Regenerative Potential of Granulocyte Colony-Stimulating Factor Immobilized by Using Electron-Beam Synthesis Nanotechnology in an Experimental Model of Ovarian Reserve Depletion.

The pharmacological activity of granulocyte CSF (G-CSF) immobilized using electron-beam synthesis nanotechnology (imG-CSF) was evaluated in an experimental model of ovarian reserve depletion. The effectiveness of the drug was compared with that of its unmodified form. Depletion of the ovarian follicular pool in female Sprague-Dawley rats was caused by a single intravenous injection of the antitumor drug etoposide in the maximum tolerated dose. The effectiveness of the studied drugs was assessed by serum concentration of anti-Mullerian hormone (AMH) measured by ELISA and by the number of primordial, two-layer, multilayer, and atretic follicles counted on serial sections of the ovaries (5-µm thick; through the entire organ) stained with hematoxylin and eosin. It was found that imG-CSF prevents depletion of the ovarian reserve in the model used, which was confirmed by high AMH concentration and higher numbers of primordial, two- and multilayer follicles in comparison with the corresponding parameters in the control (etoposide), and by a decrease in the severity of atretic processes. Unmodified form of the drug demonstrated lower efficiency.

Assessment of Ante- and Postnatal Development of the Offspring of Male Rats Crossed in Delayed Periods after Treatment with Methotrexate in Low Doses.

We studied ante- and postnatal development of the offspring of intact female rats crossed with males injected with low doses of methotrexate 3 and 6 months before mating. The time of crossing corresponded to the manifestation of the cytostatic effect on spermatogonial stem cells. The offspring of methotrexate-treated males was characterized by increased preimplantation losses and fetal growth restriction in the antenatal period and inhibition of physical development, delayed formation of sensory-motor reflexes, and impaired learning abilities in the postnatal period.

Experimental Evaluation of the Effectiveness of Isobornylphenols in the Model of Pathospermia and Their Effect on the Antioxidant-Prooxidant Balance of Male Germ Cells.

We studied the effect of antioxidants dibornol (2,6-diisobornyl-4-methylphenol) and its derivative (4-hydroxymethyl-2,6-diisobornylphenol), members of the alkylated phenols group, on the redox potential of male germ cells and their morphological and functional state in the rat model of pathospermia. Pharmacological effect was observed in animals treated with dibornol. The studied compounds led to the normalization of the antioxidant-prooxidant balance. However, the value of this indicator against the background of treatment with dibornol derivative attested to a shift in the redox balance of cells towards reduction reactions.

Delayed Consequences of the Toxic Effect of Paclitaxel on the Testes of Prepubertal Rats and Their Correction with p-Tyrosol.

Delayed gonadotoxic effects were revealed in outbred male sexually mature rats (SD) after exposure to paclitaxel in the prepubertal period, and the possibility of their correction with p-tyrosol was shown. It was found, that administration of paclitaxel does not inhibit the ability of animals to conceive, but impairs the reserve capacity of the testicular tissue. In intact female rats crossed with male rats receiving paclitaxel, increased post-implantation fetal death was observed. Combined administration of paclitaxel and p-tyrosol alleviated the delayed effects of the cytostatic treatment on the prepubertal testis.

Estimation of the Regenerative Potential of para-Tyrosol in the Model of Testicular Insufficiency Caused by Damage to Spermatogonial Stem Cells.

The regenerative properties of p-tyrosol were investigated in a model of testicular insufficiency caused by a toxic effect on spermatogonial stem cells (single administration of paclitaxel in the maximum tolerable dose). Against the background of p-tyrosol administration, we observed an increase in the number of normal spermatogonia and Sertoli cells, stimulation of spermatogenesis, and renewal of the spermatogenic tissue. The treatment with p-tyrosol also led to a decrease in DNA damage in cells of the testicular tissue. These changes were accompanied by a decrease in the level of free radicals, an increase in antioxidant protection, and normalization of the redox potential.

Experimental Evaluation of Long-Term Toxic Effects of Methotrexate on Male Reproductive System.

The morphological and functional state of the reproductive system was studied in male outbred rats (SD stock) and male F1(CBA×C57BL/6) mice after long-term (3 months) methotrexate administration. The drug was administered subcutaneously once a week for 4 weeks, the dose for male rats was 1 mg/kg, for male mice 2.2 mg/kg. It was found that male rats retained the ability to conceive, their reproductive potential was not limited by increased risk of embryo death. At the same time, signs of astheno- and pathospermia were revealed. The testicular tissue was characterized by reduced content of the sources of the proliferative pool of spermatogenesis. In mice treated with methotrexate, increased content of DNA breaks was detected in the testicular cells.

Evaluation of the Effect of p-Tyrosol on the Level of DNA Damage in the DNA Comet Assay In Vivo.

