 ± 4.05 0.055 cHCO3 - (mmol/L) 17.90 ± 1.13 16.05 ± 2.63 0.034 WBC (cells/ml) 26.36 ± 11.65 15.79 ± 9.22 0.021 LYM (cells/ml) 7 (1 - 18) 5 (2 - 15) 0.247 MON (cells/ml) 1 (0.14 - 12) 0.99 (0.51 - 4) 0.437 GRA (cells/ml) 14.12 ± 8.28 8.67 ± 7.80 0.105 RBC (x103 cells/ml) 10.66 ± 1.93 8.72 ± 2.72 0.051 Hct (vol%) 44.07 ± 9.65 36.12 ± 11.26 0.070 Hgb (g/dl) 12 (8 - 45) 10 (7 - 16) 0.022 Platelets (cells/ml) 199.58 ± 101.84 214.76 ± 76.02 0.679 Table 2: Mean trauma scores (mGCS, ATT), endothelial glycocalyx layer data (syndecan-1, thrombomodulin), and hematologic values were statistically compared between nonsurviving and surviving cats in cats after head trauma (19, 20). In addition, Tundo et al (20) stated that fractures involving the skull in cats after head trauma had a common and predictable distribution pattern in the midface (nose, maxilla, intermaxillary suture, orbit, nasopharynx, and zygomatic arch), with a high incidence of TMJ fractures. According to the CT scans in this study, the most common injuries were the separation of the mandibular symphysis, separation of the palatal bone, and TMJ fractures. The results are consistent with those of recent studies (19, 20). It is important to point out that one of the main problems that limited our CT imaging method was the inadequacy of the older generation of equipment, which prevented us from scanning thinner sections and obtaining detailed views of small structures. Some retrospective studies have been performed to determine the association between Evaluation of trauma scoring and endothelial glycocalyx injury in cats with head trauma 191 the severity of injury and hyperglycemia in cats and dogs with head trauma. Thus, some studies have indicated that cats and dogs with head trauma may have hyperglycemia, the extent of which is related to the severity of head trauma (16, 21). However, in the present study, no statistically significant difference was found between hyperglycemia and mortality, based on the statistical analyses between non-surviving and surviving cats with head trauma. However, blood glucose levels were found to be higher than normal in the cats with head trauma. In this study, Hgb, HCO3, and K + levels decreased below normal values on laboratory examination of non-surviving cats at the time of admission. Their white blood cell counts, although at normal levels, were lower than those of the surviving cats. In addition, a decrease in venous pO2 was observed in all cats with head trauma, including both non-surviving and surviving cats. Previous studies have shown that such a decrease, especially in dogs with head trauma, is due to impaired hemodynamic stability, which may lead to secondary brain damage (16). According to the data of the present study, this situation may also occur in cats with head trauma. Previous studies have shown an association between the detachment of glycocalyx components (syndecan-1 and thrombomodulin) and increased morbidity and mortality (22, 23). A previous study by Albert et al (24) examined the effects of endothelial damage on mortality by focusing on syndecan-1 and thrombomodulin levels in human patients with a head injury. They found that there was no significant increase in syndecan-1 levels associated with traumatic brain injury (TBI), which is a determinant of endothelial glycocalyx damage, and no significant change in thrombomodulin levels. However, they found a significant association between increased syndecan-1 levels and mortality (24). In a related study, Rodriguez et al (25) examined endothelial glycocalyx dysfunction using syndecan-1 and thrombomodulin levels in isolated TBI, polytraumatic TBI, and TBI-free trauma patients. They determined that syndecan-1 levels were highest in the polytraumatic TBI group, whereas they were lowest in the isolated TBI group. They also pointed out that thrombomodulin levels, although higher than normal, were similar in the three groups. In addition, the isolated TBI patients reported experiencing less glycocalyx destruction, as measured by their circulating syndecan-1 levels. Again, increased syndecan-1 concentrations were associated with increased 72 hours mortality in the isolated TBI patients (25). Because there have been no previous studies of syndecan-1 and thrombomodulin levels in cats with head trauma, the results of the present study were compared with those of human studies. According to studies, the syndecan-1 levels were increased in the endothelial glycocalyx damage of patients with head trauma, and this was more strongly associated with mortality than the increase in thrombomodulin levels. However, in our study, there was no statistically significant result related to syndecan-1 and thrombomodulin levels in the cats that did not survive after head trauma. Therefore, the results of this study differ from those of human studies and the use of different biomarkers related to endothelial glycocalyx injury may lead to more efficient results. Our study had several limitations: 1) the study population was relatively small, and reevaluation of the hypothesis of endothelial glycocalyx damage in larger sample populations is warranted. 2) Histopathological examinations were not performed on cats that did not survive after head trauma. Conclusion This was the first study to perform prognostic assessments based on the mGCS and ATT scoring systems and endothelial glycocalyx layers in cats with head trauma. Overall, no statistically significant difference was found between the syndecan-1 and thrombomodulin levels of the healthy cats and those of the cats with head trauma. In addition, linear regression analysis showed that the mGCS, potassium, bicarbonate, white blood cell, and hemoglobin levels were independent mortality factors in the head trauma group. Although the mGCS and ATT scoring systems are the most common scoring systems used to assess cats with trauma, the former is believed to be one step ahead of the latter. However, the markers syndecan-1 and thrombomodulin were not found to be useful for prognosis or choice of treatment options in cats with head trauma. K. Parlak, A. Naseri, M. Yalcin, E. T. Akyol, M. Ok, M. Arican192 Acknowledgements Supported by the Coordination of Scientific Research Projects of Selçuk University (BAP) (No. 19401043). The authors thank N. Zamirbekova and all staff from the Department of Surgery for animal care. Ethical approval: This study was approved by the Ethics Committee (03/2019) of the Faculty of Veterinary Medicine and Experimental Animal Production and Research Centre, Selçuk University, Turkey. Informed owner consent was also obtained from the owners and the ethical guidelines of the institution were followed. References 1. Kolata RJ. Trauma in dogs and cats: an overview. Vet Clin North Am Small Anim Pract 1980; 10: 515–22. 2. Rochlitz I. Study of factors that may predis- pose domestic cats to road traffic accidents: Part 2. Vet Rec 2003; 153(19): 585–8. 3. 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Scand J Trauma Resusc Emerg Med 2018; 26(1): e102. doi: 10.1186/s13049-018- 0565-3 VREDNOTENJE SISTEMA OCENJEVANJA TRAVME IN POŠKODB ENDOTELIJSKEGA GLIKOKALIKSA PRI MAČKAH S POŠKODBO GLAVE K. Parlak, A. Naseri, M. Yalcin, E. T. Akyol, M. Ok, M. Arican Izvleček: Namen te študije je bil ovrednotiti spremenjeno glasgowsko lestvico kome (angl. Glasgow Coma Scale, GCS) in ocene ATT (Animal Trauma Triage), laboratorijske spremenljivke in prognostične značilnosti s travmo povzročene poškodbe endotelijskega glikokaliksa pri mačkah s poškodbo glave. V študijo je bilo vključenih 25 mačk s poškodbo glave in 10 zdravih mačk. Ravni sindekana-1 in trombomodulina v serumu 25 mačk s poškodbo glave (v 48 urah) in 10 zdravih mačk smo merili z encimsko imunoadsorpcijsko preiskavo (ELISA). Ocene mGCS, ocene ATT, laboratorijske vrednosti ter ravni sindekana-1 in trombomodulina smo primerjali med mačkami, ki so po zdravljenju preživele, in mačkami, ki kljub zdravljenju niso preživele. Vrednosti sindekana-1 in trombomodulina se med zdravimi mačkami in mačkami s poškodbo glave niso statistično razlikova- le. Pri mačkah s poškodbo glave se je sistem točkovanja mCGS izkazal za občutljivejšega od sistema točkovanja ATT. Zaklju- čili smo, da vrednosti sindekana-1 in trombomodulina pri mačkah s poškodbo glave niso statistično pomembne. Ključne besede: triaža poškodb živali; mačka; poškodba glave; spremenjena glasgowska lestvica kome; endotelijski glikokaliks Received: 22 September 2022 Accepted for publication: 13 October 2022 Slov Vet Res 2022; 59 (4): 195–209 DOI 10.26873/SVR-1591-2022 UDC 615.9:546.81:616-091.8:615.035:547.458.88:599.323.452 Original Research Article Introduction One of the most serious environmental medicine issues is lead poisoning. Lead contamination in the environment caused by industrial lead production and metal recycling (1). Due to its harmful cumulative action in the environment, lead can affect all biological systems by exposure from PECTIN IMPROVES HEMATO-BIOCHEMICAL PARAMETER, HISTOPATHOLOGY, OXIDATIVE STRESS BIOMARKERS, CYTOKINES AND EXPRESSION OF HEPCIDIN GENE IN LEAD INDUCED TOXICITY IN RATS Sabry M. El-Bahr1,2*, Saad Al-Sultan1,3, Ahlam F. Hamouda4, Shimaa. A. E. Atwa5, Seham Y. Abo-Kora6, Aziza A. Amin7, Saad Shousha1,8, Sameer Alhojaily1, Aymmen Alnehas9, Rabab R. Elzogby10 1Department of Biomedical Sciences, 3Department of Public Health, 9Department of Clinical Sciences, College of Veterinary Medicine, King Faisal University, Al-Ahsa, 31982, Saudi Arabia, 2Department of Biochemistry, Faculty of Veterinary Medicine, Alexandria University, Alexandria, 21523, Egypt, 4Department of Forensic Medicine and Toxicology, Teaching Hospital, 5Department of Biochemistry, 6Department of Pharmacology, 7Department of Histopathology, 8Department of Physiology, Faculty of Veterinary Medicine, Benha University, Benha, 13736, 10Department of Pharmacology, Faculty of Veterinary Medicine, NewVally University, Egypt *Corresponding author, E-mail: selbahar@kfu.edu.sa Abstract: Publications concerning the protective effect of pectin against lead induced toxicity in rats are not available. In order to study such effect, 40 male rats were divided into 3 groups. The first group was contained 10 rats that kept as control group. The second group was contained 10 rats that received pectin at dose of 100 mg/kg BW during experimental period (8 weeks). The third group was contained 20 rats that received 400mg/kg BW of lead