Background: The lithium-pilocarpine (Li-Pc) model of status epilepticus (SE) is the most convenient and is frequently used for pathophysiological and management strategies in search of new, safe, and effective therapeutic agents including natural remedies for SE. Oral administration of cinnamon (CIN), Nigella sativa (NS), and curcumin (CUR) can reduce neuroinflammation which is a common feature of neurodegenerative disorders. Although many studies on CUR and NS effects on SE have been undertaken, to our best knowledge no study has been conducted on studying the effect of CIN on SE.

Purpose: The present study explores the neuroprotective effects of natural food products CUR, NS, and CIN on Li-Pc-induced SE in rats. This study may help to understand the neurotherapeutic effects of these natural food products on cognitive dysfunctions and brain oxidative stress which have a correlative perspective with SE.

Methods: SE was induced in experimental rats using the lithium–pilocarpine model. Besides control groups, the animals were also grouped as treatment groups which received CIN, NS, and CUR pre-treatment before induction of SE. Besides the severity of the seizures, and cognitive dysfunctions, the oxidative stress parameters were also estimated in the fore-brain tissue of all groups of animals.

Results: Treatment with CIN, NS, and CUR significantly lessened the frequency and severity of epileptic seizures in a dose-dependent manner. The cognitive dysfunctions and the oxidative stress indices (enzymatic and non-enzymatic) were ameliorated significantly and dose-dependently by these natural food products pre-treatments in the order NS<CIN<CUR in all parameters.

Conclusion: Possible therapeutic applications of CIN, NS, and CUR as antiepileptics and as antioxidants for the treatment of SE in the order NS<CIN<CUR, have great potential and warrant further studies. Most importantly, involving such natural food products for neurotherapeutic applications bears great significance from a healthcare point of view for neurodegenerative disorders.

• Natural food products
• Status epilepticus
• Neuroprotection
• Neurotherapeutics
• Curcumin
• Nigella sativa
• Cinnamon

Ahmad M, Alharbi H, Alshehry AS, Alsadoun A (2024) Comparative Evaluation of the Ameliorating Effects of Some Natural Products as Neu- rotherapeutic Interventions for a Neurodegenerative Disorder in Status Epilepticus Model. Ann Neurodegener Dis 8(1): 1035.

The animal models serve a great purpose because we do not have to expose human subjects to health risks for observing the course of a disease and to study the effects of interventions by natural or synthetic compounds through reliable, ethical, responsible, economical, and safe preclinical assays in early stages of screening processes in search of newer natural food products as neurotherapeutic compounds for treating various diseases of the nervous system.

Status epilepticus (SE) is a neurodegenerative condition causing neuronal injuries in the brain [1], due to neurochemical imbalances in affected brain regions [2]. Ample evidences exist for the association of such chemical imbalances with excessive generation of free radicals suggesting vulnerability of the brain tissue to the oxidative stress [3,4]. Lithium (Li) Pilocarpine (PC) induced SE in rodent models has provided information regarding oxidative stress-related epileptic activity [5-7]. Furthermore, oxidative stress has also been related to cognitive impairment [7-9]. SE affects brain cell survival and function by targeting multiple adversities in the neuronal microenvironment such as inflammation, oxidative stress, mitochondrial alterations, calcium excitotoxicity, and bioenergetic challenges. The Li–Pc model of SE reproduces most clinical, temporal, and neuropathological features of SE [2,10]. Although the anticonvulsant effect of several agents having antioxidant properties has been demonstrated in various studies [2,11,12], the unsatisfactory outcomes of available SE pharmacotherapy necessitate the search for alternative natural resources that can target the various underlying mechanisms of SE pathology and reduce disease occurrence and/or progression. Among the natural resources, many studies on Nigella sativa (NS) and curcumin (CUR) effects on SE have been undertaken [7,13- 15], but no study as such has been conducted on studying the effect of cinnamon (CIN) on SE for having antiepileptic potential in experimentally induced SE.

Many recent studies have reported protective effects of Cur against oxidative damage and antioxidant and anticonvulsant properties exerting powerful oxygen free radical scavenging effects, anti-inflammatory, anticarcinogenic, antitumoral, antiviral, antifungal, antiparasitic, antimutagen, anti-infectious and antihepatotoxic compound [5,6,8,9,16-21].

