Assessment of the in luence of cilostazol on learning-memory and motor co-ordination by rodent models

Sadgunottama goud kamparaj1, Kudagi B. L.*2, Muthiah N. S.3, Pravin Kumar R.4 1Research scholar, Department of Pharmacology, Bharath Institute of Higher Education and Research (BIHER), Selaiyur, Chennai, Tamil Nadu, India 2Department of Pharmacology, Narayana Medical College, Nellore, Andhra Pradesh, India 3Department of Pharmacology, Sree Balaji Medical College, BIHER, Chennai, Tamil Nadu, India 4Department of Pharmacology, Government Medical College, Government Estate wallaja road, Chennai, Tamil Nadu, India


INTRODUCTION
According to the world health organisation, dementia is a set of symptoms that include deterioration of the performance of cognitive tasks, learning, and reproducibility beyond the predictable from typical ageing. This is frequently associated with the worsening of emotional control, inspiration, creativeness, social performance, wondering, struggle in walking. The primary form of dementia is Alzheimer's disease in 60 to 70% of patients where the progressive loss of memory and loss abilities on language can be appreciated (WHO, 2020). There are several forms of dementia which include fron-totemporal, vascular, stroke, Lewy body, syphilitic, senility, mixed dementias (Al, 2018). The global prevalence of dementia is nearly 50 million, with increasing 10 million every year and may reach 82 million in 2030 and 152 million 2050 (WHO, 2020). Every 3 seconds there is an occurrence of a new case, which may increase the economic burden up to 2 Trillion USD by 2050 for the medical needs of the patients (Rao et al., 2020). Dementia may be reversible and irreversible types according to their nature. Regain of memory can be observed in revisable dementia after the leading cause vanished. Irreversible dementia needs medical care where there is no treatment to cure, but galantamine, donepezil, rivastigmine (anticholinergic agents) and memantine (NMDA glutamate receptor blockers) are available (WHO, 2017;Shaji et al., 2018). Apart from pharmacological approach reminiscence therapy, cognitive stimulation therapy may also improve the quality of life (Shaji et al., 2018). The above facts may lead to a new search for an effective therapeutic agent. Dementia may be associated with a low physical performance which may include gait disorder, irregular automatic actions, seizures, metabolic disorders, skin diseases, visual abnormalities, anaemia (Draper and Withall, 2016). In elderly patients, there are precise shreds of evidence to establish co-occurrence of sluggish walking and diminished cognition performance (Wennberg et al., 2017). The gait of an individual can be performed by the neuromusculoskeletal system, which can be impaired in neurological diseases (e.g. Alzheimer's disease, Parkinson's disease) (Ferrucci et al., 2016). Activated cyclic adenosine monophosphate (cAMP) can produce anti-apoptosis activity, neuroprotective activity, motor improvement, and cognitive enhancement activity.
Phosphodiesterase (PDE) inhibitors raise intracellular cAMP levels and promote its mediated activities by inhibiting the breakdown of cAMP into its metabolites. PDE 3 inhibition by cilostazol may increase cAMP levels and approved as an antiplatelet agent (Chattipakorn et al., 2014). There is no adequate research on the cilostazol role in cognition and motor co-ordination. So the present study aimed to assess the in luence of cilostazol on learning-memory and motor co-ordination by rodent models.

Animals
150g -200g weighed Wister strain albino rats of both genders were used in the experiment. The rats were served with adlibitum, water, and standard pellet diet in the animal house. They were kept in the central animal house, which was established with the standard laboratory conditions (12/12 h dark-light cycle, 25 o c ± 1 o c temperature). All the experiments were conducted by following the guidelines of the Committee for Control and Supervision of Experiments on Animals (CPCSEA), a regulatory body on the animal experiments in India. Rejection criteria to exclude from the experiment consist of any variation in weight and signs of diseases. The institutional ethical committee (IAEC) approved to carrion the experiment.

Grouping of animals
The experiment was conducted 5 and 6 groups (n=6) for evaluation of learning & memory property, motor co-ordination, respectively, Rats were allocated randomly.