acetate daily for 4 weeks then divided into two subgroups (3A and 3B). Subgroup 3A contained 10 rats that still receive lead acetate in the same dosage whereas, subgroup B co-treated with lead acetate and pectin daily for another 4 weeks. Blood samples were collected after 2, 4 and 8 weeks from the start of the experiment. Liver, kidney and bone marrows were collected only at the end of the experiment. Lead acetate induced anemia only after 4 weeks of administration as reflected on decreased values of Hb, PCV, MCV, MCH and MCHC. These indices remained at lower levels in lead acetate treated groups until the end of the experiment. Concentrations of serum ferritin, iron, total antioxidant capacity (TAC) and reduced glutathione (GSH) and the expression of hepatic hepcidin gene were decreased significantly in lead acetate intoxicated rats compared to control. Activities of ALT and AST and concentrations of urea, creatinine, Nitric oxide (NO), TNF-α, IL-6, total iron binding capacity (TIBC) and lead were increased significantly in lead acetate intoxicated group compared to control. Hepatic degeneration and hemorrhage, renal lytic necrosis and apoptosis of myeloid cells were most prominent changes in lead intoxicated rats. Lead acetated related changes were improved by co-treatment with pectin however; normal control val- ues have not been achieved. Conclusively, pectin is recommended to protect against lead acetate toxicity in rats. Key words: lead acetate; toxicity; pectin; hepcidin; oxidative stress biomarkers; histopathology multiple sources such as air, water, and food. Lead has the ability to migrate up the food chain, causing harm to humans and other animals. Lead induced microcytic hypochromic anemia in mammals due to its interaction with iron and copper metabolism (2). Lead may disrupt the integrity of the RBC membrane, making it more fragile, resulting in a disorder of red blood cell metabolism in the bone marrow or mature erythrocytes, inhibit the enzyme ferrochelatase and reducing iron (Fe) incorporation into heme and disrupting heme synthesis (3). 196 S. M. El-Bahr, S. Al-Sultan, A. F. Hamouda, S. A. E. Atwa, S. Y. Abo-Kora, A. A. Amin, S. Shousha, S. Alhojaily, A. Alnehas, R. R. Elzogby Lead exposure has been observed to reduce serum iron and transferrin saturation levels in rats (4). Lead acetate has hepatoxic effect which increase hepatocyte permeability in rats. The damaged hepatocyte cell membrane of hepatocytes leading to escape of liver enzymes to blood. Lead toxicity produces an increase in cellular basal metabolic rate, irritability, and destructive alteration of liver cells due to its oxidative effect (5). Oral dose of lead acetate caused a significant rise in blood urea and serum creatinine in rats (6). The main mechanism responsible for lead toxicity is oxidative stress. This type of stress causes changes in the composition of fatty acids in cells membrane (affecting processes such as exocytosis and endocytosis, as well as signal transduction processes). The production of reactive oxygen species (ROS), the activation of lipid peroxidation, and the depletion of antioxidant reserves are all factors that contribute to lead exposure (3, 7). Hepcidin gene expression is reduced after experimentally induced anemia and hypoxia, which could explain the increased (Fe) release from reticuloendothelial cells and higher (Fe) absorption normally observed in these conditions, suggesting hepcidin involvement in anemic states. Lead has also been found to prevent (Fe) from being transferred from endosomes to the cytoplasm (8). Hepcidin expression was found to be lower in people with anemia of chronic disorders (ACD) and in mammalian models that resembled ACD (9, 10). In patient and mammals with (ACD), iron deficiency anemia, serum hepcidin levels and/ or liver mRNA expression were both decreased considerably (9). Additionally, Hypoxia may diminish hepcidin expression while increasing serum iron and transferrin saturation, allowing massive erythropoiesis to compensate for tissue hypoxia (11). Pectin is a galacturonic acid polymer found mostly in plant walls. It can be isolated from fruit pips, apple pulp, and peal (12). Pectin rich in galacturonic acid (GalA) are effective at chelating heavy metals (13). Pectin’s ability to chelate metals in the digestive system and inhibit absorption while aiding their removal in the faces, toxic metal absorption and bioaccumulation were reduced with its administration (14). Oral administration of pectin resulted in decrease lead absorption (15). In industrial settings, commercial apple pectin is an excellent agent for preventing lead incorporation (16). The goal of this work was to evaluate the effect of pectin on hemato-biochemical parameter, histopathology, oxidative stress biomarkers, cytokines and expression of Hepcidin gene in lead intoxicated rats. Materials and methods Chemicals Lead acetate obtained from Al Gomhoria company, Egypt. Pectin obtained from Sigma Company, Egypt. Experimental animals and design This experiment was carried out according to the guidelines of the Institutional Animal Ethics Committee, Benha University, Egypt, and Approval (Permission # BUFVTM 05-12-21). Forty male albino rats were purchased from Lab Animal House at Vet College Benha University; their average weight was (160±10g). They kept in well-ventilated metal cages throughout the study and acclimatized for one week at a temperature of 18-24°C with 12 hours of light and darkness, on normal feed diet and water ad libitum. The experimental design illustrated obviously at Table 1. This design showed that, 40 male rats were divided into 3 groups. The first group was contained 10 rats that kept as control group and received normal physiological saline daily during experimental period (8 weeks). The second group was contained 10 rats that received pectin at dose of 100 mg/kg BW (17) during experimental period (8 weeks). The third group was contained 20 rats that received 400mg/kg BW of lead acetate daily (18) for 4 weeks then divided into two subgroups (3A and 3B). Subgroup 3A contained 10 rats that still receive lead acetate in the same dosage whereas, subgroup B co-treated with lead acetate and pectin daily for another 4 weeks. Blood samples were collected after 2, 4 and 8 weeks from the start of the experiment. The whole blood was used or the detection of hematological indices (Hb, PCV, MCV, MCH and MCHC). The obtained serum was used for the estimation of the activity of ALT and AST and the concentration of urea, creatinine, ferritin, iron, TIBC TAC, GSH, NO, TNF-α, IL-6 and lead. Liver, kidney and bone marrows collected only at the end of the experiment and subjected to histopathological examination. Portion of liver tissues was frozen by liquid nitrogen until used for detection of expression of hepatic hepcidin gene. 197Pectin improves hemato-biochemical parameter, histopathology, oxidative stress biomarkers, cytokines… Time Samples Parameters measured Groups Group 1 Group 2 Group 3 After 2 weeks blood Hb, RBCs, PCV, MCV, MCH, MCHC ✓ ✓ ✓ After 4 weeks blood Hb, RBCs, PCV, MCV, MCH, MCHC, ✓ ✓ ✓ 3A 3B After 8 weeks Blood, serum, liver, kidney, bone marrow Hb, RBCs, PCV, MCV, MCH, MCHC, Basophilic Stippling cell, Ferritin, Iron, TIBC, AST, ALT, urea, creatinine, TAC, GSH, NO, TNF-α, IL-6, lead, Histopathology ✓ ✓ ✓ ✓ Table 1: The experimental design of the study Assessment of Complete blood count Assessing of complete blood count was performed using an electronic cell counter (VetScan HM5 Hematology system, Abaxis, Inc., Union City, CA, USA). Assessment of liver and kidney function tests and iron profile Activities of ALT and AST were performed as described earlier (19). Serum urea and creatinine were performed as described previously (20, 21), respectively. Serum iron, ferritin and Total iron Binding Capacity (TBIC) were performed as described earlier (22, 23, 24), respectively. Assessment of serum oxidative stress biomarker and cytokines concentrations The total antioxidant capacity (TAC), reduced glutathione (GSH) and nitric oxide (NO) were performed as described in previous researches (25, 26, 27), respectively. TNF-α and IL-6 concentrations were determined by using ELISA kit that described earlier (28). Detection of lead residues in blood Detection of lead residues in blood was performed as described earlier (29). Briefly, 1ml whole blood was measured into clean test tubes, followed by 1 ml concentrated nitric acid containing 0.1 percent triton-100, which was mixed carefully. Cotton wool was used to plug the test tubes, which were then placed on the bench overnight. The mixture was then cooked in a water bath at 100°C for 20 minutes on the second day, and then allowed to cool. The digested blood samples were transferred to a measuring cylinder and filled with distilled water to a volume of 25 ml. Lead residue was determined by using Perkin-Elmer 2380 Atomic absorption spectrophotometer. Investigation of mRNA expression of Hepcidin gene This stage was completed at Benha University’s Central Laboratory, Faculty of Veterinary Medicine. The following primer sets were used to dissect liver samples from all rat groups (30). Hepcidin, sense (5'-GAAGGCAAGATGGCACTAAGCA-3') and anti-sense (5'-TCTCGTCTGTTGCCGGAGATAG-3'), and actin as a housekeepin gene, sense (5'-AGAAGAGCTATGAGCTGCCTGACGCG-3') and anti-sense (5'-CTTCTGCATCCTGTCAGCGATGC-3'). The Blood Smear (Field stain) Blood smears are used to look at single-cell spread in blood components (31). Histopathological examination Specimens were taken immediately from liver and kidneys of all groups, fixed in 10% buffered neutral formalin for 24 hours. After proper fixation, the specimens were washed in running tape water, dehydrated in different grades of ethyl alcohol, cleared in xylol and embedded in paraffin, then blocked and sectioned as 5 mm thickness. Then stained by hematoxylin and eosin and examined microscopically (32). Pathological alterations were examined using an Olympus light microscope, femur bones were collected, decalcified, fixed and samples were processed (33). All pathological markers were measured using a standard semi quantitative scoring approach to compare the severity of lesion severity between groups. The following is a five-point ordinal scale: (0) no 198 S. M. El-Bahr, S. Al-Sultan, A. F. Hamouda, S. A. E. Atwa, S. Y. Abo-Kora, A. A. Amin, S. Shousha, S. Alhojaily, A. Alnehas, R. R. Elzogby changes, (1) mild 