Recent studies have shown significant potential of pharmacological, prophylactic, or therapeutic use of Nigella sativa (habba sauda) seed extract (NS) in many beneficial activities in the body including neuroprotection from neurodegenerative diseases and antioxidant properties [22-30]. A literature review has also shown that NS and its components can be considered as promising agents in the treatment of nervous system disorders [14].

Although CIN is widely used as a spice and as a traditional medicine for blood sugar control [31], anti-oxidant [32], anti- inflammatory [33], antimicrobial activities [34], an inhibitory effect on Tau aggregation related to AD [35], anti-cancer [36,37], cardiovascular disease [38], wound healing [39], inflammatory syndromes, cholesterol levels, immunomodulatory diseases [40,41], neuroprotective/neurodegenerative purposes [35,42,43], to the authors best knowledge, no studies have been undertaken to see effects of CIN on epilepsy or SE as anticonvulsant.

In the light of the above, the present study explores the neuroprotective effects of natural food products CUR, NS and CIN on Li-Pc induced SE in rats. This study may encourage to understand cognitive dysfunctions and brain oxidative stress which have a correlative perspective with SE. Furthermore, such research works can help in to understand indirectly the importance of screening natural food products that are beneficial as neurotherapeutic agents in ameliorating the convulsions, brain oxidative stress and cognitive dysfunctions in SE.

Experimental Animals

Adult male Sprague Dawley rats (weighing 200-250g, 2 months old) were used in the present study. The animals were taken care of under controlled conditions at 22 ± 1°C, humidity at 50-60% with 12 hours light-dark diurnal cycle with free access to food and water except during experimental handlings. All animal handling procedures and study protocols were followed as according to the approved directions from the Research and Ethics Committee of King Saud University, Riyadh, Saudi Arabia for executing the experiments.

Induction of Status Epilepticus (SE) and Treatment Groups

Animals were randomly assigned into twelve groups. Groups 1, 2, 3, 4, and 5 served as controls receiving saline, Li (3mEq/ml/ kg, i.p.), Pc (20mg/ml/kg, s.c.), 50 % dimethyl sulfoxide (DMSO) and 0.5% methylcellulose (MC) respectively. SE was induced in groups 6-12 by administering an aqueous (saline) solution of Li (BDH Laboratory Supplies, Poole, England in a dose as in control), followed by (20 h later) Pc (Sigma Chemical Co, St. Louis, MO, USA, in the dose as used for control). Group 6 served as the experimental control of the SE group and groups 7-12 served as the CUR (groups 7 and 8), NS (groups 9 and 10), and CIN (groups 11 and 12) test groups. CUR (Sigma, USA), was dissolved in 50% DMSO, and was administered at doses of 50 and 100 mg/kg body weight/ml orally; powdered seeds of NS (100 g), were purchased locally and were extracted in a Soxhlet extractor with ethanol (70%). The extract obtained was concentrated under reduced pressure and kept at-20 oC until being used. The NS extract was dissolved in saline and used at doses of 100 and 200 mg/kg body weight /ml orally; whereas CIN (purchased locally) was dissolved in 0.5% MC, and was administered at doses of 25 and 50 mg/kg body weight/ml orally, for seven days before the administration of Li and Pc for the induction of SE as described above. After the Pc injections, the animals (n=20 per group) were observed for a sequence of convulsive behavioral alterations, including peripheral cholinergic signs (PCS), stereotyped movements (STM), clonic movements of forelimbs, head bobbing, tremors and seizures, which developed progressively within 1-2 h into SE [44]. All seizure activities were presented as latencies to develop seizure and SE. Mortality (if any) within 24 h, was also recorded.

Cognitive Performance in Morris Water Maze

After induction of SE, the animals (n=10 to 12 per group) were subjected to cognitive behavioral studies over a period of 6 days according to the method of the Morris water-maze test [45], which has been extensively used to assess cognitive functions in a variety of epilepsy models [46,47], for visual-spatial memory performance. Animals were allowed to acclimatize to the testing room for 2 h before testing all tests were performed between 10:00 and 15:00 h of the lighted phase. The details of test are described elsewhere [2,7]. The testing procedure used during the four days of locating the hidden platform provides a measure of hippocampal-dependent spatial reference memory [48], whereas the probe trials of the water maze test (measured on the 5th day of the test for 120 s in which the platform was removed from the pool) measures the strength of spatial learning or memory recall, the closest parallel to episodic memory in humans [49].

Biochemical Studies in the Fore-Brain Tissue

Based on our pilot studies and literature survey [50-52], biochemical studies were undertaken 1 h after Li-Pc treatment.

