Experimental Model of Cerebral Arterial Air Embolism in Conscious Rats.

In this work, an optimal air supply mode was selected to create a model of cerebral arterial air embolism (CAAE) on conscious male Sprague-Dawley rats (n=49). The efficacy of the selected model (administration of 100 µl/kg of air at a rate of 10 µl/min with an infusion pump) was determined by changes in serum biochemical parameters (cholesterol, alkaline phosphatase, inorganic phosphates, AST, and triglycerides), impaired motor functions in the Rotarod test, and visual assessment of the ischemic foci (staining of frontal sections with 1% triphenyltetrazolium chloride solution) at different terms after AAE. The model of AAE created by us confirmed impairment of coordination and motor function in conscious animals and reproduced the lethal consequences of this condition. The obtained results can serve as the basis for drug testing and the development of new approaches to the treatment of ischemic stroke.

Modeling of Cerebral Ischemic Stroke in Conscious Rats via Arterial Air Embolization.

The etiological factor of cerebral ischemia in the vast majority of cases is vascular embolism. In the present study we investigated embolism caused by atmospheric air bubbles injected into the internal carotid artery of conscious rats. Immediately after embolism modeling, behavioral abnormalities were observed in the animals, and after 24 h, foci of brain damage were detected. The death of animals was observed within 5 days after embolism. The proposed experimental model of cerebral ischemia in conscious rats is more relevant and better corresponds to real conditions than the model on narcotized animals and allows to perform physiological tests immediately after modeling.

Study of PT1 Peptide Analgesic and Anti-Inflammatory Activity on a Local Inflammation Model in Mice CD-1.

PT1 peptide isolated from the venom of spider Geolycosa sp. is a modulator of P2X3 receptors that play a role in the development of inflammation and the transmission of pain impulses. The anti-inflammatory and analgesic efficacy of the PT1 peptide was studied in a model of complete Freund's adjuvant-induced paw inflammation in CD-1 mice. The analgesic activity of PT1 peptide was maximum after intramuscular injection at a dose of 0.01 mg/kg, which surpassed the analgesic effect of diclofenac at a dose of 1 mg/kg. The anti-inflammatory activity was maximum after intramuscular injection at a dose of 0.0001 mg/kg; a decrease in paw thickness was observed as soon as 2 h after the administration of the PT1 peptide against the background of inflammation development. All tested doses of PT1 peptide showed high anti-inflammatory activity 4 and 24 h after administration. PT1 peptide at a dose of 0.01 mg/kg when injected intramuscularly simultaneously produced high anti-inflammatory and analgesic effects compared to other doses of the peptide. Increasing the dose of PT1 peptide led to a gradual decrease in its analgesic and anti-inflammatory activity; increasing the dose of intramuscular injection to 0.1 and 1 mg/kg is inappropriate.

Pathological Changes in the Gastrocnemius Muscle throughout Hind Limb Ischemia Modeling in Rats.

We present a two-stage model for the study of chronic hind limb ischemia in rats. In the area of ischemia, sclerotic changes with atrophic rhabdomyocytes and reduced vascularization were revealed. CD31 expression in the endothelium increased proportionally to the number of vessels in the ischemic zone, and at the same time, focal expression of ßIII-tubulin was detected in the newly formed nerve fibers. These histological features are equivalent to the development of peripheral arterial disease in humans, which allows using our model in the search for new therapeutic strategies.

The Study of TRPV1 Channels of the Central Nervous System and Their Effect on Anxiety in ICR Mice.

The TRPV1 channel is actively involved in various neuronal processes and is found in various structures of the nervous system, including peripheral and central neurons, sensory ganglia, spinal cord, and various parts of the brain. Due to its ability to respond to various stimuli, TRPV1 can have a significant impact on the body's responses to stress. Studies indicate the involvement of TRPV1 in the regulation of anxiety behavior. Suppression of TRPV1 activity leads to a decrease in the level of anxiety in animals, which indicates the importance of this channel in psychoemotional regulation. A promising compound for inhibiting this channel is the APHC3 peptide, which is a selective receptor antagonist. The results obtained this study show that this peptide has a pronounced anxiolytic effect, reducing the level of anxiety in the studied animals.

Effect of a helium and oxygen mixture on physiological parameters of rats with cerebral arterial air embolism.

