The study investigated the impact of selective agonists and antagonists of adrenergic receptors at different concentrations on the behavior of the unicellular organism Paramecium caudatum. It was demonstrated that exposure to methoxamine at a concentration of 10-3 M increases the movement speed of Paramecium caudatum cells but reduces the number of motile cells by activating various intracellular processes. The presence of phenylephrine at a concentration of 10-3 M in the environment decreases motor activity and exhibits similar effects to adrenaline.

The introduction of terbutaline at a concentration of 10-3 M leads to a sharp and prolonged increase in cell movement speed and overall activity, while the addition of dobutamine at a concentration of 10-3 M reduces motor activity. The study also showed the effects of selective blockers prazosin and atenolol at a concentration of 10-5 M, which manifested in the complete correction of the effects induced by the corresponding selective agonists on all investigated parameters of motor activity.

As of today, amidst the tightening of bioethical control over preclinical studies, the use of various cell cultures for screening pharmacological compounds has become more popular in the scientific community compared to research involving higher vertebrates and endeavors focused on predicting effects through computer modeling. However, pharmacological studies on cell cultures, particularly on unicellular organisms, are characterized by the diversity of employed methods and are complex in the interpretation of obtained results, leading to significant debates and discussions within this field [1]. 

Previously, in pioneering studies aimed at developing new methods for utilizing Paramecium caudatum as a model organism for primary screening of pharmacological compounds, we demonstrated and described a method that allowed the registration of changes in swimming behavior in response to the administered drug. This method is based on the variability of the motor activity of eukaryotic cells of Paramecium caudatum [2]. This method further confirmed its effectiveness in subsequent studies involving agonists and blockers of GABA-A receptors [3].

In our recent study, we decided to present the results obtained after optimizing our method, enabling the parallel comparison of investigated drugs, specifically selective agonists of adrenergic receptors and their blockers widely used in medical cardiological practice. This significantly accelerated data processing time and improved the quality of the results obtained, as simultaneous registration of movements placed all experimental groups in equal conditions.

Study design was randomized controlled trial and study duration was July 2018 to June 2020 at Neonatal Ward, Dhaka Shishu Hospital among all preterm babies admitted in neonatal ward of Dhaka Shishu Hospital during the study period. Data were collected using a structured questionnaire (research instrument) containing all the variables of interest. A standard questionnaire including cardinal points of the history, examination findings and pre & post medication investigation results prepared by the investigator was used to collect data. Sample was collected by simple random sampling and randomization was done by lottery method. At first 8 sealed envelopes were kept in a small box at a time, 4 containing name of study group in a paper inside and 4 of control group. One envelope was randomly opened during enrolment and the case was assigned to that group. This process was repeated until expected number of samples were enrolled. After admission of preterm neonates in SCABU and NICU were assessed by modified new ballard scoring and history of last menstrual period (LMP) for gestational age. Preterm neonates who were between 32-34 weeks and also those neonates whose 1st ROP screening showed peripheral avascular zone were selected. The parents or guardians of the neonates who met the inclusion criteria were informed about retinopathy of prematurity. They were also explained about the purpose, procedure, importance and benefit of the study. Informed written consent was secured from them and then enrollment was done. After enrollment, baseline assessment was done and subjects were randomized by block randomization on a 1:1 basis to either the active treatment or control arm. In vitamin A supplementation group, preterm infants were given the daily dose of 5000 IU/Kg/day vitamin A drop perorally as soon as enteral feeding was started and continued from starting point to 4 weeks. Vitamin A was given by insulin syringe as Cap. Ratinol Forte, marketed by Drug International, Bangladesh. DAR (Drug administration Registration) no (210-47-078), Mfg. Lic. (Manufacturing license) no (127 & 389) and expired date of drug ensured before giving drug. Each capsule of Ratinol Forte contains 50,000 IU retinyl palmitate. At first, squeezed vitamin A capsule (50000 IU) and made sure the entire content fell into the insulin syringe of 100 unit and upto 20 unit of insulin syringe filled with vitamin A. So each unit of insulin syringe contained 2500 IU vitamin A. Multivitamin drop was started from 2 weeks of age in both groups. In case of vitamin A group multivitamin drop was given as drop V-plex perorally along with vitamin A capsule and dose of vitamin A adjusted with multivitamin drop. Each ml of V-plex drop contain 5000 IU vitamin A along with other vitamins. Dose of vitamin A also adjusted with multivitamin drop when vitamin A group had to be given multivitamin drop. Control group had been given standard treatment and multivitamin drop from 2 weeks of age. Daily follow up regarding clinical course, oxygen supplementation along with other complication like respiratory distress syndrome, asphyxia, apnea, hyper bilirubinemia and septicemia were recorded in the questionnaire. On every Thursday, ROP screening was done in our NICU ward. During discharge I counselled both groups for ROP screening and contacted with them over phone. I ensured their 1st ROP screening which was done at 4 weeks of age. First ROP screening was done at 4 weeks of age. It was performed by Associate Professor Dr. Shamima Jahan and researcher assisted her. Pupils were dilated with 0.5% tropicamide eye drops and was instilled twice 1 to 5 minutes apart. The examination was performed 20 to 25 minutes later using a binocular indirect ophthalmoscope and +20D lens. Follow up schedule was planned according to location and severity of ROP. One week follow up: zone I stage 1 or 2 ROP and two weeks follow up: zone II stage 1 ROP. Lowest the zone and highest the stage of ROP considered as highest risk for severe ROP. Patient who had ROP, classified into stage (1- 5), zone (I-III) and presence or absence of plus disease. Findings were analyzed between vitamin A and control group. Preterm neonates whose ROP screening showed zone I any stage with plus disease or zone I stage 3 without plus disease or zone II stage 2 or 3 with plus disease assigned for laser therapy or intravitreal anti VEGF therapy (Bevacizumab) or combination of both. Regression of ROP in any stage and any zone were noted and follow up of every 1-2 weeks was carried out up to 45 weeks of post menstrual age.