25 percent tissue damage, (2) moderate 25 percent: 50 percent tissue damage, (3) severe 50 percent: 75 percent tissue damage, and (4) extensive severe >75 percent tissue damage (34). Statistical analysis The data was analyzed using SPSS 15.0 statistical software and provided as mean SD (SPSS Inc., Chicago, IL). One analysis of variance (ANOVA) was used for statistical analysis of the current data. Results Complete blood count The mean and standard deviation values of complete blood count of the different experimental groups after 2, 4 and 8 weeks were depicted in Table 1, 2 and 3, respectively. After 2 weeks from the start of the experiment, all measured hematological indices (Hb, PCV, MCV, MCH and MCHC) remained unchanged significantly in both pectin and lead acetate treated groups compared to the control (Table 2). However, after 4 weeks from the start of the experiment, HB, RBCs, PCV, MCH, MCHC values were decreased significantly in lead acetated treated group compared to control (Table 3). These values remained unchanged significantly in pectin treated rats compared to control (Table 3). After 8 weeks from the start of the experiment, all hematological indices were decreased significantly in lead acetate rats (group 3A) compared to control (Table 4). In addition, basophilic stippling cell noticed in this compared to control (Table 4). Co-treatment of rats with lead acetate and pectin recovered all measured hematological indices into normal control values except for MCV and MCHC that remained lower than that of control values (Table 4). Furthermore, basophilic stippling cell was disappeared in rats co-treated with lead acetate and pectin compared to control (Table 4). Group 1: contained 10 rats that kept as control group. Group 2: contained 10 rats that received pectin at dose of 100 mg/kg BW during experimental period (8 weeks). Group 3: contained 20 rats that received 400mg/kg BW of lead acetate daily for 4 weeks then divided into two subgroups (3A and 3B). Subgroup 3A: contained 10 rats that still receive lead acetate in the same dosage. Subgroup B: contained 10 rats that co-treated with lead acetate and pectin daily for another 4 weeks. Table 2: effect of lead administration on blood indices in different experimental groups after 2 weeks from the start of the experiment Table 3: effect of lead administration on blood indices in different experimental groups after 4 weeks from the start of the experiment. Parameters Groups Group 1 Group 2 Group 3 Hb (mg/dl) 14.18±0.52a 12.15±2.41a 12.30±3.70a RBCs (×106 /µml) 6.71±0.20a 6.32±1.30a 6.42±1.15a Pcv (%) 36.60±2.35a 37.40±2.24a 39.09±7.54a MCV (fl/cell) 60.5±1.15a 62.1±2.05a 60.62±2.04a MCH (pg/cell) 20.43±0.38ab 21.34±2.24a 16.09±4.41a MCHC (g/dl) 33.90±1.22b 32.80±1.18a 34.77±1.50a The values represent Mean ± SD. Means within the same row followed by different letters are significantly different (P ≤ 0.05). Parameters Groups Group 1 Group 2 Group 3 Hb (mg/dl) 14.03±0.30a 13.07±0.51a 9.64±0.07b RBCs (×106 /µml) 6.83±0.06a 6.99±0.12a 5.12±.0.08b PCV (%) 34.66±2.52a 33.87±2.49a 26.60±0.2b MCV (fl/cell) 53.71±1.48a 52.69±1.60a 45.94±2.49b MCH (pg/cell) 20.97±0.44a 19.88±1.50a 16.01±0.71b MCHC (g/dl) 38.28±0.37a 36.40±2.40a 31.89±0.49b The values represent Mean ± SD. Means within the same row followed by different letters are significantly different (P ≤ 0.05). 199Pectin improves hemato-biochemical parameter, histopathology, oxidative stress biomarkers, cytokines… Table 4: Effect of lead acetate on blood indices in different experimental groups after 8 weeks from the start of the experiment Table 5: Effect of lead acetate on liver and kidney function tests and iron profile in different experimental groups Table 6: Effect of lead acetate on oxidative markers, cytokines and lead in different experimental groups Parameters Groups Group 1 Group 2 Group 3 3A 3B Hb (mg/dl) 14 ±0.95a 13.45 ±1.67a 9.01±0.11c 13.3± 0.55a RBCs (×106/µml) 6.00± 0.6a 5.88± 0.68a 4.50± 0.13b 6.22± 0.34ab PCV (%) 35.63± 2.41a 32.99± 2.37a 28.29 ±0.77c 32.36 ±2.43b MCV (fl/cell) 61.48± 1.20a 57.88± 1.29a 52.13± 0.27b 52.08±0.98b MCH (pg/cell) 21.13± 0.4a 22.15± 0.51a 18.96±0.39b 21.26±0.35a MCHC (g/dl) 35.89±1.19a 37.99±1.21a 31.68±0.51b 31.23±1.45b Basophilic Stippling cell - - + - The values represent Mean ± SD. Means within the same row followed by different letters are significantly different (P ≤ 0.05). Parameters Groups Group 1 Group 2 Group 3 group A group B AST (U/ml) 30.10± 3.18c 32.21± 2.87c 145.62± 4.10a 103.7± 1.53b ALT (U/ml) 29.8 ±1.71c 31.66 ±2.01c 59.33± 0.88a 35.91± 3.00b Urea (mg/dl) 25.98± 1.96c 27.79± 1.10c 131.7± 2.94a 42.30± 0.36b Creatinine (mg/dl) 0.68± 0.02c 0.64± 0.05c 2.9±0.34a 0.83± 0.02b Ferritin (ng/dl) 0.7±0.057a 0.81±0.07a 0.47±0.028b 0.70±0.054a Iron (µg/dl) 2.31±0.089a 2.42±0.06a 1.29±0.10b 2.25±0.07a TIBC (mcg/dl) 6.01±0.51b 6.06±0.49b 8.53±0.24a 6.87±0.53b The values represent Mean ± SD. Means within the same row followed by different letters are significantly different (P ≤ 0.05). Parameters Group 1 Group 2 Group 3 3A 3B TAC (µmol/L) 2.45± 0.22a 2.51± 0.30a 0.99± 0.10c 1.93± 0.10b GSH (mg/g) 56.18 ±3.59a 55.22±3.60a 33.98±2.14c 41.45±1.73b NO (µmol/L) 19.06±2.25c 20.10±2.40c 42.24±1.57a 30.11±1.05bc TNF-α (pg/ml) 19.85±5.59c 21.99±5.64c 59.84 ±4.59a 47.45±4.07b IL6 (pg/ml) 9.87±0.12 c 9.67±0.24c 45.72±1.67a 19.64±1.27b Serum lead (mg/dl) 0.95±0.06a 0.98±0.07a 11.65±0.63c 5.95±1.54b The values represent Mean ± SD. Means within the same row followed by different letters are significantly different (P ≤ 0.05). TAC (total antioxidant capacity), GSH (reduced glutathione), NO (nitric oxide). 200 S. M. El-Bahr, S. Al-Sultan, A. F. Hamouda, S. A. E. Atwa, S. Y. Abo-Kora, A. A. Amin, S. Shousha, S. Alhojaily, A. Alnehas, R. R. Elzogby Figure 1: Effect of lead acetate on liver hepcidin mRNA expression level and ameliorative effect of Pectin in male albino rats Table 7: The score of histopathological lesions in different experimental groups Lesion score Group 1 Group 2 Group 3 3A 3B Liver Congestion of hepatic blood vessels 0 0 4 1 Activation of Vonkupfer cell 0 0 3 0 Perivascular Leukocytic infiltration 0 0 3 0 Degenerative changes 0 0 4 1 Necrosis of hepatic cells 0 0 3 0 Nuclear changes 0 0 3 0 Kidney Congestion of renal blood vessels 0 0 3 1 Degeneration of blood vessels wall 0 0 2 0 Perivascular edema 0 0 3 0 Tubular epithelial degeneration and necrosis 0 0 4 1 Hyaline and cellular casts 0 0 2 0 Interstitial leukocytic infiltration 0 0 3 0 necrosis of glomerular tuft 0 0 2 0 Bone marrow Reduction in erythropoiesis 0 0 3 1 Degeneration of myeloid 0 0 2 0 Liver and kidney function tests and iron profile Activities of ALT and AST and concentrations of urea, creatinine and total iron binding capacity (TIBC) were increased significantly in lead acetate intoxicated group (subgroup 3A) compared to control (Table 5). However, concentrations of serum ferritin and iron, were decreased significantly in lead acetate intoxicated rats (subgroup 3A) compared to control (Table 5). These parameters were improved in rats co-treated with lead acetate and pectin than that of lead acetate treated rats but the normal control values have not been achieved for ALT, AST, urea and creatinine (Table 5). Oxidative stress biomarkers, cytokines and lead concentrations The concentrations of NO, TNF-α, IL-6 and lead concentrations were increased significantly in lead acetate intoxicated group (subgroup 3A) compared 201Pectin improves hemato-biochemical parameter, histopathology, oxidative stress biomarkers, cytokines… to control (Table 6). However, concentrations of serum TAC and GSH were decreased significantly in lead acetate intoxicated rats (subgroup 3A) compared to control (Table 6). These parameters were improved in rats co-treated with lead acetate and pectin than that of lead acetate treated rats but the normal control values have not been achieved (Table 6). mRNA gene expression The hepcidin mRNA gene expression of different experimental groups was illustrated in Figure 1. The expression of hepcidin gene in rats fed pectin alone remained unchanged significantly compared to the control (Fig. 1). The expression of hepcidin gene in liver tissue were decreased significantly in lead acetate intoxicated rats (subgroup 3A) compared to control (Fig. 1). Co-treatment of rats with lead acetate and pectin (subgroup B3) up- regulated the expression of this gene to normal control value (Fig. 1). Histopathological examination The score of lesions of histopathological examination were seen in the liver, kidneys, and bone marrow of rats in different experimental groups as shown in Table 7. The histopathoogical picture of rats tissues that received pectin only (group 2) were not showed in the current study because it looks like the control group (group 1) as illustrated in Table 7. The histological examination of the liver in subgroup 3A showed that there were congestion of the hepatic blood arteries and blood sinusoids, as well as portal distension and leukocytic cellular infiltration, primarily lymphocytes and macrophages (figure 2A). Hepatocytes also showed significant degeneration in the form of diffuse hydropic degeneration (figure 2B) with diffuse hemorrhage in the hepatic parenchyma, as well as multifocal patches of coagulative necrosis with pyknotic nucleus. Furthermore, diffuse regions of lytic necrosis in the hepatic parenchyma were detected, that consisted of necrotic debris mixed with erythrocytes and leukocytes (figure 2C&D). The hepatic tissue collected from rats in subgroup 3B showed an improvement in the hepatocellular architecture when compared with rats in subgroup 3A. In comparison to the control, the liver tissue returned to its normal histological structure. The majority of the hepatic parenchyma had recovered, and only minor hepatocyte vacuolar degeneration (figure 2E) was observed, while the portal area appeared normal. The microscopic analysis of hepatic tissue from control and pectin treated groups showed normal histological structure and showed in the figure. Figure 2: Liver of rats of subgroup 3A (A-D) and subgroup 3B (E). The tis- sue showed (A) distension of the por- tal area with mononuclear leukocytic cells (arrow), (B) diffuse hydropic de- generation of hepatocytes, (C) multi- focal areas of coagulative necrosis of hepatocytes with pyknotic nuclei in the hepatic parenchyma (arrow), (D) extensive diffuse area of lytic necrosis reprsented by necrotic