The effect of p-tyrosol on the spontaneous level of DNA damage in the cells of the bone marrow, liver, kidney, and rectum of mice (series I) and on the genotoxic effects of cytostatic drugs with different mechanisms of action in rat testicular cells (series II) was studied by DNA comet assay on C57BL/6 mice. p-Tyrosol was administered in a dose of 40 mg/kg once (series I) or for 5 days before and 5 days after cytostatic exposure (busulfan, paclitaxel, methotrexate; series II). It was found that p-tyrosol reduced spontaneous level of DNA damage in all studied organs. p-Tyrosol exhibited an antigenotoxic effect with respect to the DNA-damaging action of methotrexate and produced no genoprotective effect in case of busulfan and paclitaxel.

Effectiveness of Phenolic Antioxidants in Experimental Model of Benign Prostatic Hyperplasia.

Experimental model of sulpiride-provoked benign prostatic hyperplasia was employed to comparatively assess the effect of phenolic antioxidants (dihydroquercetin, p-thyrozol, dibornol, and prostagenin) on prostate morphology. All examined agents decreased the degree of hyperplasia in acinar epithelium; the greatest efficacy was demonstrated by prostagenin. Moreover, dihydroquercetin and p-thyrozol increased the cross-section area of acinar lumina and prostate volume, which is inadmissible in this pathology. These results suggest that the use of phenolic antioxidants in the therapy of benign prostatic hyperplasia should be strictly controlled.

Evaluation of Kagocel Genotoxicity.

Antiviral drug Kagocel in concentrations of 0.0008, 0.004, 0.02, 0.1, 0.5, and 2.5 mg/ml with or without metabolic activation does not induce gene mutations in S. typhimurium strains ТА98, ТА100, ТА1535, and ТА1537 and in a combination of E. coli strains pKM101 and uvrA. A single intragastric administration of Kagocel in a daily therapeutic dose and a 10-fold daily therapeutic dose to male mice or multiple administrations in daily therapeutic dose to male and female mice did not led to a significant increase in the percentage of chromosomal aberrations in the bone marrow cells. DNA comet assay revealed no significant increase in the incidence of DNA breaks in cells of mouse testes after single or multiple administration of Kagocel at daily therapeutic and 10-fold daily therapeutic doses. Our results indicate that Kagocel exhibits no genotoxic activity in the studied dose range.

Experimental Study of the Effectiveness of Phenolic Antioxidants in Male Infertility Caused by Pathospermia.

The effect of phenolic antioxidants (dihydroquercetin, p-tyrosol, dibornol) on the morphology, functions, and redox processes in the reproductive cells of male rats was studied on the model of experimental pathospermia. All antioxidants reduced the percentage of degenerative forms of spermatozoa. Dibornol was most effective. Dihydroquercetin and p-tyrosol did not increase the total number of spermatozoa and the percentage of their mobile forms. These indicators were improved only by dibornol. After administration of all test drugs, the antioxidant potential of spermatozoa increased and did not significantly differ from the baseline values.

Protective Effect of Polysaccharides from Tussilago farfara L. on Bone Marrow Cells and Small Intestinal Epithelium Under Conditions of Polychemotherapy Evaluated by DNA Comet Assay.

Using DNA comet assay we found that polysaccharides from Tussilago farfara L. reduced the intensity of polychemotherapy-induced apoptosis and DNA damage in bone marrow cells and small intestinal epithelium of C57Bl/6 mice, which attested to genoprotective properties of these polysaccharides.

Experimental Analysis of the Efficacy of Dihydroquercetin on the Model of Chronic Nonbacterial Inflammation of the Prostatic Gland.

We studied the efficiency of dihydroquercetin on the model of chronic nonbacterial inflammation of the prostatic gland in rats. It was found that administration of dihydroquercetin was followed by a significant decrease in the area of the connective tissue in the prostatic gland to initial levels, which attested to antifibrotic properties of this oxidant. Additionally, the substance prevented the development of atrophy of acinus epithelium. After administration of reference drug Prostamol Uno, only moderate antifibrotic effects were observed.

Experimental Assessment of the Effect of Kagocel on Reproductive Function in Pubertal Male Rats.

We studied possible toxic effects of antiviral drug Kagocel on reproductive function in pubertal male rats. The drug was administered in therapeutic and 10-fold higher doses throughout the spermatogenesis cycle (48 days). Kagocel did not reduce mating and fertilizing capacities, did not suppress spermatogenesis, and had no toxic effects on the offspring. The results characterize Kagocel as a drug with a broad reproductive safety profile and demonstrate that the age limits for using Kagocel in pediatric practice can be extended.

Effect of Granulocyte Colony-Stimulating Factor Immobilized by the Electron-Beam Synthesis Nanotechnology on Reparative Regeneration of Spermatogenous Tissue.