Training procedure
Rats of all the groups were subjected to conditioned avoidance response before the induction of Maximal Electric Shock (MES) seizures except group 1. Each rat was positioned individually in a pole climbing apparatus provided with a grid loor which was electri ied and conditioned with a buzzer sound. Rats were trained thrice in a day initially to avoid the electroshock (80 V, 5 pulses/sec) applied to the grid loor, intermittently preceded by the beep (conditioned stimulus). Rats that were learned to escape the shock by climbing the pole after training for 14 days were used in the study. Group 1, Group 3, Group 4, Group 5 rats were treated with normal saline, Cilostazol 50 mg/kg p.o, cilostazol 75 mg/kg p.o, cilostazol 100 mg/kg p.o respectively. Rats in all groups were induced seizures with an electroconvulsive meter except Group 1 (Shibnath et al., 2015).

Conditioned avoidance response: Performance after seizure induction
After ensuring recovery from seizures, each rat was positioned exclusively in the pole climbing apparatus for determining the memory retention effect. A ixed number (i.e. 10) of shocks was applied to the electric grid loor, and the number of shocks escaped by each animal of a group was noted and tabulated (Gupta, 2004;Vogel, 2008).

Evaluation of the effects of treatments on motor co-ordination
Group I -Normal saline

Procedure
Rats were trained 15 minutes per day on the Rotarod for one week (12 RPM). Rats were positioned on the rod, and the animals that remained for 5 minutes were selected for the study. Rats were accustomed to the experimental environment 30 min before the start of the experiment. The rats were evaluated for motor co-ordination at an interval of 30, 60 minutes after administration of the experimental interventions as mentioned in the grouping of animals. The rotating rod accelerated at a speed of 4-40 RPM for 5 min. If the animal fails more than once to remain on the rotating rod for 5 minutes, then the test was considered positive. The rod was cleaned with a cotton ball soaked in spirit between each procedure. The latency time of each rat fall off from the rod for the irst time was noted and tabulated (Gupta, 2004;Kulakarni, 2007;Abada et al., 2013).

Statistical analysis
The outcomes of the experiments were noted and presented as Mean ± SD. The outcomes were tested by ordinary two way ANOVA followed by Tukey's multiple comparisons test. A p < 0.05 was considered statically signi icant.

Effects of treatment on learning-memory
The Table 1 re lects that group 5 demonstrated a signi icant difference in NSA out of 10S compared to other groups (p<0.001). Group 4 demonstrated a signi icant difference over group 2 (p<0.05) but not over group 3 (p=0.834) in NSA out of 10S. Group 3 did not exhibit statistically signi icant variance over group 2 (p<0.26) in NSA out of 10S.

Effects of treatment on motor-coordination 30 min after the drug administration
The Table 2 re lects that the group 4 exhibited a signi icant variance in latency period of the irst fall of the rod over the group 1 (p<0.001) and 3 (p<0.01). Group 3 did not demonstrate a signi icant variance in the latency period of the irst fall of the rod over group 1 (p=0.552). Group 2 demonstrated a signi icant difference over group 6 (p<0.001) but not with group 5 (p=0.696). Group 6 demonstrated statistically signi icant variance over group 2 and 5 (p<0.001).

min after the drug administration
Table no 2 re lects that group 4 exhibited a significant variance in the latency period of the irst fall of the rod over group 1, 3 (p<0.001). Group 3 did not demonstrate signi icant variance over group 1 (p=0.981). Group 5 and 6 were exhibited a signi icant difference in relation to group 2 (p<0.001).

