UVOD Etilen-glikol (EG) je alkohol koji se široko koristi u domaćinstvima i industriji, pre svega kao glavni sastojak antifriza. Zbog bezbojnosti, slatkastog ukusa i lake dostupnosti, često predstavlja izvor trovanja kod kućnih ljubimaca, posebno u zimskom periodu. Nakon oralnog unosa, EG se brzo apsorbuje i metaboliše u jetri i bubrezima u toksične metabolite, uključujući oksalnu kiselinu, koja izaziva teška oštećenja bubrežnog parenhima i dovodi do akutne bubrežne insuficijencije (ABI). Klinička slika trovanja razvija se kroz tri faze i uključuje neurološkim, gastrointestinalnim i bubrežnim simptomima. Smrtonosna doza za mačke iznosi približno 1,5 mL/kg, a stopa smrtnosti izuzetno visoka. Intoksikacija EG kod mačaka je klinički značajan problem, sa posebnim izazovima u dijagnostici i lečenju. MATERIJAL I METODE Dve gravidne vlasničke mačke, stare oko 1,5 godinu, dovedene su u lošem opštem stanju sa znacima konvulzija i dehidratacije. Zbog viskog stepena dehidratacije nije bilo moguće uzeti uzorke krvi za laboratorijsku analizu. Terapija je sprovođena subkutanim putem i obuhvatala je infuziju, antiemetičku terapiju (maropitant, pantoprazol - nakon uspostavljanja venske linije) i nadoknadu vitamina B1, B6, B12 i C. Dijagnostičke procedure obuhvatile su ultrazvučni pregled abdomena (korišćen je aparat Esaote MyLab Seven, Esaote, Genova, Italija) i mikroskopski pregled urina. Uprkos sprovedenoj terapiji, obe mačke su uginue. Obdukcija je izvršena na Fakultetu veterinarske medicine, uz fotodokumentaciju patomorfoloških promena. Uzorci bubrega su fiksirani u formalinu, obrađeni standardnom histološkom procedurom i uklopljeni u parafinske kalupe. Preseci tkiva debljine 5 mm dobijeni rotacionim mikrotomom (Cut 5062, Slee, Mainz, Nemačka) obojeni su hematoksilin/eozinom, kao i kompletom za Masson-Goldner bojenje (Merck KGaA, Darmstadt, Nemačka). Histološka analiza je sprovedena svetlosnim mikroskopom, uz fotodokumentaciju najreprezentativnijih struktura (mikroskop CX3; kamera UC 50, oba Olympus, Münster, Nemačka). REZULTATI Na kliničkom pregledu obe mačke su pokazivale akutne neurološke simptome, jake konvulzije, uz tešku dehidrataciju i opštu slabost. Telesna temperatura bila je u granicama normale, dok je disanje bilo ubrzano. Vlasnici su prijavili da su mačke bile nestale dva dana pre pojave simptoma, a po njihovom pronalasku posumnjali su na kontakt sa antifrizom. Klinička slika je brzo progresirala ka znacima sistemske intoksikacije, sa izraženim indikatorima ABI. Ultrazvučnim pregledom abdomena utvrđeno je da su obe mačke bile gravidne, ali u različitim fazama graviditeta - jedna u ranoj, a druga u kasnoj fazi. Kod obe su uočene izražene promene u bubrežnom parenhimu, sa hiperehogenim korteksom i gubitkom kortikomedularne diferencijacije. Kod starijih fetusa primećena je difuzna hiperehogenost bubrežnog parenhima, edematozno širenje intersticijuma i gubitak normalne anatomije bubrega. Prisutnost hiperehogene "zvezdaste" strukture unutar parenhima ukazivala je na akutne toksične promene, koje su bile u skladu sa nalazima akutne tubularne nekroze (ATN). Mikroskopski pregled urina otkrio je prisustvo umerene količine granularnih i hijalinih cilindara. U sedimentu su uočene brojne deskvamirane tubularne epitelne ćelije, manja količina leukocita i pojedinačni eritrociti. Primećena je značajna količina kalcijum-oksalat monohidratnih kristala. Bakterije nisu uočene, dok su u manjoj meri zapažene amorfne proteinske granule. Makroskopski pregled kod uginulih mačaka otkrio je niz karakterističnih promena koje ukazuju na sistemsku toksičnost, sa najizraženijim lezijama u bubrezima i drugim visoko vaskularizovanim organima. Bubrezi su bili uvećani, bledosive do voštane boje, sa glatkom, ali zrnastom površinom. Na preseku su korteks i medula bili slabo diferentovani, uz prisustvo sitnih, svetlijih tačkica u kortikalnom delu. Jetra je bila uvećana, bledoružičaste boje, sa glatkom i zategnutom kapsulom. Sluznica usne duplje bila je izrazito bleda, dok je rožnjača bila zamućena. Na površini pluća i epikardu uočena su brojna punktiformna krvarenja. U telesnim šupljinama, naročito u peritonealnoj i pleuralnoj, nalazila se veća količina serozne, žućkaste tečnosti. Histološki pregled bubrega otkrio je izražene promene koje ukazuju na ATN. Epitelne ćelije proksimalnih tubula bile su otečene, delimično deskvamirane, sa izraženom vakuolizacijom citoplazme i piknotičnim jedrima. U lumenima tubula bili su prisutni hijalini i granularni cilindri, kao i brojni kristali kalcijum-oksalata u obliku igličastih i rozetastih formacija, koje su pokazivale dvolomnost pod polarizovanom svetlošću. Glomeruli uglavnom nisu pokazivali značajne promene. DISKUSIJA Intoksikacija EG predstavlja hitno stanje koje se kod mačaka retko dijagnostikuje na vreme zbog nespecifičnih simptoma i brzog progresivnog kliničkog toka. Neurološki znaci, koji su bili inicijalno prisutni kod mačaka, karakteristični su za prvu fazu trovanja i rezultat su direktnog delovanja EG i njegovih metabolita na centralni nervni sistem. Ubrzano disanje i izražena slabost upućuju na metaboličku acidozu i hipoksiju, koje prate dalju razgradnju toksina. Ultrazvučni nalazi ukazivali su na izražene strukturne promene u bubrezima, kao što su hiperehogenost korteksa i gubitak kortikomedularne diferencijacije. Takvi nalazi su u skladu sa prethodno opisanim slučajevima...