Immediately after killing the animals (n=8 from each group) by decapitation, brains were dissected on ice. The fore-brain (which includes the hippocampus and striatum in the cerebral areas) was removed and frozen in liquid nitrogen and stored at -70°C for determination of some nonenzymatic and enzymatic oxidative stress indices.

Determination of Nonenzymatic OS Indices

Lipid Peroxides: Lipid peroxides (LP) were determined spectrophotometrically as thiobarbituric acid-reactive substances (TBARS) according to the method of Ohkawa et al. [53]. Tissue lipid peroxide levels were quantified using an extinction coefficient of 1.56×105m-1 cm-1 and expressed as nanomoles of TBARS formed per g tissue weight. The results are expressed as nmol/g wet weight.

Glutathione: Reduced glutathione (GSH) level was measured enzymatically in the brain tissues by a slightly modified method of Mangino et al. [54]. The slope of the change in absorbance was used to quantify total GSH by comparing the slope of the samples with a standard curve prepared with pure glutathione (Sigma). The specific activity is expressed into umol/g tissue weight.

Determination of Enzymatic OS Indices

Glutathione-S-Transferase: Glutathione S-transferase (GST) was estimated by the method of Habig et al. [55], by using 1-chloro-2,4-dinitrochlorobenzene (CDNB) as substrate at 340 nm. The GST activity is expressed as U/g tissue weight.

Catalase: Catalase (CAT) activity was measured by the method of Aebi [56], by tracking the decomposition of hydrogen peroxide by measuring the decrease in extinction of H2O2 at 240 nm. The activity of CAT is expressed as the rate constant of first- order reaction K per gram tissue weight.

Superoxide Dismutase: Superoxide dismutase (SOD) activity was estimated by the method of Misra and Fridovich [57]. Activity is expressed as the amount of enzyme that inhibits the oxidation of epinephrine by 50% which is equal to U per gram tissue weight.

Statistical Analysis

The data were analyzed by Bartlett’s test for equal variance and by Gaussian-shaped distribution for normality using the Kolmogorov-Smirnov goodness-of-fit test. As the data passed the normality test (????>0.10), group means were compared with one-way ANOVA with post hoc testing using Tukey-Kramer Multiple Comparisons Test or Student-Newman-Keuls Multiple Comparisons Tests. Differences in seizure and SE incidences and mortality were tested by Student-Newman-Keuls Multiple Comparisons Tests. All results were expressed as means ± SEM and the significance were defined as ????<0.05 for all tests.

Behavioral features of Li-Pc induced SE

After injection of Pc, all animals started developing a gradual and significant change in behavior within 5 min. Changes in behavioral features included PCS (miosis, piloerection, diarrhea, mild tremors, scratching, and salivation) and STM (sniffing, paw licking, and rearing). Seizures in 100% of animals (mean latency to develop seizures was 9.62 ± 1.2 min) were developed following the behavioral changes [Table 1]. The convulsions in the animals consisted of head bobbing with intermittent clonus of forelimbs and hind limbs, hyperextension of tails, loss of posture, falling back, and myoclonic jerks building up to SE in 100% of tested animals. The mean latency to onset of SE was 23.86 ± 1.54 min [Table 1], and on average, the SE lasted for more than one hour. Mortality of 10% was observed within 24 h following Pc injections [Table 1].

Effect of CUR, NS, and CIN pretreatment on Li-Pc induced SE

Pretreatment of animals with CUR, NS, and CIN increased the latencies to seizure and SE and decreased the percentages of seizures and SE significantly in a dose-dependent manner [Table 1]. Furthermore, it also reduced the intensity and frequency of seizure, PCS, and STM episodes [not shown in Table 1]. The three natural food compounds were effective in the order NS<CIN<CUR. The higher dose (100mg/kg) of CUR completely abolished SE [Table 1]. No mortality was observed in the rats pretreated with all three natural food products, as compared to 10% mortality in the Li-Pc treated (SE) group [Table 1]. The control groups that received saline, Li, DMSO, and MC alone did not show any signs of seizure or SE.

Morris Water-Maze Test

Animals treated with Li-Pc only (SE group) exhibited longer escape latencies to reach the platform as compared to the control group [????<0.01; Figure 1]. However, all groups developed a gradual improvement in such performance over the 4 days of testing (training) period. The number of successful animals to reach the platform was significantly higher in the CUR, NS, and CIN pretreated groups as compared to Li-Pc (SE) group on all the four testing days and the effectiveness was in the order NS<CIN<CUR [???? < 0.001; Figure 1]. The probe trial studies showed that CUR, NS, and CIN pretreated animals spent more time in the target (platform) quadrant as compared to the Li-Pc only (SE) group and the effectiveness was in the order NS<CIN<CUR [p<0.001; Figure 2].