debris admixed with erythrocytes and leukocytes (ar- row), (E) mild vacuolar degeneration of hepatocytes. H&E stained x 400 202 S. M. El-Bahr, S. Al-Sultan, A. F. Hamouda, S. A. E. Atwa, S. Y. Abo-Kora, A. A. Amin, S. Shousha, S. Alhojaily, A. Alnehas, R. R. Elzogby Figure 3: kidney of rats for subgroup 3A(A-D) and subgroup B (E). tissue showed (A) perivascular edema admixed with erythrocytes (arrow) with vacuolar degeneration of the lining epithelium of some renal tubules and its necrosis with pyknotic nuclei in other tubules, (B) necrosis of the glomerular tuft (asterisk) and with thrombosis of the renal blood vessels (T) with extensive focal area of lytic necrosis represented by necrotic debris admixed with erythrocytes and leukocytes (arrow), (C) necrosis of the lining epithelium of the con-voluted tubules (zigzag arrow), inter tubular mononuclear leukocytic cellular infiltrations (arrow) with precip-itation of lead pigment renal tubules (asterisk), (D) precipitation of lead pigment in the renal tubules of renal medulla (zigzag arrow) with the presence of intra-nuclear eosinophilic inclusion bodies (arrow), (E) mild vacuolation of the endothelial cell lining of glomerular tuf with cloudy swelling of the lining epithelium of some renal tubules. H&E stain x 400 Figure 4: Bone marrow of rats for control (A-B), subgroup 3A (C-E) and subgroup 3B (F-G), showing (A-B) nor-mal bone marrow (A, x400, B, x1000), (C) degeneration and apoptosis of myeloid cells (arrow, x1000), (D) intra-nu- clear eosinophilic inclusion bodies (arrow, x1000), (E) degeneration of megakaryocytes (x1000), (F) hyperplasia of megakaryocytes (x1000), (G) restoring of normal histological structure of bone marrow (H&E-stained x1000) 203Pectin improves hemato-biochemical parameter, histopathology, oxidative stress biomarkers, cytokines… Figure 5: Blood smear of rats. (A) normal RBCs of control group, (B-E) RBCs of subgroup 3A (B) basophilic gran-ules in RBCs (C) hypo chromatic RBCs, (D) tear drops RBCs (E) hemolysis of RBCs. (F) RBCs of group B blue arrow nor- mal RBCs and black arrow hypo chromatic RBCs The renal blood vessels, inter-tubular and glomerular blood capillaries, and perivascular edema mixed with erythrocytes were all congested in the kidneys retrieved from subgroup 3A (figure 3A). Thrombosis of the renal blood vessels with vacuolation of the glomerular endothelial cells, as well as necrosis of the glomerular tuft in some cases in association with focal area of lytic necrosis characterized by complete absence of renal tissue and replaced by eosinophilic debris with erythrocytes and few leukocytes (figure 3B) were also detected. In addition, extensive degenerative changes in the lining epithelium of the renal tubules were observed in the renal cortex, including vacuolation, hydropic degeneration, desquamation and necrosis with pyknotic nuclei in association with eosinophilic hyaline casts in the lumen of some renal tubules, mononuclear leukocytic cellular infiltrations in interstitial tissue with precipitation of lead pigment In most deteriorated epithelial cells, clumps of amorphous blue staining lead pigment were precipitated in varying quantities in the cytoplasm of the degenerated tubules of the renal medulla in conjunction with intra-nuclear eosinophilic inclusions (figure 3D). While in subgroup 3B showed improvement in the degenerative changes in the kidneys caused by lead acetate. A microscopical examination of 204 S. M. El-Bahr, S. Al-Sultan, A. F. Hamouda, S. A. E. Atwa, S. Y. Abo-Kora, A. A. Amin, S. Shousha, S. Alhojaily, A. Alnehas, R. R. Elzogby the kidney from subgroup 3B demonstrated a significant improvement in renal tissue histology when compared to subgroup 3A. There was mild congestion of the renal blood arteries and glomerular blood capillaries with normal histological structure of the glomeruli. Meanwhile, mild vacuolation of the endothelial cell lining of the glomerular tuft was observed in some cases (Figure 3E), along with mild degenerative changes in the lining epithelial cell of the renal tubules in the form of cloudy swelling, while control and pectin treated group’s revealed normal renal histological structure. Compared to the control group bone marrow (Figure 4a-b), a marked reduction in erythropoiesis was demonstrated in the bone marrow of lead intoxicated rats (subgroup 3A) as well as degeneration of myeloid cells, especially megakaryocytes, apoptosis of myeloid cells with the presence of intranuclear eosinophilic inclusion bodies was detected (Figure 4C-E). On the other side, a reduction in the pathological alterations induced by lead toxicity was observed in the bone marrow of rats in subgroup B as an increase in cell density affecting erythroid and myeloid cells with megakaryocytic hyperplasia in proportion to the other cells types (Figure 4F-G). The Blood Smear (Field stain) Blood smears were used to look for abnormal red blood cells as basophilic granulation, hypo chromatic, tear drops and hemolysis of RBCs Figure 5(B-E) which considered as important marker for lead toxicity, this may explain that lead causes anaemia and there were basophilic stippling cell noticed in subgroup 3A compared to other experimental groups. Discussion Lead is one of the most hazardous heavy metals on the human and animals. It is one of the most serious environmental pollutants. It used by humankind for many years due to its wide range of applications. Lead enters the body through a variety of routes, including the air, food, dust, soil, and water (35). Adult Wister rats were exposed to lead, had toxic effects in their blood, liver, and kidneys. Oxidative stress is a primary mechanism of metal toxicity, and it was identified as a significant factor in our investigation when we discovered an altered redox state in treated rats’ tissues as well as hematological problems (5). The activity of Aminolevulinic acid dehydratase (ALAD) is severely inhibited by lead, which disrupts haeme anabolism (36). The significant decrease of hematological indices (RBCs, Hb, MCH and MCHC) in lead acetate intoxicated rats indicated a state of anemia. This finding may be attributed to chelating properties of lead acetate (37). Lead can bind to essential minerals in the body, producing a variety of physiological problems in addition to affecting protein production and inhibiting hemoglobin formation (37). The protective effect of pectin as demonstrated in the current study was consistent with previous findings (18) which recorded the pectin‘s ability to chelate metals in the digestive system and inhibit absorption while aiding their removal in the faces (14). RBCs, Hb, PCV %, MCV, MCH, and MCHC were all lower in microcytic hypochromic anemia (39). In the current study, subgroup 3A showed that lead toxicity to rats induced microcytic hypochromic anemia, as it decreased RBCs, Hb, PCV%, MCV, MCH and MCHC, which in accordance with the results reported previously (40). Lead inhibits several enzymes that are important for haeme synthesis (41), and lead suppresses enzymatic activities such amionlevulinic acid dehydratase (ALAD), and ferochelatase, which are all important for haeme production, this suppression leads to a problem with iron metabolism (18). However, on the other hand, subgroup 3B showed an improvement in blood indices (RBCs, Hb, PCV%, MCV, MCH, and MCHC) when compared to subgroup 3A which in the same line of previous work (38) showed that date pectin extract was effective when taken orally for one month boosted RBC, Hb, MCV and MCH levels significantly (P ≤ 0.05). previous work (42) demonstrated that low- esterified pectin quickly forms complexes with divalent metals, including ions of hazardous elements (mercury, lead, and cadmium), hence, reduced the cytotoxic effects of heavy metals. Histopathological examination of bone marrow in subgroup 3A (Figure 4c, e) explained that a marked reduction in erythropoiesis as well as degeneration of myeloid cells, especially megakaryocytes and apoptosis of myeloid cells were observed. While in subgroup 3B (Figure 4f, g) 205Pectin improves hemato-biochemical parameter, histopathology, oxidative stress biomarkers, cytokines… hyperplasia of megakaryocytes and restoring of normal histological structure of bone marrow was observed and this explained the anemic picture improvement that appeared in blood indices of rats co-treated with lead acetate and pectin. Lead toxicity in subgroup 3A causing abnormal red blood cells which clear in blood smear (field stain) shown in figure 5 B1-B4) development of basophilic granules, hypo chromatic, tear drops and heamolyzied RBCs, which are characteristics of anemia caused by lead poisoning (43, 44), as previously noted (45). While in subgroup 3B, figure (5C) showed, only hypo chromatic RBCs due to protective effect of pectin. Our investigations (Figure 1) revealed significant (P ≤ 0.05) decrease in hepcidin gene expression of subgroup 3A in comparison by control and subgroup 3B. This finding is parallel to previous work (9, 10) reported that Hepcidin gene expression is reduced following experimentally induced anemia and hypoxia. This could explains the increased iron release from reticuloendothelial cells and Hepcidin participation in anemic conditions is revealed by increased iron absorption in these settings and iron transfer from endosomes to the cytoplasm has also been found to be inhibited by lead. Subgroup 3B showed significant elevation in hepcidin expression gene (P ≤ 0.05) which attributed to Pectin‘s ability that chelate metals in the digestive system and inhibit absorption while aiding their removal in the faces (14). In present study, subgroup 3A showed that, serum ferritin (iron store) and iron levels decreased significantly, in despite of increment of TIBC levels compared to the control. This result is in agreement with previous work (46) revealed that lead poisoning can cause anemia by interfering with haeme production, resulting in iron shortage. More recently (4), rats exposed to lead were found to have lower serum iron and transferrin saturation levels. The significant increase of TIBC could be related to higher production of transferrin by the liver in an attempt to make the most of the iron that is available (47). More over subgroup 3B showed an increasing of serum iron and ferritin and decreased TIBC as pectin rich in galacturonic acid (GalA) effectively chelate heavy metals (13, 48) suggesting that iron (Fe) bound to pectin