DISCUSSION
The present preclinical study was aimed to assess the in luence of cilostazol on learning-memory and motor co-ordination by cook's pole climbing technique (condition avoidance re lex), rotarod technique, respectively. The outcomes of the study revealed that cilostazol 100 mg/kg p.o demonstrated a greater signi icant memory enhancement activity in conditioned avoidance re lex (CAR) (cook's pole climbing) technique when compared to cilostazol 50 mg/kg p.o., cilostazol 75 mg/kg p.o., and MES-control. Cilostazol 50 mg/kg p.o did not produce a signi icant effect on memory enhancement activity in CAR when compared to MEScontrol. Still, Cilostazol 75 mg/kg p.o demonstrated a less signi icant memory enhancement activity in CAR. Cilostazol 20 mg/kg i.p displayed better motor co-ordination performance in both sessions (30 nim and 60 min after the drug administration) of rotarod test when related to cilostazol 10 mg/kg i.p and normal saline. Cilostazol 10 mg/kg i.p did not display a signi icant response in both sessions of the rotarod test. Diazepam 2 mg/kg i.p demonstrated signi icant muscle relaxant property in the rotarod test. In relation to diazepam, concomitant use of cilostazol 10 mg/kg i.p and diazepam demonstrated improved motor co-ordination performance in one session that was 60 min after the drug administration. Cilostazol 20 mg/kg i.p + diazepam 2 mg/kg i.p demonstrated a signi icant improvement  in motor coordination performance in both session of the study in relation to diazepam 2 mg/kg i.p and Cilostazol 10 mg/kg i.p + diazepam 2 mg/kg i.p. So this re lected the signi icant relaxant effect of diazepam. Concomitant use of cilostazol 20 mg/kg with diazepam 2 mg/kg was signi icantly improved the motor co-ordination performance as cilostazol 20 mg/kg itself improved motor co-ordination over normal saline in both sessions.
The improvement in the learning and memory of cilostazol may be due to increase in cyclic adenosine monophosphate (cAMP) levels which ultimately activates a series of the cascade by phosphorylated signalling pathways like CREB-BDNF-TrkB-PI3K/Akt (cAMP response elementbinding protein-Brain-derived neurotrophic factor -phosphoinositide 3 kinase -protein kinase B) (Jiang et al., 2017). These are responsible for neuron survival, maturation and neuronal transmission in the hippocampus was demonstrated in ischemia/reperfusion-induced cognitive de icits rats via PI3K-Akt1/caspase-3 pathways (Qi et al., 2016). The cAMP/PKA/CREB is responsible for Neuroprotection by inhibiting neuronal cell apoptosis and post-stroke axonal regeneration (Zheng et al., 2019;Gao et al., 2020). These signalling pathways also up-regulate Bcl-2 and (cyclooxygenase-2) COX-2 expressions leading to improvement in learning and memory (Watanabe et al., 2006). Insulin-like growth factor I (IGF-I) play a signi icant role in angiogenesis and neurogenesis in the hippocampus, which was elevated by cilostazol, can improve memory (Zhao et al., 2010). An increase in glucose uptake by cilostazol via vasodilatation may prevent amyloid β-protein-induced impairment of glucose transport in neurons of mild Alzheimer disease receiving acetylcholinesterase inhibitors (Lee et al., 2019). Rababa et al. study results revealed that cilostazol could normalise biomarkers levels like (superoxide dismutase (SOD) and glutathione peroxidase (GPx)) in hippocampus without improvement in the learning and memory functions due to inadequate memory impairment by streptozotocin administration (Rababa'h et al., 2019). In contrast, the present study supported by Kyoung JaKwon et al. study demonstrated improvement of spatial memory in hypoperfused diabetes mellitus-induced dementia or vascular dementia by activation of CREB phosphorylation and BDNF expression (Kwon et al., 2015). Improvement in motor function by cilostazol may result due to an increase in cAMP-induced monoaminergic facilitation (Hedya et al., 2018;Mitoma and Konishi, 1996). The present study was supported by Shuichi Yanai et al., where cilostazol was tested against normal ageing in mice. According to Yanai et al., cilostazol improved spatial memory in 23 months aged mice with no difference in memory of cilostazol-treated aged mice and untreated middle-aged mice (Yanai et al., 2018). A cohort study conducted by shu-yu Tai et al. also supported the present study and revelled that cilostazol may reduce dementia by in luencing cerebral circulation and Aβ metabolism (Tai et al., 2017). In luence of cilostazol on memory impairment and hyperactivity was demonstrated through oral administration in a well-established Ts65Dn mouse model of Down syndrome with several symptoms of patients of a down syndrome like cognitive impairment, an undersized span of attention, low muscle tone and supported the present study (Tsuji et al., 2020).

CONCLUSION
The present self-funded study concludes that cilostazol has improved the learning and memory performance after MES convulsions. It also improved motor co-ordination against diazepam induced sedative muscle relaxation.
Further extensive clinical research should be done to evaluate the safety and ef icacy of cilostazol use as a Neuroprotective agent.