Piracetam Pakistan

[Caleb Nelands]

Temporallobe epilepsy (TLE) is a chronic neurological disorder characterized by the frequent incidences of spontaneous recurrent seizures (SRSs) and 30% of individuals show pharmacoresistance with the typical available anti-seizure drugs (ASDs) (1). The prevalence of SRSs in TLE is high in children and elderly patients due to various precipitators (1). The most common precipitators of TLE are high-grade fever, stroke, hypoxia, metabolic acidosis/alkalosis, brain infections/tumors and injury which may lead to a series of events in the development of a normal brain into an epileptic one (2). The most observed anomalies in the autopsy of TLE brain are the sclerosis of hippocampal cornu ammonis and modification in the dentate gyrus region (3). The phenomenon of transforming a healthy brain into an epileptic one is termed as epileptogenesis and a chain of events over time such as hyper-excitability, neuronal network rewiring, oxidative stress, neurotransmitters imbalance provoke ictogenesis to SRSs in TLE individuals (4). Experimentally, the TLE model in rodents (mimics the TLE in humans) can be induced by chemoconvulsants such as kainic acid, pilocarpine and lithium-pilocarpine. Subsequent to chemoconvulsant administration, a rapid acute status epilepticus (SE) develops which, after the span of 4 to 6 weeks (epileptogenesis), transforms into full-blown TLE (2).

ASDs are mostly used for the management of epilepsy, but occasionally the single drug fails to sufficiently control the incidences of seizures and comorbid behavioral conditions such as anxiety, depression, agitation and cognitive impairments associated with TLE (5). Therefore, the reasonable use of drug combinations or polypharmacy has emerged with substantial ameliorative outcomes over the last decade in TLE patients and associated comorbid conditions (6, 7). A generally accepted idea is to combine two or more drugs (cocktail) with the dissimilar mechanism of actions for effective management of seizures and affiliated behavioral anomalies (6, 8). However, a cocktail regimen should be used with care keeping in mind the factors of drug additive toxicities and dose overload (7).

Moreover, usage of piracetam (nootropic) alone and in combination with old generation ASDs for the improvement of cognitive function in experimental animal models has been demonstrated although piracetam possesses minimal anticonvulsant potential (15). Piracetam also improves the function of neurotransmitters such as glutamate and acetylcholine which are related to memory function (16). A real-time microdialysis study has demonstrated the involvement of the cholinergic system in acute SE (17, 18). Furthermore, a scientific study justified the use of scopolamine in drug cocktails (muscarinic receptor antagonist) for the effective termination of acute SE and subsequent damage (8). However, the role of scopolamine is not clear in epileptogenesis and TLE.

The use of multi-targeted drugs in clinical studies and experimental models of seizure are widely debated. However, there is inadequate information available regarding the implication of polypharmacy pertaining to behavioral consequences and neurochemical dynamics in lithium-pilocarpine induced SE model. Keeping this aspect in view, we used lacosamide as a single drug and in combination with other drugs (lacosamide-pregabalin; lacosamide-piracetam; lacosamide-pregabalin-scopolamine) for the assessment of recurrence of seizures, behavioral changes and biochemical studies in cholinomimetic induced TLE model.

To see the impact of drug cocktails on the behavioral changes of animals during the time course of 6 weeks post-SE , various behavioral parameters such as anxiogenic, depression and cognitive disturbance were noted. For behavioral evaluation, all experimental animals were transported to a behavioral room at least 1 hour before the commencement of experiments and the environment was kept very calm (with balanced light, visuals and adequate room temperature).

In our experimental settings, we assessed the anxiety that the animals were exhibiting post-SE on day 10, 20, and 30 of the study and the protocol used was adopted by our reported method previously (27). A plus-shaped apparatus with a dimension of 50 10 cm for an open arm (L W) and 50 10 30 cm for a closed arm (L W H) raised 60 cm above the ground was used for this experiment in a soundproof room. The experimental rats were placed in the plus-maze facing towards the open arm in the central arena of 10 cm and 5 min was allowed for each rat. The whole experiment was video recorded with the help of C310 camera (Logitech, Inc. Switzerland) and videos were evaluated with ANY-MazeTM (version 6.11, Stoelting Co., USA) behavioral assessment software to find out the parameters for the instance the time spent and the number of entries in the open or enclosed mazes.

Day 11, 21, and 31 of the study was reserved for the evaluation of anxiety-like behavior in experimental rats by L/D box (28). The two-dimensional light and dark chambers of (40 20 20 cm) were interlinked with a small opening (10 10 cm) in the middle for free crossings. Each animal was positioned in the light chamber and allowed to move freely for 5 minutes between the chambers. Whole experiments were video recorded with the help of C310 camera (Logitech, Inc. Switzerland) and videos were evaluated with ANY-MazeTM (version 6.11, Stoelting Co., USA). Behavioral parameters ranging from the time spent in each chamber and the number of transitions between chambers were considered as standard criteria.