is utilized by rats and enhancing the final Hb content. The current study demonstrated that liver functions in lead acetate intoxicate group had significantly higher AST and ALT activities than that of the control. These differences could be due to the toxic effect of lead acetate, which causes those enzymes to be released by increasing hepatocyte permeability or damaging the cell membrane of hepatocytes. In addition, lead toxicity produced an increase in cellular basal metabolic rate, irritability, and destructive alteration of liver cells (5, 49). As well as the creation of free radicals by lead caused harmful effect on hepatocytes, which reinforced by our histopathological picture showing in fig (5A-D) in subgroup 3A. This figure showed diffuse hydropic degeneration of hepatocytes in the hepatic parenchyma, multifocal regions of coagulative necrosis of hepatocytes with pyknotic nuclei and extensive diffuse regions of lytic necrosis with mild vacuolar degeneration of hepatocytes. The same picture showed mild amelioration of histpathological alternation in subgroup B (fig5 E), which showed mild vacuolar degeneration of hepatocytes, as majority of the hepatic parenchyma appeared to be improved may be due to treatment with Low-esterified pectin rapidly forms complexes with divalent metals as lead which are poisonous decreasing heavy metal cytotoxicity (42, 50). Previous study (38) found that ALAD activities were boosted by pectin treatment and decreased lipid peroxidation product in rat, as well as a significant increase in erythrocyte-SOD (Super oxide dismutase) and GSH activities, indicating that pectin protects body cells from oxidative radicals caused by lead. The significant increase of urea and creatinine lead acetate treated group compared to the control agrees with previous work (6) which recorded oral dose of lead acetate caused a significant rise in blood urea and serum creatinine. These results confirmed histopathologically (fig6 A-D), showed perivascular edema admixed with erythrocytes with necrosis of the glomerular tuft and thrombosis of the renal blood vessels with severe localized lytic necrosis, necrosis of the convoluted tubule lining epithelium, and moderate vacuolation of the glomerular tuft endothelial cell lining with cloudy swelling of the lining epithelium of some renal tubules. Slight improvement was observed in subgroup 3B as reflected on significant decrease of these parameters compared to that of subgroup 3A. These findings have been confirmed by hispathological picture (fig 6 E), that revealed improvement in renal tissue histology. This improvement was in the forms of mild vacuolation of the endothelial cell lining of the glomerular tuft 206 S. M. El-Bahr, S. Al-Sultan, A. F. Hamouda, S. A. E. Atwa, S. Y. Abo-Kora, A. A. Amin, S. Shousha, S. Alhojaily, A. Alnehas, R. R. Elzogby with mild degenerative changes in the lining epithelial cell of the renal tubules epithelium of some renal tubules, mild congestion of the renal blood vessels and glomerular blood capillaries with normal histological structure of the glomeruli. These findings were hand in hand with the improvement of functions the liver and kidney recorded earlier (50) with aresinic toxicity and co- treatment with pectin. This may be due to pectin hastened the elimination of arsenic in faces by lowering intestinal absorption, preventing buildup, and reducing arsenic toxicity. The exposure to lead acetate in subgroup 3A reduced the GSH activities and increased Nitric Oxide (NO) activity when compared to control. This result is comparable to that of a previous study of lead toxicity (38) caused oxidative stress, depletion of rapid antioxidants, higher production of reactive oxygen and nitrogen species and activation of lipid peroxidation. Thus, increasing oxidative stress induced a decrease in GSH levels, resulting in a decrease in glutathione concentration (3, 7). While in subgroup 3B, there were an improvement, as TAC and GSH were significantly elevated while Nitric Oxide activity was reduced. This finding may attributed to ROS scavenging activity of pectin, which is known to be reliant on pectin structural properties (51, 52). Present study showed that serum TNF-α and IL-6 in subgroup 3A increased significantly compared to the control group. Smilar results of (53) found that exposure to low lead level caused an increase of pro- inflammatory cytokines, such as TNF-α with a corresponding increase in other cytokines, such as IL-10, a T cell cross-regulatory factor, suggesting possible interference of lead in the immunophlogosis system. Lead has been found to enhance TNF- α production in vitro by human peripheral mononuclear cells (54). TNF-α is made primarily by activated macrophages and lymphocytes at the site of inflammation, and it plays a role in local and systemic inflammatory reactions with IL-6 and IL-1. It participates in local and systemic inflammatory reactions. Previous work (55) found that lead boosted total TNF- α cell expression in PBMC (+1ng/mL LPS). Pervious study (56) showed that blood levels of interleukin 6 (IL-6) and tumor necrosis factor (TNF- α) of 56 male workers chronically exposed to lead were significantly higher than that of the control group. In the current study, both TNF-α and IL-6 showed significant reduction in subgroup B compared to the control. This explained previously that commercially available Pectin had a pro-inflammatory effect in the spleen of BALB/c mice, up regulating cytokine release, including IL- 17, IFN-, and (TNF- α), independent of Gal-3 inhibition (57). Moreover, pectin had a similar reducing effect on (TNF- α) and IL-10 secretion resulting in a higher survival rate in endotoxin- shocked mice (58). Citrus pectin has also been proven to inhibit the production of interleukin-6 (IL-6) and lowered the inflammatory cytokine gene expression (59). Conclusion Present study on lead toxicity on rats demonstrated a hazards effect on Hepcidin expression gene, serum iron profile, blood indices, and liver and kidney functions, oxidative and pro inflammatory effects. So to manage lead toxicity it is necessary to use a specific chelating agent and in the same time. Pectin may be regarded nutritional items that could be utilized to reduce lead intestine absorption, minimize lead buildup, and ameliorate lead poisoning because they are both selective and effective in interacting with lead. We concluded that daily pectin ingestion is recommended in highly polluted areas with lead. 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Citrus pectin attenuates endotoxin shock via suppres- sion of toll-like receptor signaling in peyer’s patch myeloid cells. J Nutr Biochem 2017; 50: 38–45. PEKTIN VPLIVA NA IZBOLJŠANJE HEMATOLOŠKIH IN BIOKEMIJSKIH PARAMETROV, HISTOPATOLOGIJE, BIOLOŠKIH OZNAČEVALCEV OKSIDATIVNEGA STRESA, CITOKINOV TER IZRAŽANJE GENA ZA HEPCIDIN PRI S SVINCEM POVZROČENI TOKSIČNOSTI PRI PODGANAH S. M. El-Bahr, S. Al-Sultan, A. F. Hamouda, S. A. E. Atwa, S. Y. Abo-Kora, A. A. Amin, S. Shousha, S. Alhojaily, A. Alnehas, R. R. Elzogby Izvleček: Objav o zaščitnem učinku pektina pred toksičnostjo svinca pri podganah ni na voljo. Da bi proučili ta učinek, smo 40 samcev podgan razdelili v 3 skupine. V prvi, kontrolni skupini je bilo 10 podgan. V drugi skupini je bilo 10 podgan, ki so v poskus- nem obdobju (8 tednov) prejemale pektin v odmerku 100 mg/kg telesne teže. V tretji skupini je bilo 20 podgan, ki so 4 tedne dnev- no prejemale svinčev acetat v odmerku 400 mg/kg telesne teže. Tretja skupina je bila nato razdeljena v dve podskupini (3A in 3B). V podskupini 3A je bilo 10 podgan, ki so še naprej 4 tedne prejemale svinčev acetat v enakem odmerku, v podskupini 3B pa je 10 podgan prejemalo svinčev acetat in pektin. Vzorci krvi so bili odvzeti po 2, 4 in 8 tednih od začetka poskusa. Ob koncu poskusa so bili odvzeti še jetra, ledvice in kostni mozeg. Svinčev acetat je povzročil anemijo šele po štirih tednih, kar se je kazalo v zmanj- šanih vrednostih Hb, PCV, MCV, MCH in MCHC. V skupini podgan, ki so prejemale svinčev acetat, so te vrednosti do konca po- skusa ostale nizke. Koncentracije serumskega feritina, železa, skupne antioksidativne kapacitete (TAC), reduciranega glutationa (GSH) in izražanje jetrnega gena za hepcidin so se pri podganah, ki so prejemale svinčev acetat, znatno zmanjšale v primerjavi s kontrolo. Aktivnosti ALT in AST ter koncentracije sečnine, kreatinina, dušikovega oksida (NO), TNF-α, IL-6, skupne kapacitete vezave železa (TIBC) in svinca so se v skupini, ki je prejemala svinčev acetat, znatno povečale v primerjavi s kontrolno skupino. Najvidnejše spremembe pri podganah, ki so prejemale svinec, so bile jetrna degeneracija in krvavitve, ledvična nekroza in apop- toza mieloidnih celic. Spremembe, povezane s svinčevim acetatom, so se izboljšale s sočasnim zdravljenjem s pektinom, vendar normalne kontrolne vrednosti niso bile dosežene. Zaključili smo, da je pektin priporočljiv za zaščito pred toksičnostjo svinčevega acetata pri podganah. Ključne besede: svinčev acetat; toksičnost; pektin; hepcidin; biološki označevalci oksidativnega stresa; histopatologija Received: 25 January 2021 Accepted for publication: 28 November 2022 Slov Vet Res 2022; 59 (4): 211–17 DOI 10.26873/SVR-1286-2022 UDC 599.742.28:591.2:616-091.5(513.1) Case Report Introduction The giant panda (Ailuropoda melanoleuca) is an enigmatic carnivore, adapted to a highly specialized ecological niche (1). To date, the phylogeny, demographic history, genetic variation, population structure and adaptive evolution of the giant panda have been extensively documented (2). However, wild giant pandas remain endangered and threatened by human interference, climate change, disease, and food shortages (1, 3). Although China established its first panda sanctuary in 1987, captive breeding, especially of old animals, is a major problem for captive giant pandas (4-6). Although there are pathological studies on geriatric PATHOLOGICAL FINDINGS IN AN OLD FEMALE GIANT PANDA – A CASE REPORT Bangyuan Wu1,2,3, Juan Wang3, Tong Cai3, Chengdong Wang5, Desheng Li5, Linhua Deng5, Xi Peng4* 1Key Lab of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sci-ences, 1-5 Beichenxi Road, Chaoyang, 100101 Beijing, 2Key Laboratory of Southwest China Wildlife Re-sources Conservation (Ministry of Education), 3College of Life Science, China West Normal University, Nanchong, 637009 Sichuan, 4College of pharmacy, Chengdu University, Chengdu, 