Introduction: Cerebral arterial air embolism (CAE) is a serious and potentially dangerous condition that can interrupt the blood supply to the brain and cause stroke. One of the promising gas mixtures for emergency treatment of air embolism is an oxygen-helium mixture. Methods: We modeled CAE in awake rats by injecting air into the common carotid artery. Immediately after CAE, animals were either untreated or underwent hyperbaria, oxygen inhalation, heated air inhalation, or helium-oxygen mixture inhalation. Body temperature, locomotor activity, respiratory and cardiovascular parameters were monitored in the animals before CAE modeling, and 3 and 24 h after CAE modeling. Results: After 3 hours of CAE modeling in awake rats, depression of the nervous, cardiovascular and respiratory systems, as well as decreased body temperature were observed. 24 h after CAE modeling multifocal cerebral ischemia was observed. Normobaric helium-oxygen mixture inhalation, on par with hyperbaric treatment, restored body temperature, locomotor activity, respiratory volume, respiratory rate, and blood pressure 3 hours after CAE, and prevented the formation of ischemic brain damage lesions 24 h after CAE. Discussion: Thus, inhalation of a heated oxygen-helium gas mixture (O2 30% and He 70%) immediately after CAE improves the physiological condition of the animals and prevents the foci of ischemic brain damage formation.

Experimental Approaches to Scar Modeling through Mechanical Skin Damage and Chemical Burn in Sprague Dawley Rats.

Creating of a scar model in laboratory animals is the most acceptable option for the preclinical search of scar treatment. However, due to high skin regeneration rate in laboratory rodents, creating an optimal animal model of scar formation is a challenge. Here we describe five methods for modeling a scar tissue in rats that we have tested. These methods allowed achieving different histopathological features and different stages of skin scar formation.

Selective targeting of α7 nicotinic acetylcholine receptor by synthetic peptide mimicking loop I of human SLURP-1 provides efficient and prolonged therapy of epidermoid carcinoma in vivo.

α7-Type nicotinic acetylcholine receptor (α7-nAChR) promotes the growth and metastasis of solid tumors. Secreted Ly6/uPAR-Related Protein 1 (SLURP-1) is a specific negative modulator of α7-nAChR produced by epithelial cells. Here, we investigated mechanisms of antiproliferative activity of recombinant SLURP-1 in epidermoid carcinoma A431 cells and activity of SLURP-1 and synthetic 21 a.a. peptide mimicking its loop I (Oncotag) in a xenograft mice model of epidermoid carcinoma. SLURP-1 inhibited the mitogenic pathways and transcription factors in A431 cells, and its antiproliferative activity depended on α7-nAChR. Intravenous treatment of mice with SLURP-1 or Oncotag for 10 days suppressed the tumor growth and metastasis and induced sustained changes in gene and microRNA expression in the tumors. Both SLURP-1 and Oncotag demonstrated no acute toxicity. Surprisingly, Oncotag led to a longer suppression of pro-oncogenic signaling and downregulated expression of pro-oncogenic miR-221 and upregulated expression of KLF4 protein responsible for control of cell differentiation. Affinity purification revealed SLURP-1 interactions with both α7-nAChR and EGFR and selective Oncotag interaction with α7-nAChR. Thus, the selective inhibition of α7-nAChRs by drugs based on Oncotag may be a promising strategy for cancer therapy.

Modeling of Chronic Lung Inflammation in Rats by Repeated Intratracheal Administration of LPS.

A model of a chronic lung inflammation in SPF Sprague-Dawley rats was developed by repeated intratracheal administration of LPS in a dose of 0.4 mg/kg. On day 22 of the study, male rats treated with LPS have relative monocytopenia and reduced mean concentration of hemoglobin in the erythrocyte and the mean platelet volume in comparison with the control animals (saline). Intratracheal administration of LPS induced an inflammatory process in the lungs characterized by focal atelectasis, compensatory emphysematous expansion of subpleural pulmonary acini, focal mononuclear and neutrophilic perivascular and peribronchial infiltration, and minor focal mononuclear and neutrophilic infiltration of the alveolar walls. Against the background of LPS administration, germinal centers appeared in the lymphoid follicles of the white pulp of the spleen, and focal mononuclear infiltration of the tracheal mucosa and/or submucosa was observed in some animals.

Taxifolin Reduces Blood Pressure in Elderly Hypertensive Male Wistar Rats.

Male Wistar rats aged 10 months were assigned to groups according to the initial level of systolic BP: hypertensive (systolic BP >115 mm Hg) and normotensive (systolic BP <115 mm Hg). The animals were injected intraperitoneally with 100 µg/kg taxifolin daily for 7 days. Systolic BP and HR were measured on the next day after single taxifolin administration and on the next day after 7-day injection course. In the group of hypertensive animals, systolic BP markedly decreased on the next day after the first injection; this decrease became even more pronounced (to the level of normotensive animals) at the end of the taxifolin course. In the group of normotensive animals, systolic BP remained unchanged. Hence, we demonstrate the possibility of course administration of taxifolin for BP normalization in hypertensive patients.