Effectiveness of the granulocyte colony-stimulating factor immobilized by using electronbeam synthesis nanotechnology was investigated on the model of experimental testicular failure caused by the toxic effect on stem spermatogonia. Administration of the drug to experimental paclitaxel-treated animals increased the number of sources of the proliferative pool of spermatogenesis and its productivity. The effectiveness of immobilized granulocyte colony-stimulating factor was based on its ability to stimulate reparative regeneration of the spermatogenic tissue, which manifested in a decrease in spermatogenic layer maturity and increase in the number of microenvironment cells. Effectiveness of the immobilized form of the drug was superior to that of non-immobilized form.

Plant Polysaccharides Attenuate Fluorouracil Toxicity for the Small Intestinal Epithelium.

Polysaccharides from Tussilago farfara L., Acorus calamus L., and Echinacea purpurea (L.) Moench attenuated the toxic effect of fl uorouracil on the small intestinal epithelium of mice with Lewis lung carcinoma. Addition of polysaccharides to chemotherapy protocols stimulated reparative regeneration processes in the small intestine damaged by the cytostatic treatment. No stimulating effects of the polysaccharides on tumor growth and metastasizing were revealed.

Quantitative Evaluation of Primordial Follicles in Rat Ovaries during the Early and Delayed Terms after Different Cytostatic Exposures.

Experiments on female Wistar rats showed that cytostatic agents (farmorubicin, platidiam, carboplatin, and etoposide) induce an initial significant decrease in the number of primordial follicles. Over the next 2-3 estrous cycles after administration of farmorubicin, platidiam, and carboplatin, this index practically did not differ from the control. The number of primordial follicles in the third and fourth estrous cycles after farmorubicin administration, as well as in the second and sixth estrous cycles after etoposide administration was much higher than the follicular reserve after cytostatic treatment (first estrous cycle). The ovarian reserve was exhausted in the delayed period after the start of the experiment. This dynamics of the pool of primordial follicles suggests that the ovary of rats in the postnatal period of life contains a limited number of female germline stem cells.

Dihydroquercetin effects on the morphology and antioxidant/prooxidant balance of the prostate in rats with sulpiride-induced benign hyperplasia.

A course of dihydroquercetin (antioxidant) injections to 5-month-old Wistar rats with sulpiride-induced benign prostatic hyperplasia led to reduction of proliferative activity in the glandular structures and to attenuation of the inflammatory reaction in the tissue. Prostatic antioxidant/prooxidant balance returned to normal after the treatment.

Pharmacological stimulation of the regenerative capacity of rat testes damaged by paclitaxel.

Productivity of spermatogenesis and the population of spermatogonial cells were substantially reduced in male Wistar rats 3 months after administration of cytostatic drug paclitaxel, damaging stem spermatogonia. However, signs of reparative regeneration appeared in the testicular tissue. In animals receiving paclitaxel in combination with granulocyte CSF, the same period of observation, the number of spermatogonia and the efficiency of spermatogenesis at the same terms of the study did not differ from those in intact animals. Intensive recovery processes started 1 month earlier than after administration of paclitaxel alone. These data attest to pronounced potentiating effect of granulocyte CSF on reparative regeneration of the testicular tissue.

Mechanisms of reparative regeneration of rat testis after injection of paclitaxel.

Population of spermatogonia was reduced in 2, 3, and 6 months after single intravenous injection of antitumor drug paclitaxel in maximum tolerated dose (MTD). The count of Sertoli cell increased in 3 months after the start of the experiment. The maturity of the seminiferous tubule epithelium was lower than in intact rats. Spermatogenesis productivity did not differ from that in intact animals 6 months after start of the experiment. These data indicate that regeneration of the spermatogenous tissue after paclitaxel treatment is realized via renewal of the spermatogenic epithelium, but considering the amount of spermatogonial cell population, the recovery rate would be low.

[An experimental study of the effectiveness of the drug afalaza in chronic aseptic prostatitis].

A pilot study evaluated the efficacy of the drug afalaza (mixture of affinity purified antibodies to PSA and endothelial NO-synthase) compared with the Serenoa repens extract in a model of chronic abacterial prostatitis in Wistar rats caused by suturing of prostate tissue by silk thread. Except for the animals of intact group, rats (n = 13 in each group) underwent intraperitoneal injection of distilled water (10 ml/kg), afalaza (at a doses of 5, 7.5 and 10 ml/kg) or an Serenoa repens extract (50 mg/kg) 1 month after surgery for 45 days. After infusion, the mass, volume, and prostate weighting factor were evaluated, and prostate tissue was examined histologically. 2.5 months after surgery, development of chronic abacterial prostatitis was observed in the control group. Compared with intact group, significant increase in weight, weighting factor, and volume of prostate were detected in control group. Against the background of administration of Serenoa repens extract and afalaza, these parameters were not significantly different from control values. The use of Serenoa repens extract prevented the development of atrophic processes and slowed the development of sclerotic processes. Administration of afalaza at all studied doses prevented the development of sclerotic changes, and a dose of 7.5 ml/kg prevented the development of atrophic processes with the effectiveness matching to Serenoa repens extract. Taking into account the high safety of afalaza, this drug is a promising treatment for chronic prostatitis.