Caenorhabditis elegans is a nematode that is known as an alternative model for research. Adult forms are about 1mm long, while larvae reach a length of 0.25 mm. This nematode is easily grown in laboratories in a special nematode growth medium (NGM) with a layer of the bacterium Escherichia coli, on which C. elegans feeds. The worms exist in two natural sexes. C. elegans is one of the most frequently used experimental model organisms in biomedicine. The main advantages of C. elegans as a model organism are its small size, high reproductive capacity, simple cultivation, low maintenance costs, the possibility of long preservation at low temperatures, very short reproductive time and good visibility. C. elegans has been used for analysis of toxicants, investigation of cancer, discovery of antiparasitics and anticancer drugs, and investigation of the functions of the neuromuscular system. The C. elegans model is successful for identifying and characterizing molecular drug targets of all classes of anthelmintics acting on the nervous system. All these characteristics make C. elegans a valuable and prospective model organism in biomedical research. This review article primarily focuses on the utility of C. elegans as a model organism in the evaluation of anthelmintic substances, with particular emphasis on essential oils and natural products, while briefly highlighting its general biomedical applications. The main goal of this paper is to demonstrate our application of this model in some antinematodal research.

Background/Objectives: Drug repurposing is one of the possible sources of new anthelmintics. Afoxolaner is a insecticide/acaricide that acts on the insect GABA receptor and regulates uptake of chloride ions. We examined the interaction of afoxolaner with GABA receptors of nematodes. Methods: We tested the effects of afoxolaner on the motility of the free-living nematode C. elegans and its effect on the contractions/relaxation of the parasitic nematode A. suum. By means of structural bioinformatics, we analyzed the potential binding sites of afoxolaner on the nematode synaptic/exrasynaptic GABA receptor. Results: Afoxolaner inhibited the development of adult C. elegans, significantly reduced the distance traveled, movement speed, and motility score. Afoxolaner significantly inhibited the contractions of the A. suum neuromuscular preparation by increasing the EC₅₀ value of ACh but not changed the E_max. However, afoxolaner neutralized the inhibitory effect of GABA, significantly reduced the EC₅₀ of ACh and increased E_max. In the relaxation study, afoxolaner potentiates GABA-evoked relaxation but inhibited piperazine-induced relaxation. Conclusions: Pharmacological effect of afoxolaner on GABA receptors in nematodes can be defined in two ways: when contractions were induced by ACh, afoxolaner behaves as a partial agonist/antagonist, which would involve binding to an orthosteric site on the receptor. Probably this effect is realized on synaptic GABA receptors between inhibitory interneurons and motoneurons. However, when relaxation is induced by activation of an extrasynaptic GABA receptor, afoxolaner exhibits the characteristics of positive allosteric modulator, it does not cause relaxation by itself, but enhances the effect of GABA and antagonizes the action of piperazine.