730000 Sichuan, 5Key-laboratory of Endangered Animal Reproduction and Conservation and Genetics, China Conservation and Research Center for the Giant Panda, Wolong, 623006 Sichuan, China *Corresponding author, E-mail: pengxi197313@163.com Abstract: The giant panda (Ailuropoda melanoleuca) is one of the most endangered species in the world. Climate change and susceptibility to disease are two of the greatest threats to this species. We performed a necropsy and histopathological exami- nation of the organs of an old panda and investigated the pathogenesis associated with death. Necropsy and histopathological observation revealed some typical age-related lesions, such as cataract, atherosclerosis, renal insufficiency and splenic atrophy. We also confirmed hepatic lesions associated with parasitic infection. Overall, our observations revealed that the predominant cause of mortality in this panda was multiple organ dysfunction (MOD). Key words: aged; giant panda; multiple organ dysfunction; pathology diseases in humans and domestic animals (7-9), pathological lesions in old giant pandas have been reported only once (5). Therefore, in this study, we investigated pathological changes associated with mortality of a deceased geriatric giant panda. The results could make an important contribution to the limited literature in this field and help to improve the welfare of giant pandas in captivity. Materials and methods History and observed clinical signs of the panda At this stage, we examined the life history of the giant panda, including sex and age, living conditions, treatment situation and course of the giant panda. Pathological examination revealed 212 B. Wu, J. Wang, T. Cai, C. Wang, D. Li, L. Deng, X. Peng that the panda was in an emaciated state and died. In addition, clinical signs, mental status, nutritional status, fur, skin, eyes, visible mucous membranes and the condition of other body surfaces, and physiological indices were observed. Necropsy The body of the giant panda was thoroughly vi- sually examined. After the external visual exami- nation, a necropsy was immediately performed, which revealed changes in various organs. The or- gans with visible pathological lesions were photo- graphed with a color video camera (Nikon 3 CCD) and further examined. Histopathology After thorough observation at a gross visual level, we selected tissue samples for histopathology from the lung, heart, aorta, liver, kidneys, spleen, digestive tract (including esophagus, duodenum, jejunum, ileum, colon, and rectum), mesenteric lymph nodes, ovaries, subcutaneous nodular lesion, and tissue from the decubitus. Tissue samples were then fixed in 4% paraformaldehyde (PFA) solution, dehydrated in a series of alcohols (at concentrations ranging from 70% to 100% ) and embedded in paraffin wax. Tissue sections (5 μm) were prepared, stained with hematoxylin and eosin (H&E) and examined under a light microscope. Histological changes were photographed using a digital camera (Olympus, Japan). Results History and clinical signs The case presented for necropsy involved a female giant panda, 15 years old, rescued from a nature reserve in China in 2005. Her age was estimated based on the degree of wear of the molars and skull growth (10). As she was unable to chew bamboo, she was fed a mixture of minced bamboo leaves and concentrated feed daily. Clinical examination revealed severe cataract (Fig. 1A), which indicated that the panda was suffering from anaemia and severe cardiac, hepatic and renal insufficiencies. After timely rescue and treatment, physiological values were essentially back to normal. In 2010, she went completely blind. Later, she gradually became emaciated and Figure 1: Gross pathological findings A. A cataract in the right eye. B. A necrotic decubital tissue in the left gluteal region. C. Massive mucous secretion in the pharynx (arrow). D. Yellowish-white nodules on the liver surface and a nematode found in a nodule (arrow). E. The spleen shows a shrunken appearance (arrow). F. A urinoma in the left dilated kidney (arrow) 213Pathological findings in an old female giant panda – a case report lost her fur, resulting in bald skin. In September 2013, decubital ulcers (Fig. 1B) appeared on the left buttock as a result of prolonged sleeping, which were difficult to treat. In late 2013, she fell into a deep coma and eventually died despite emergency rescue measures. Necropsy findings At necropsy, a large amount of viscous secretion was noted in the pharynx (Fig. 1C). The heart was enlarged and filled with blood clots. In addition, several pieces of a semitransparent jelly-like substance were seen adhering firmly to the aortic wall, showing signs of atherosclerosis. Multiple yellowish-white nodules of various sizes were observed on the surface of the liver, which contained caseous and purulent exudate and nematodes (Fig. 1D). The capsule of the spleen was contracted giving it a shrunken appearance (Fig. 1E). Both kidneys were swollen, and there were many yellowish-white mottled lesions on the surfaces. The left kidney had an enlarged pelvis filled with urine. (Fig. 1F). A single solid nodule (14×10×8 mm) was also found under the skin of the left abdomen. Figure 2: Histopathological changes, tissue sections stained with H&E A. Lung. Lymphocyte infiltration and thickening of alveolar walls. B. Aorta. Atheromatous plaque. C. Liver. A proliferative nodule (square) and inflammatory cell infiltration (circle). D. Liver. Fatty degeneration (black arrow) and necrosis of hepato-cytes (blue arrow). E. Kidney. Glomerular atrophy (black arrow) and proteinaceous casts in the tubule lumina (blue arrow). F. Kidney. Hyperplasia of connective tissue (black arrow) and infiltration of inflammatory cells (blue arrow). G. Spleen. Age-related atrophy of the spleen (black arrow). H. Mesenteric lymph node. Lymphocytes (black arrow), macrophages (blue arrow), and erythrocytes (green arrow) in a lymphatic sinus. I. Decubital tissue. Numerous inflammatory cells infiltrating the necrotic muscle tissue of the decubital ulcer 214 B. Wu, J. Wang, T. Cai, C. Wang, D. Li, L. Deng, X. Peng Histopathological Findings Histopathological examination of the lungs revealed slight thickening of the alveolar walls. This could be due to congestion and infiltration of inflammatory cells. Small scattered foci of inflammation were also observed, consisting mainly of lymphocytes and plasma cells, or macrophages phagocytosing black granules (Fig. 2A). In the aorta, an atheromatous plaque was noted that contained lipid-laden macrophages and proliferated connective tissue (Fig. 2B). In the liver, nodular cirrhosis with pseudohepatic lobules, fatty degeneration, and necrosis of hepatocytes was observed. Connective tissue hyperplasia, inflammatory cell infiltration, and a small focal abscess were also found in the examined tissue section of the liver (Fig. 2C and 2D). In the kidney, chronic sclerosing glomerulonephritis was diagnosed, characterized by glomerular atrophy, necrosis of the tubular epithelium, formation of proteinaceous casts, connective tissue hyperplasia, and infiltration of inflammatory cells (lymphocytes and neutrophils) (Figs. 2E and 2F). Hyperemia, abundant hemosiderin, and macrophages phagocytosing hemosiderin/erythrocytes were observed in the red pulp of the spleen (Fig. 2G). The splenic trabeculae were relatively enlarged due to age-related atrophy. Mild acute serous lymphadenitis was observed in the mesenteric lymph nodes, characterized by accumulation of lymph, fibrin, and inflammatory cells in the slightly enlarged sinuses of the lymph nodes (Fig. 2H). Only mild edema and mild exfoliation of the mucosal epithelium were noted in the digestive tract (images not shown). The single subcutaneous nodule was a benign fibroma composed of fibrocytes and desmocytes. The muscle tissue at the site of the pressure ulcer showed coagulation necrosis and inflammatory cell infiltration composed predominantly of neutrophils and macrophages (Fig. 2I). Discussion According to medical records, this female giant panda was about 23 years old when she died in 2013 after 8 years in captivity (11). At necropsy, massive pharyngeal mucous secretion was noted, possibly leading to the panda’s asphyxiation and death. Previously, it was reported that difficulty in coughing up sputum in old pandas was due to decreased intrathoracic negative pressure, weakening of respiratory muscles and elastic retraction of the lungs (7). Lung function is known to deteriorate with age (12), and pulmonary alveolar epithelium permeability has been found to be higher in the elderly than in adults (13). It has been reported that age may play an important role in certain diseases, such as acute respiratory distress syndrome and chronic obstructive pulmonary disease (12), which is consistent with our findings in this report. In addition, some studies suggest that the lung plays an important role in the development of multiple organ dysfunction (MOD) (14, 15). MOD is more commonly reported after trauma and is associated with high mortality (16, 17). It has also been reported that the elderly are more susceptible and at higher risk of MODS (14). MOD is defined as a group of various chronic diseases, including chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis (7), decreased glomeruli and tubulointerstitial fibrosis in the kidney (8), immune dysfunction and degeneration of the spleen(18), chronic heart disease (19), presbycusis (20), cataracts, and cognitive impairment (3). In our case of old giant panda, several chronic pathological changes such as cataracts, lung and kidney lesions, adipose tissue atrophy, atheromatous plaques in the aorta, and splenic atrophy were observed and reported, which developed along with parasitic infection of the liver and consequent emaciation. Histopathologically, the main findings that could be age-related were atheromatous plaques, reduction of renal glomeruli, age-related atrophy of the spleen, thickening of alveolar septa in the lungs, and connective tissue hyperplasia in the liver and kidney. These lesions were consistent with the pathological manifestations of ageing described in humans and other mammals, including giant pandas (5, 7, 8). A typical age- related change, “cataract”, was due to the gradual loss of elasticity of the lens (21, 22). In addition, oxidised low-density lipoprotein (ox-LDL) has been reported to lead to endothelial