Prior to working with selective adrenergic receptor agonists, it was observed that the introduction of adrenaline at concentrations of 10-3 M into the medium with Paramecium caudatum cells reduces the motor activity of Paramecium caudatum (Fig. 1a, b).

Figure 1: Changes in the speed of movement and activity of Paramecium caudatum in response to the administration of adrenaline and methoxamine. a – Changes in the average speed of cell movement under the influence of adrenaline at concentrations of 10-3, 10-5, 10-7 M during a 25-minute registration period. The arrow indicates the moment of adding the drugs to the well (fifth minute of registration). Cell concentration - 600±50 cells/mL. * - significant difference from the speed of cells in the intact group, p <0>

The concept of motor activity encompasses three parameters: swimming speed, the number of actively moving cells, and the smoothness of Paramecium trajectories. Thus, the presence of adrenaline at a concentration of 10-3 M in the medium reduces the swimming speed of Paramecium caudatum by 0.7 mm/s, while lower concentrations of adrenaline did not lead to changes in this parameter (Figure. 1a). The alteration in swimming speed may be associated with a disruption in the sequence and rhythmicity of cilia beating in Paramecium. [5].  In response to the activation of adrenergic receptors by administered adrenaline, there is a manifestation of intermittent jerky movements in different directions. It is also noteworthy that there is a 50

The use of the primary screening method of pharmacological agents on the cellular model of Paramecium caudatum has demonstrated its effectiveness in determining the most active concentration of adrenoreceptor agonists. Analysis of changes in cell motility revealed a divergent effect upon activation of β-1 and β-2 adrenoreceptors in relation to the two investigated parameters. Activation of α-adrenoreceptors leads to a reduction in the number of mobile cells, corresponding to the cellular response to the presentation of adrenaline. Additionally, the study demonstrated that the introduction of selective blockers, prazosin and atenolol, completely blocks the action of selective adrenoreceptor agonists. This suggests the targeted action of these drugs on the investigated adrenoreceptors.

Gruzdev G. А “conducting an experiment, obtaining and processing data, writing a draft of the article”; Karpukhina O. V. “supervision and explanation of results”; Inozemtsev A. N. “writing – review and editing”; Povarnina P. Yu “project administration”.

All applicable international, national, and/or institutional principles for the care and use of animals were followed. This article does not contain any studies with human participants as subjects.

The research was conducted as part of the scientific project under the state assignment of Lomonosov Moscow State University (Theme №. 121032500080-8), with additional support from the Interdisciplinary Scientific and Educational School of Moscow University, "Brain, Cognitive Systems, Artificial Intelligence".

The authors declare no explicit or potential conflicts of interest associated with the.