The active constituents of essential plant oils (EOAIs), monoterpenoid carvacrol and monoterpene p-cymene, are widely distributed in many aromatic plants and their products. They differ in that carvacrol has a phenolic functional group. The numerous pharmacological effects of these two EOAIs are well known. In different doses/concentrations, they exhibit analgesic, neuroprotective, vasorelaxant, antiinflammatory, antiviral, antibacterial and antiparasitic effects. The acute toxicity of carvacrol and p-cymene in rats and the free-living nematode Caenorhabditis elegans was investigated. Furthermore, the impact of subacute administration of these two ter-penes on general health, CNS integration, i.e. motor coordination and balance of rats, as well as their effects on the movement of adult C. elegans, was also examined. The aim was to compare the effects and describe in more detail the selective toxicity of carvacrol and p-cymene. The calculated LD50 value of carvacrol was 790.15±1.15 mg/kg, while the LD50 value of p-cymene is above 3000 mg/kg. Tested doses of carvacrol and p-cymene administered for 28 days (50, 100, and 200 mg/kg) did not exert any effect on the CNS of rats or cause any clinical disorders. LC50 value of carvacrol for adult C. elegans was 184.13±1.51 micM and for p-cymene 1268±1.65 micM. In subacute testing, carvacrol showed negative effects on C. elegans reproduction, distance traveled, movement speed and rotational index at lower concentrations than p-cymene, indicating higher toxicity. On the other hand, although less toxic to C. elegans, p-cymene exhibited a specific effect on worm motility, with more rolling which should be further investigated, and can be a consequence of cuticle damage or loss of orientation.

The active constituents of essential plant oils (EOAIs), monoterpenoid carvacrol and monoterpene p-cymene, are widely distributed in many aromatic plants and their products. They differ in that carvacrol has a phenolic functional group. The numerous pharmacological effects of these two EOAIs are well known. In different doses/concentrations, they exhibit analgesic, neuroprotective, vasorelaxant, anti-inflammatory, antiviral, antibacterial and antiparasitic effects. The acute toxicity of carvacrol and p-cymene in rats and the free-living nematode Caenorhabditis elegans was investigated. Furthermore, the impact of subacute administration of these two terpenes on general health, CNS integration, i.e., motor coordination and balance of rats, as well as their effects on the movement of adult C. elegans, was also examined. The aim was to compare the effects and describe in more detail the selective toxicity of carvacrol and p-cymene. The calculated LD₅₀ value of carvacrol was 790.15 ± 1.15 mg/kg, while the LD₅₀ value of p-cymene is above 3000 mg/kg. Tested doses of carvacrol and p-cymene administered for 28 days (50, 100, and 200 mg/kg) did not exert any effect on the CNS of rats or cause any clinical disorders. LC₅₀ value of carvacrol for adult C. elegans was 184.13 ± 1.51 μM and for p-cymene 1268 ± 1.65 μM. In subacute testing, carvacrol showed negative effects on C. elegans reproduction, distance traveled, movement speed and rotational index at lower concentrations than p-cymene, indicating higher toxicity, which may be due to its phenolic structure. On the other hand, although less toxic to C. elegans, p-cymene exhibited a specific effect on worm motility, with more rolling which should be further investigated, and can be a consequence of cuticle damage or loss of orientation.

Isoxazoline insecticides, such as fluralaner, afoxolaner, and sarolaner, are widely used for pest control in agriculture and veterinary medicine due to their effectiveness against arthropods like fleas, ticks, and mites. These compounds work by targeting GABA receptors and chloride ion channels in insects and arachnids. Recent studies, however, suggest that isoxazolines may also have potential in combating nematodes, which pose significant challenges in agriculture and human health. Nematodes, including parasitic and free-living species, can cause severe damage to crops and livestock. Isoxazolines may exert antinematodal effects by interacting with GABA receptors in certain nematodes, disrupting their nervous systems and leading to paralysis or death, much like their effects on arthropods. Research indicates that some isoxazolines may inhibit nematode motility and viability, particularly in species like Heterodera (cyst nematodes) and Meloidogyne (root-knot nematodes). This potential is linked to the structural similarity of isoxazolines to other anthelmintic compounds targeting nematode neuromuscular junctions. Despite these promising results, challenges remain in applying isoxazolines as antinematodal agents. Nematodes show significant diversity in their physiological traits, which may affect the efficacy of isoxazolines. Additionally, resistance to chemical treatments is a concern, requiring further research into sustainable use. The potential for non-target effects and environmental toxicity also necessitates careful consideration, especially in ecosystems where non-arthropod invertebrates are essential. In this work we will represent some of our findings about antinematodal effects of afoxolaner. Based on our preliminary results, we can recognize the repurposing potential of certain isoxazoline derivatives. While isoxazolines show promise as antinematodal agents, more research is needed to optimize their use, minimize resistance, and understand their environmental impact. Continued development of isoxazoline-based treatments could offer a valuable tool in managing nematode-related damage.