Intermittent OFT was performed on the animals from control (normal control and post-SE control) and drugs treated groups on day 12, 22 and 23 of the study by employing an open field arena of the dimension of 80 80 45 cm (L W H) to examine the level of anxiety in the rats on the basis of their exploratory behavior. In the ANY-Maze software settings, the 6400 cm2 area of the maze was divided into 64 small squares of equal dimension (10 10 cm) for the allocation of central and corner zones. The box was properly illuminated with 775 lux light intensity. Animals from each group were placed in the middle of the maze one by one and allowed to roam for 300 s based on their natural exploratory tendency. Between the sessions of the experiment, the maze was cleaned (debris) each time and further sprayed with 70% isopropyl alcohol to remove any residual smell of the previous session (27, 28). Selection criteria of the maze tests were (a) Time spent in the central and corner zone, (b) the number of entries in either zones and (c) distance traveled by each rat was video recorded.

From day 35 to 42, Morris water maze was performed on experimental rats for the evaluation of cognitive performance (spatial learning and memory deficit). Protocol for the water maze has been adopted and modified from our previous study and mainly consists of three phases i.e. training, acquisition and probe trials (27). A large circular water-filled tank (Diameter 150 Height 50 cm) was used and divided into four equal quadrants (NE, SE, SW, NW). A square-shaped platform 10 10 30 cm was placed at the middle of one of the four quadrants (SW). The temperature of the water was kept constant (25 2C) throughout the experiment in order to prevent distortion with the physical/swimming activity of the rats. For the facilitation of the animals to remember the coordinates of the tank, the proximal cues of different geometrical shapes and colors were mounted on the interior of the tank whereas distal cues were placed around the 4 poles of the tanks on wooden standees and were sufficiently visible to animals inside the tank.

(a) Training session: an adequate training is required for the effectual performance of the animals in the water maze and therefore in this context, beforehand all animals were put into a two-day training session. The platform was placed in the SW quadrant 2 cm above the water level. Each rat was placed in the tank facing towards the wall and was allowed 120 seconds to locate the platform with a minimum 4 trials/day. If the rat was not able to find the platform in 120 seconds, then the rat was guided towards the platform and allowed to stay there for 20 seconds for memorization of cues/coordinates.

(c) Probe day: with the removal of the platform, on day 42 of the experiment, each rat was engaged once from the north pole and allowed to swim for 120 s to judge its retrieval memory on the basis of its (1) time spent, (2) number of entries and (3) distance swum in the SW quadrant. Recorded videos were subsequently analyzed by behavioral tracking software.

An ELISA based kit method (8-isoprostane express; Cayman Chemicals, USA) was employed for the quantification of isoprostanes in the collected microdialysates. The kits were used in accordance to the companys guidelines. The limit of detection was approximately 10 pg/ml.

Combination of lithium-pilocarpine induced successful SE in 93/100 animals (limbic seizures of stage 4 of the Racine scale). Seven rats did not develop SE and remained in stage 2 or 3 of Racine scale, therefore excluded from the study. Furthermore, 18/93 rats expired within 24 hours of pilocarpine administration. The surviving 75 rats were distributed into 5 group as elaborated in the methods part. The control epileptic group remained aggressive/hyperactive and mortality persisted throughout the study (8/15 animals survived). Nevertheless, in drug-treated groups, a variable rate of mortality was observed over time; the highest in L/P/S group (4/15 expired).

Prevalence of any existing SRSs in all groups was observed from day 23 to day 29 of the study (8 hours daily) by visual and video recordings. In the span of 8 hours, only seizures of stage 3 and beyond (Racine scale) were counted as an index of seizure severity by the experimenter. As evident from Fig. 2, the least number of seizures per day (1.14 0.14 seizure/day) was observed in L/P treated group in comparison to the control (P < 0.001 versus post-SE control control). The majority of the animals in L/P group remained protected from progression of seizures beyond stage 3 of Racine scale (head nodding, tremors). Furthermore, L/Pi group (50/140 mg/kg) demonstrated a significant reduction in the occurrence of seizure (Stage 3 according to Racine scale, 1.85 0.3 seizures/day; P < 0.001) in comparison to lithium-pilocarpine induced SE group. However, in L group many animals exhibited stage 4 or stage 5 seizures (Racine scale; 2.14 0.26 seizures/day) and animals displayed typical generalized seizure patterns i.e. tonic-clonic or clonic seizures. In the L/P/S treated group, the observed number of seizures/days were 2.57 0.37 (P < 0.05) and animals were in agitation, circular movement, and tonic-clonic seizures (at the time of seizure-onset).

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