dysfunction, which is considered to be an initial step in the formation of atheroma (23, 24). Ox-LDL has been shown to play an important role in the formation of lipid-laden macrophages, the primary cellular component of atherosclerotic lipid lesions (25). This may be the reason why we found the atheromatous aortic plaque in this case. We believe that the decrease in renal glomeruli in this 215Pathological findings in an old female giant panda – a case report giant panda case is related to tubular necrosis and fibrosis, similar to some previous reports (26, 27). Age-related changes are well-known factors that influence the susceptibility to disease of almost all vital organs. The elderly individuals often show a markedly exaggerated host immune response to inflammatory stimuli (14). In the present study, the pathological lesions associated with inflammation, particularly the infiltration of inflammatory cells in the lung, kidney, liver, and lymph nodes, were possibly due to the spread of inflammation from bedsores or parasitic hepatitis to these organs. In general, systemic inflammatory response syndrome (SIRS) has been shown to have an adaptive survival function for the host, but in critically ill patients, uncontrolled production of inflammatory mediators can lead to MODS (28). The results of this study were consistent with the “2-hit” hypothesis in the development of MODS, which states that an initial insult primes the host such that a subsequent impairment, such as infection or surgery, greatly enhances the host response (18). We believe that in our case a combination of age-related pathological lesions and chronic parasitic infection led to MOD, and that MOD was the main cause of death in this old giant panda. The parasitic hepatitis could be the initial impairment, and the decubitus ulcer could be the subsequent impairment leading to increased damage, as the aging can increase susceptibility to organ dysfunction and systemic inflammation, as shown by previous reports (29, 30). The macroscopic and microscopic lesions in the liver were consistent with chronic verminous hepatitis, and it could be hypothesised that this giant panda already suffered from a parasitic infection such as the nematode Baylisascaris schroederi in the wild (31). In an anatomical study of 33 wild giant pandas, a 100% lumbricoid infection rate indicated the prevalence of parasitic infections in wild animals (32). A study examining causes of death in wild giant pandas from 1971 to 2005 found that the greatest threat to wild giant pandas is migration of visceral larvae (33). Consistent with the anatomical location of the nematode, the lesions found in the liver and the life cycle of the parasite (34), the parasite may have migrated from the bile duct to the liver. Currently, research in China mainly focuses on parasite species and associated morbidity in wildlife. However, in the future, more attention should also be paid to the transmission and control strategies of parasitic wildlife diseases to increase the overall life expectancy of wildlife. In addition, many infectious diseases of humans have hosts or vectors in animals, which places greater demands on research and control of animal diseases (35). In conclusion, we believe that MOD was the main reason for the death of this old female giant panda. A series of age-related pathological lesions, supported by pre-existing pathological conditions such as liver dysfunction due to parasite infestation, eventually led to poor physical condition, emaciation, respiratory failure and death. Acknowledgments Authors wish to express gratitude to China Conservation and Research Center for the Giant Panda for their cooperation and assistance in conducting the study. Supported by the program for the Education Department of Sichuan Province (project no. 17ZB0425), the Meritocracy Research Funds of China West Normal University (project no. 17YC349) and the Fundamental Research Funds of China West Normal University (project no. 20A003). The datasets supporting the results of this document are contained within the article. Any additional data may be requested to the corresponding author. The datasets supporting the results of this document are contained within the article. Any additional data may be requested to the corresponding author. The authors declare no conflict of interest. Conceptualization and supervision: W.B.Y. and P.X., Methodology, investigation, data curation, W.B.Y., W.J., C.T., W.C.D., L.D.S., D.L.H, writing- original draft preparation: W.B.Y. and W.J., writing–review and editing: P.X. All authors have read and agreed to the published version of the manuscript. References 1. Lu Z, Warren EJ, Marilyn MR, et al. Patterns of genetic diversity in remaining giant panda pop- ulations. Conserv Biol 2001; 15(6): 1596–607. 2. Wei FW, Yibo H, Lifeng Z, Michael WB, Zan XJ, Lei Z. Black and white and read all over: the 216 B. Wu, J. Wang, T. Cai, C. Wang, D. Li, L. Deng, X. Peng past, present and future of giant panda genetics. Mol Ecol 2012; 21(23): 5660–74. 3. Steinmeyer C, Pennings PS, Foitzik S. Mul- ticolonial population structure and nestmate recognition in an extremely dense population of the European ant Lasius flavus. Insect Soc 2012; 59(4): 499–510. 4. Burrell C, Hemin Z, Desheng L, Chengdong W, Caiwu L, Aitken-Palmer C. 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The age of determination for giant panda. Acta Theriol Sin 1989; 8(3): 161–5. 11. Zhou X, Yan H, Jingyan H, Shiqiang Z, Dian L. Behavioral development of gaint panda and influencing factors in husbandry manage- ment. J Heilongjiang Univ Sci Technol 2013; 34(2): 106–10. 12. Sevransky JE, Haponik EF. Respiratory failure in elderly patients. Clin Geriatr Med 2003; 19(1): 205–24. 13. Yang MF, He ZX, Wang SW. Evaluation of pulmonary epithelial permeability with 99Tc- m-DTPA radioaerosol. Chin J Nucl Med 2002; 22(4): 250–2. 14. Wang XP, Zhu QL, Xue Q, et al. Role of the lung in the progression of multiple organ dysfunction syndrome in ageing rat model. Chin Med J 2012; 125(015): 2708–13. 15. Wang SW, Han YL, Qian XS, et al. Clinical features of multiple organ failure in the elderly: a report of 1605 cases. Chin J Mult Organ Dis Elderly 2002; 1(1): 7–10. 16. Durham RM, Moran JJ, Mazuski JE, Sha- piro MJ, Baue AE, Flint LM. Multiple organ fail- ure in trauma patients. 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Int Urol Nephrol 2010; 42(2): 425–33. 217Pathological findings in an old female giant panda – a case report 28. Nathens AB, Marshall JC. Sepsis, SIRS, and MODS: What’s in a name? World J Surg 1999; 20(4): 386–91. 29. Nin N, Lorente JA, De Paula M, et al. Aging increases the susceptibility to injurious mechan- ical ventilation. Intensive Care Med 2008; 34(5): 923–31. 30. Eachempati SR, Hydo LJ, Shou J, Barie PS. Outcomes of acute respiratory distress syn- drome (ARDS) in elderly patients. J Trauma 2007; 63(2): 344–50. 31. Zhou X, Yu H, Wang N, Xie Y, Yang GY. Molecular diagnosis of Baylisascaris schroed- eri infections in giant panda (Ailuropoda mela- noleuca) feces using PCR. J Wildl Dis 2013; 49(4): 1052–5. 32. Feng WH, Anju Z, eds. Giant panda reproduc- tion and disease research. Chengdu, Sichuan, China : Sichuan Science and Technology Press, 1991: 244–8. 33. Zhang JS, Peter D, Huali H, Guangyou Y, A Marm K, Shuyi Z. Parasite threat to panda con- servation. EcoHealth 2007; 5(1): 6–9. 34. Blouin MS, Liu J, Berry RE. Life cycle variation and the genetic structure of nematode populations. Heredity 1999; 83(3): 253–9. 35. Kaupke A, Rzezutka A. Epidemiology of the invasion of Cryptosporidium parvum in farm and wild animals. Med Weter 2017; 73(7): 387–94. PATOLOŠKE UGOTOVITVE PRI SAMICI VELIKEGA PANDE – POROČILO O PRIMERU B. Wu, J. Wang, T. Cai, C. Wang, D. Li, L. Deng, X. Peng Izvleček: Veliki panda (Ailuropoda melanoleuca) je ena najbolj ogroženih vrst na svetu. Vrsto najbolj ogrožajo podnebne spre- membe in dovzetnost za bolezni. Opravili smo nekropsijo in histopatološki pregled organov starega pande ter raziskali patoge- nezo, povezano s smrtjo. Z nekropsijo in histopatološkim opazovanjem smo odkrili nekatere značilne starostne spremembe, kot so katarakta, ateroskleroza, ledvična insuficienca in atrofija vranice. Potrdili smo tudi jetrne spremembe, povezane s parazitsko okužbo. Naša opažanja so pokazala, da je bil prevladujoči vzrok smrti tega pande disfunkcija več organov (MOD). Ključne besede: star; veliki panda; disfunkcija več organov; patologija Slov vet Res, Author Index Volume 59, 2022 219 AUTHOR INDEX VOLUME 59, 2022 Abo-Kora SY, see El-Bahr SM, Al-Sultan S, Hamouda AF, Atwa SAE, Abo-Kora SY, Amin AA, Shousha S, Alhojaily S, Alnehas A, Elzogby RR ..................................................... 195 Aja PM, see Alum EU, Ibiam UA, Ugwuja EI, Aja PM, Igwenyi IO, Offor CE, Orji OU, Aloke C, Ezeani NN, Ugwu OPC, Egwu CO..........................31 Akbalık ME, see Topaloğlu U, Ketani MA, Akbalık ME, Sağsöz H, Saruhan BG, Bayram B ..... 99 Akyol ET, see Parlak K, Yalcin M, Akyol ET, Ok M, Arican M .............................................185 Alakuş İ, see Deveci MZY, Yurtal Z, İşler CT, Emiroğlu SB, Alakuş İ, Altuğ ME .................... 