Discerning Comparison of 1 and 0.5% Ethylene Glycol in Sprague-Dawley Rats with Modeled Urolithiasis.

A common method of modeling urolithiasis is the use of 1 and 0.75% ethylene glycol, or a combination of ethylene glycol with other lithogens, but too rapid progression of the disease and multiple organ toxicity have been reported. We developed a urolithiasis model in Sprague-Dawley rats, in which the animals received a relatively low concentration of ethylene glycol (0.5%), but for a long-term period (6 weeks) followed by animal observation during the 6-week recovery period. In urine samples, signs of the urolithiasis development were observed starting from the sixth week: the presence of ketones, decrease in diuresis and urine pH; in the blood, urea, protein, and hematocrit were elevated. However, no leukocytes were detected in the urine; in the blood, no shifts in differential leukocyte count and no elevation in ALT, creatinine, cholesterol, and triglycerides were observed, which indicates the absence of multiple organ failure while using 1% ethylene glycol. In addition, the animals receiving 0.5% ethylene glycol were followed up to 12 weeks in contrast to animals receiving 1% ethylene glycol (the experiment in this case was stopped during the third week for ethical reasons).

Anxiolytic Effect of Peptides from Sea Anemone Heteractis crispa, Modulators of TRPV1 and ASIC Channels.

A number of studies confirmed the involvement of transient receptor potential vanilloid (TRPV) and acid-sensing (ASIC) ion channels in the physiological processes associated with the development of anxiety disorders. This makes their ligands new potential anxiolytic agents. We examined the efficacy of two peptides from the sea anemone Heteractis crispa, Hcr 1b-2 and HCRG21, affecting ASIC1a and TRPV1 channels, respectively, in the open field and elevated plus maze tests. According to the obtained data, HCRG21 significantly decreases both the level of anxiety and stimulates the activity of animals at doses of 0.01-1 mg/kg, whereas Hcr 1b-2 has a weak anxiolytic effect only at a dose of 0.1 mg/kg. The pharmacodynamic study showed that the HCRG21 has an anxiolytic effect for 2 h, and its effectiveness is higher than that of the reference drug.

Comparison of Biochemical Parameters and Pathomorphological Changes in Rats Receiving Standard and High-Fat Diets during Modeling of Streptozotocin Diabetes.

The use of a high-fat diet, along with streptozotocin administration, can provide more profound insight into the mechanism of development of complications in diabetes, as well as their treatment. High-fat diet given over 3 weeks before intraperitoneal injection of streptozotocin in a dose of 40 mg/kg promoted the appearance of hyperglycemia in Wistar rats. The biochemical analysis of blood serum revealed increased levels of urea, triglycerides, cholesterol, AST, ALT, and concentration of inorganic phosphates and K+ ions in the high-fat diet group in comparison with the control. Both the biochemical analysis of the blood and histological analysis showed more pronounced abnormalities in rats receiving high-fat diet in comparison with animals receiving standard ration. These changes are the early markers for the development of nephropathy, impaired liver function, and microvascular disorders typical of patients with diabetes mellitus.

α-Conotoxin RgIA and oligoarginine R8 in the mice model alleviate long-term oxaliplatin induced neuropathy.

Оligoarginines were recently discovered (Lebedev et al., 2019 Nov) as a novel class of nicotinic acetylcholine receptors (nAChRs) inhibitors, octaoligoarginine R8 showing a relatively high affinity (44 nM) for the α9/α10 nAChR. Since the inhibition of α9/α10 nAChR by α-conotoxin RgIA and its analogs is a possible way to drugs against neuropathic pain, here in a mice model we compared R8 with α-conotoxin RgIA in the effects on the chemotherapy-induced peripheral neuropathy (CIPN), namely on the long-term oxaliplatin induced neuropathy. Tests of cold allodynia, hot plate, Von Frey and grip strength analysis revealed for R8 and α-conotoxin RgIA similar positive effects, expressed most prominently after two weeks of administration. Histological analysis of the dorsal root ganglia sections showed for R8 and RgIA a similar partial correction of changes in the nuclear morphology of neurons. Since α9/α10 nAChR might be not the only drug target for R8, we analyzed the R8 action on rat TRPV1 and TRPA1, well-known nociceptive receptors. Against rTRPV1 at 25 µM there was no inhibition, while for rTRPA1 IC50 was about 20 µM. Thus, involvement of rTRPA1 cannot be excluded, but in view of the R8 much higher affinity for α9/α10 nAChR the latter seems to be the main target and the easily synthesized R8 can be considered as a potential candidate for a drug design.

Effect of Standard and High-Fat Diets during Modeling of Streptozotocin-Induced Diabetes in Rats on the Development of Complications.