47 Alhojaily S, see El-Bahr SM, Al-Sultan S, Hamouda AF, Atwa SAE, Abo-Kora SY, Amin AA, Shousha S, Alhojaily S, Alnehas A, Elzogby RR...........195 Alkan S, see Karabağ K, Alkan S, Karslı T, İkten C, Şahin İ, Mendeş M .............................89 Alnehas A, see El-Bahr SM, Al-Sultan S, Hamouda AF, Atwa SAE, Abo-Kora SY, Amin AA, Shousha S, Alhojaily S, Alnehas A, Elzogby RR.....................195 Aloke C, see Alum EU, Ibiam UA, Ugwuja EI, Aja PM, Igwenyi IO, Offor CE, Orji OU, Aloke C, Ezeani NN, Ugwu OPC, Egwu CO...........................31 Al-Sultan S, see El-Bahr SM, Al-Sultan S, Hamouda AF, Atwa SAE, Abo-Kora SY, Amin AA, Shousha S, Alhojaily S, Alnehas A, Elzogby RR......195 Altuğ ME, see Deveci MZY, Yurtal Z, İşler CT, Emiroğlu SB, Alakuş İ, Altuğ ME.......................47 Alum EU, Ibiam UA, Ugwuja EI, Aja PM, Igwenyi IO, Offor CE, Orji OU, Aloke C, Ezeani NN, Ugwu OPC, Egwu CO. Antioxidant effect of Buchholzia coriacea ethanol leaf-ex- tract and fractions on freund’s adjuvant-in- duced arthritis in albino rats: A comparative stdy................................................................31 Amin AA, see El-Bahr SM, Al-Sultan S, Hamouda AF, Atwa SAE, Abo-Kora SY, Amin AA, Shousha S, Alhojaily S, Alnehas A, Elzogby RR..................195 Andrásofszky E, see Hetényi N, Andrásofszky E ...137 Arican M, see Parlak K, Yalcin M, Akyol ET, Ok M, Arican M...................................................185 Atwa SAE, see El-Bahr SM, Al-Sultan S, Hamouda AF, Atwa SAE, Abo-Kora SY, Amin AA, Shousha S, Alhojaily S, Alnehas A, Elzogby RR.......................195 Bayram B, see Topaloğlu U, Ketani MA, Akbalık ME, Sağsöz H, Saruhan BG, Bayram B...............99 Beqiraj D, see Sulçe M, Munga A, Beqiraj D, Ozuni E, Zalla P, Muça G, Koleci X...........................129 Cai T, see Wu B, Wang J, Cai T, Wang C, Li D, Deng L, Peng X...............................................211 Deng L, see Wu B, Wang J, Cai T, Wang C, Li D, Deng L, Peng X...............................................211 Deveci MZY, Yurtal Z, İşler CT, Emiroğlu SB, Alakuş İ, Altuğ ME. Herniorrhaphy and surgical outcomes of diaphragmatic hernia in cats.................................................................47 Egwu CO, see Alum EU, Ibiam UA, Ugwuja EI, Aja PM, Igwenyi IO, Offor CE, Orji OU, Aloke C, Ezeani NN, Ugwu OPC, Egwu CO...........................31 Eid HM, El-Mahallawy HS, Elsheshtawy HM, Shalaby AM, Shetewy MM, Eidaroos NH. Antimic- robial resistance and virulence-associated genes of aeromonads isolated from Lake Manzala water and wild Nile tilapia: Implications to public health and the lake microbial community..........................59 Eidaroos NH, see Eid HM, El-Mahallawy HS, Elsheshtawy HM, Shalaby AM, Shetewy MM, Eidaroos NH.....................................................59 El-Bahr SM, Al-Sultan S, Hamouda AF, Atwa SAE, Abo-Kora SY, Amin AA, Shousha S, Alhojaily S, Alnehas A, Elzogby RR. Pectin improves hemato- -biochemical parameter, histopathology, oxidative stress biomarkers, cytokines and expression of hep- cidin gene in lead induced toxicity in rats...........195 El-Ghany WAA. Avian cryptosporidiosis: a signi- ficant parasitic disease of public health hazard .....5 El-Mahallawy HS, see Eid HM, El-Mahallawy HS, Elsheshtawy HM, Shalaby AM, Shetewy MM, Eidaroos NH....................................................59 Elsheshtawy HM, see Eid HM, El-Mahallawy HS, Elsheshtawy HM, Shalaby AM, Shetewy MM, Eidaroos NH.....................................................59 Elzogby RR, see El-Bahr SM, Al-Sultan S, Hamou- da AF, Atwa SAE, Abo-Kora SY, Amin AA, Shousha S, Alhojaily S, Alnehas A, Elzogby RR...........................195 Slov vet Res, Author Index Volume 59, 2022220 Emiroğlu SB, see Deveci MZY, Yurtal Z, İşler CT, Emiroğlu SB, Alakuş İ, Altuğ ME.........................47 Ezeani NN, see Alum EU, Ibiam UA, Ugwuja EI, Aja PM, Igwenyi IO, Offor CE, Orji OU, Aloke C, Ezeani NN, Ugwu OPC, Egwu CO.........................31 Hamouda AF, see El-Bahr SM, Al-Sultan S, Hamouda AF, Atwa SAE, Abo-Kora SY, Amin AA, Shousha S, Alhojaily S, Alnehas A, Elzogby RR..........195 Hetényi N, Andrásofszky E. Evaluation of commer- cial tortoise and turtle feeds..............................137 Ibiam UA, see Alum EU, Ibiam UA, Ugwuja EI, Aja PM, Igwenyi IO, Offor CE, Orji OU, Aloke C, Ezeani NN, Ugwu OPC, Egwu CO.........................31 Igwenyi IO, see Alum EU, Ibiam UA, Ugwuja EI, Aja PM, Igwenyi IO, Offor CE, Orji OU, Aloke C, Ezeani NN, Ugwu OPC, Egwu CO.........................31 Igwenyi IO, see Alum EU, Ibiam UA, Ugwuja EI, Aja PM, Igwenyi, IO, Offor CE, Orji OU, Aloke C, Ezeani NN, Ugwu OPC, Egwu CO.......................31 İkten C, see Karabağ K, Alkan S, Karslı T, İkten C, Şahin İ, Mendeş M.......................................89 İşler CT, see Deveci MZY, Yurtal Z, İşler CT, Emiroğlu SB, Alakuş İ, Altuğ ME.........................47 Karabağ K, Alkan S, Karslı T, İkten C, Şahin İ, Mendeş M. Effects of selection in terms of meat yield traits on leptin receptor gene in Japanese quail lines.........................................................................89 Karslı T, see Karabağ K, Alkan S, Karslı T, İkten C, Şahin İ, Mendeş M...............................89 Ketani MA, see Topaloğlu U, Ketani MA, Akbalık ME, Sağsöz H, Saruhan BG, Bayram B...............99 Koleci X, see Sulçe M, Munga A, Beqiraj D, Ozuni E, Zalla P, Muça G, Koleci X...........................129 Li D, see Wu B, Wang J, Cai T, Wang C, Li D, Deng L, Peng X...............................................211 Li Y, see Munibullah M, Li Y, Munib K, Zhang Z...75 Mendeş M, see Karabağ K, Alkan S, Karslı T, İkten C, Şahin İ, Mendeş M...............................89 Milevoj N, Tozon N, Tomsič K. Use of cannabi- diol products by pet owners in Slovenia: a survey- based study....................................................149 Muça G, see Sulçe M, Munga A, Beqiraj D, Ozuni E, Zalla P, Muça G, Koleci X...........................129 Munga A, see Sulçe M, Munga A, Beqiraj D, Ozuni E, Zalla P, Muça G, Koleci X...........................129 Munib K, see Munibullah M, Li Y, Munib K, Zhang Z...........................................................75 Munibullah M, Li Y, Munib K, Zhang Z. Regional epidemiology and associated risk factors of peste des petits ruminants in Asia – A review............................................................75 Naseri A, see Parlak K, Yalcin M, Akyol ET, Ok M, Arican M..............................................185 Offor CE, see Alum EU, Ibiam UA, Ugwuja EI, Aja PM, Igwenyi IO, Offor CE, Orji OU, Aloke C, Ezeani NN, Ugwu OPC, Egwu CO.......................31 Ok M, see Parlak K, Yalcin M, Akyol ET, Ok M, Arican M........................................................185 Orji OU, see Alum EU, Ibiam UA, Ugwuja EI, Aja PM, Igwenyi IO, Offor CE, Orji OU, Aloke C, Ezeani NN, Ugwu OPC, Egwu CO.........................31 Ozuni E, see Sulçe M, Munga A, Beqiraj D, Ozuni E, Zalla P, Muça G, Koleci X...........................129 Parlak K, Naseri A, Yalcin M, Akyol ET, Ok M, Arican M. Evaluation of trauma scoring and endothelial glycocalyx injury in cats with head trauma...........................................................185 Peng X, see Wu B, Wang J, Cai T, Wang C, Li D, Deng L, Peng X...............................................211 Rajčević U, Smole A. Preclinical mouse models in adoptive cell therapies of cancer.......................173 Sağsöz H, see Topaloğlu U, Ketani MA, Akbalık ME, Sağsöz H, Saruhan BG, Bayram B........................99 Şahin İ, see Karabağ K, Alkan S, Karslı T, İkten C, Şahin İ, Mendeş M.......................................89 Saruhan BG, see Topaloğlu U, Ketani MA, Akbalık ME, Sağsöz H, Saruhan BG, Bayram B...............99 Shalaby AM, see Eid HM, El-Mahallawy HS, Elsheshtawy HM, Shalaby AM, Shetewy MM, Eidaroos NH....................................................59 Shetewy MM, see Eid HM, El-Mahallawy HS, Elsheshtawy HM, Shalaby AM, Shetewy MM, Eidaroos NH.....................................................59 Shousha S, see El-Bahr SM, Al-Sultan S, Hamouda AF, Atwa SAE, Abo-Kora SY, Amin AA, Shousha S, Alhojaily S, Alnehas A, Elzogby RR...............................................................195 Smodiš Škerl MI, Tlak Gajger I. Performance and Nosema spp. spore level in young honeybee (Apis mellifera carnica, Pollmann 1879) colonies supple- mented with candies.....................................159 Slov vet Res, Author Index Volume 59, 2022 221 Smole A, see Rajčević U,. Preclinical mouse models in adoptive cell therapies of cancer...................173 Sulçe M, Munga A, Beqiraj D, Ozuni E, Zalla P, Muça G, Koleci X. Applicability of flow cytomet- ry in identifying and staging lymphoma, leukemia and mast cell tumors in dogs: an overview......129 Tlak Gajger I, see Smodiš Škerl MI, Tlak GajgerI...........................................................159 Tomsič K, see Milevoj N, Tozon N, Tomsič K......149 Topaloğlu U, Ketani MA, Akbalık ME, Sağsöz H, Saruhan BG, Bayram B. Immunolocalization of HOXA11 and HLX prot ins in cow placenta du- ring pregnancy................................................99 Tozon N, see Milevoj N, Tozon N, Tomsič K........149 Ugwu OPC, see Alum EU, Ibiam UA, Ugwuja EI, Aja PM, Igwenyi IO, Offor CE, Orji OU, Aloke C, Ezeani NN, Ugwu OPC, Egwu CO..........................31 Ugwuja EI, see Alum EU, Ibiam UA, Ugwuja EI, Aja PM, Igwenyi, IO, Offor CE, Orji OU, Aloke C, Ezeani NN, Ugwu OPC, Egwu CO..........................31 Uršič M. Morphometrical features of the cave bear and brown bear head skeleton: A comparative study..............................................................113 Wang C, see Wu B, Wang J, Cai T, Wang C, Li D, Deng L, Peng X.......................................211 Wang J, see Wu B, Wang J, Cai T, Wang C, Li D, Deng L, Peng X.......................................211 Wu B, Wang J, Cai T, Wang C, Li D, Deng L, Peng X. Pathological findings in an old female giant panda – a case report......................................211 Yalcin M, see Parlak K, Yalcin M, Akyol ET, Ok M, Arican M.........................................................185 Yurtal Z, see Deveci MZY, Yurtal Z, İşler CT, Emiroğlu SB, Alakuş İ, Altuğ ME..........................47 Zalla P, see Sulçe M, Munga A, Beqiraj D, Ozuni E, Zalla P, Muça G, Koleci X...........................129 Zhang Z, see Munibullah M, Li Y, Munib K, Zhang Z.....................................................................75 THE SCIENTIFIC JOURNAL OF THE VETERINARY FACULTY UNIVERSITY OF LJUBLJANA SLOVENIAN VETERINARY RESEARCH SLOVENSKI VETERINARSKI ZBORNIK Slov Vet Res • Ljubljana • 2022 • Volume 59 • Number 4 • 169–222459 Volume SLOVENIAN VETERINARY RESEARCH SLOVENSKI VETERINARSKI ZBORNIK Slov Vet Res 2022; 59 (4) Review Article Rajčević U, Smole A. Preclinical mouse models in adoptive cell therapies of cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 Original Research Articles Parlak K, Naseri A, Yalcin M, Akyol E T, Ok M, Arican M. Evaluation of trauma scoring and endothelial glycocalyx injury in cats with head trauma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .185 El-Bahr SM, Al-Sultan S, Hamouda AF, Atwa SAE, Abo-Kora SY, Amin AA, Shousha S, Alhojaily S, Alnehas A, Elzogby RR. Pectin improves hemato-biochemical parameter, histopathology, oxidative stress biomarkers, cytokines and expression of hepcidin gene in lead induced toxicity in rats . . . . . . . . . . . . . . . . . . 195 Case Report Wu B, Wang J, Cai T, Wang C, Li D, Deng L, Peng X. Pathological findings in an old female giant panda – a case report . . . . . . . . . . 211 Author Index Volume 59, 2022 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219