For evaluation of the effect of high-fat diet on the development of diabetic complications, the rats were maintained on standard or high-fat diet. In 3 weeks, diabetes mellitus was modeled by single intraperitoneal injection of streptozotocin. Changes in hematological parameters, physical and biochemical parameters of the urine, and in the development of thermal allodynia were different after 15-week standard and high-fat diets.

Peptide Blocker of Ion Channel TRPV1 Exhibits a Long Analgesic Effect in the Heat Stimulation Model.

The ion channel TRPV1, which is one of the most important integrators of pain and inflammatory stimuli, is considered a promising therapeutic target in the treatment of pain conditions. In this work, we performed a comparative study of the analgesic effect in the "hot plate" test of recombinant analogues of Kunitz-type peptides from the sea anemone Heteractis crispa venom: APHC1-modulator of TRPV1 and HCRG21-a full blocker of TRPV1. As a result of biological tests, it was shown that the full blocker HCRG21, despite the higher value of 50% effective concentration of TRPV1 inhibition, had an equal analgesic ability with the APHC1 upon intramuscular administration and retained it for 13 h of observation. The analgesic effect of APHC1 at a dose of 0.1 mg/kg when administered intramuscularly developed very quickly in 5 min but lasted 3 h. The differences in the pharmacodynamic profile of the peptides are in good agreement with different mechanisms of binding to TRPV1.

Mouse Model for Assessing the Subchronic Toxicity of Organophosphate Pesticides.

The development of antidotes to organophosphate poisons is an important aspect of modern pharmacology. Recombinant acetylcholinesterase and butyrylcholinesterase are effective DNA-encoded acceptors of organophosphate poisons and, in particular, pesticides. Here, we present the results of a study on the effectiveness of recombinant butyrylcholinesterase (BChE) in modeling organophosphate poisoning caused by oral administration of paraoxon at a dose of 2 mg / kg. The study showed a high activity of BChE as a protective agent for subchronic anticholinesterase poisoning in an in vivo model. The administration of BChE in a dose of 20 mg / kg allows one to avoid mortality, and also contributed to rapid recovery after model poisoning.

Application of Tetrameric Recombinant Human Butyrylcholinesterase as a Biopharmaceutical for Amelioration of Symptoms of Acute Organophosphate Poisoning.

We present a procedure for optimizing the expression of recombinant tetrameric butyrylcholinesterase that enables large-scale production with the yield >30 mg/liter (>90 mg/roller bottle). Intravenous injection of the preparation significantly increased survival and decreased the severity of symptoms of poisoning with paraoxon, an organophosphorus toxin.

A Study of the Protective Properties of an Antibody-Based Antidote Metabolizing Organophosphorus Pesticide Paraoxon.

A catalytic antibody A17 and its mutants highly efficiently interact with organophosphorus pesticide paraoxon. In this work, we studied the protective properties of antibody A17-K47 in paraoxon poisoning using a mouse model. The optimal paraoxon dose simulating the acute toxic effect of organophosphorus compounds was 550 µg/kg. The pharmacokinetic parameters of A17-K47 antibody were t1/2distr =7.2±1.4 min, t1/2el =330±20 min. The antibody did not cause toxic effects when administered at a ten-fold calculated therapeutic dose (610 mg/kg). The drug did not reduce mortality from acute paraoxon poisoning; however, the absence of drug toxicity opens up prospects for its use in symptomatic treatment of chronic paraoxon poisoning.

Chemical Polysialylation and In Vivo Tetramerization Improve Pharmacokinetic Characteristics of Recombinant Human Butyrylcholinesterase-Based Bioscavengers.

Organophosphate toxins (OPs) are the most toxic low-molecular compounds. The extremely potent toxicity of OPs is determined by their specificity toward the nerve system. Human butyrylcholinesterase (hBChE) is a natural bioscavenger against a broad spectrum of OPs, which makes it a promising candidate for the development of DNA-encoded bioscavengers. The high values of the protective index observed for recombinant hBChE (rhBChE) make it appropriate for therapy against OP poisoning, especially in the case of highly toxic warfare nerve agents. Nevertheless, large-scale application of biopharmaceuticals based on hBChE is restricted due to its high cost and extremely rapid elimination from the bloodstream. In the present study, we examine two approaches for long-acting rhBChE production: I) chemical polysialylation and II) in-vivo tetramerization. We demonstrate that both approaches significantly improve the pharmacokinetic characteristics of rhBChE (more than 5 and 10 times, respectively), which makes it possible to use rhBChE conjugated with polysialic acids (rhBChE-CAO) and tetrameric rhBChE (4rhBChE) in the treatment of OP poisonings.