Biochemical Studies

Nonenzymatic OS Indices in the Fore-Brain

TBARS: The lipid peroxidation level (TBARS) in the forebrain was markedly (????<0.001) increased after 1h of Li-PC (SE) treatment as compared to the control group [Figure 3A]. Pretreatment with natural compounds significantly (????<0.001) and dose-dependently attenuated Li-Pc induced an increase in TBARS in the order NS<CIN<CUR [Figure 3a], as compared to Li- Pc (SE) group.

GSH: A highly significant (????<0.001) depletion of GSH was observed in the fore-brain tissue of the Li-Pc (SE) group [Figure 3B]. Pretreatment with natural compounds significantly and dose--dependently attenuated this depletion of GSH in the order NS<CIN<CUR [Figure 3B], as compared to the Li-Pc (SE) group.

Enzymatic OS Indices in the Fore-Brain

GST: A highly significant (????<0.001) depletion of GST was observed in the Li-Pc (SE) group [Figure 4A]. Pretreatment with the natural products significantly and dose-dependently attenuated this depletion of GST in the order NS<CIN<CUR [Figure 4A], as compared to the Li-Pc (SE) group.

CAT: The CAT level in the fore-brain was markedly (????<0.001) increased after 1h of Li-PC (SE) treatment as compared to the control group [Figure 4B]. Pretreatment with natural compounds significantly (????<0.001) and dose-dependently attenuated Li-Pc induced an increase in CAT in the order NS.

SOD: The SOD level was significantly (????<0.001) decrea after 1 h of Li-PC (SE) treatment as compared to the control group [Figure 4C]. Pretreatment with natural food compounds significantly (????<0.001) and dose-dependently attenuat induced a decrease in SOD in the order NS.

The present experimental findings demonstrate that Pc administration in the pretreated rats with Li causes cholinergic symptoms followed by seizures. Within 20 to 30 minutes after Pc administration, SE developed in the rats exhibiting continuous head bobbing, intermittent forelimb and hind limb clonus, hyperextension of the tail and hind limb along with loss of posture resulting in falls (fall back from the rearing postures).

Impaired visual-spatial memory and cognitive deficit were observed in the SE-induced animals when tested in the Morris water-maze test [45]. SE-induced rats took a longer time to reach the escape platform or completely failed to reach the platform and spent less time in the target quadrant as reported earlier [2,58]. The specific cause of cognitive dysfunction following SE is far from clear. However, reports suggest that neurochemical imbalance and alterations in neuronal structure in the forebrain region following SE are possibly responsible for neurobehavioral changes [2,47,51,59,60]. The biochemical results in the present study further indicate a significant disruption in the levels of oxidative indices (enzymatic as well as non-enzymatic) in the forebrain of the rats treated with the Li-Pc suggesting for a preliminary and significant level of oxidative stress. It has been reported that over activation of excitatory amino acid receptors is an important pathogenic factor that has been implicated in the mechanisms of excitotoxicity-induced neurodegeneration leading to seizures and increased oxidative stress [61], and furthermore, neuronal loss and mossy fiber sprouting in the forebrain following SE has been attributed to the excessive production of reactive oxygen species [2,62].

Use of antioxidants could be a potential approach to arresting the seizure genesis caused by excitotoxic agents [63]. Potent antioxidant activities have been reported for CUR [5,6,8,9,21], NS [29,30], as well as CIN [32]. Although CUR and NS have been reported for having a neuroprotective role in the SE-induced models, to the author’s best knowledge, no precedent has been reported for CIN. Only during the past decade, have scientists devoted to exploring the neuroprotective/neurodegenerative aspects of cinnamon, and it was shown that multidisciplinary mechanisms are involved in this regard [35,42,43]. In the SE groups of the present study, a significant disruption in levels of oxidative stress indices in the forebrain was observed. Pretreatment with CUR, NS, and CIN significantly and dose- dependently attenuated Li-Pc-induced OS-related indices (enzymatic as well as non-enzymatic) in the forebrain region as compared to the SE group. The food products tested herein were effective in the order NS < CIN < CUR. Earlier studies have also reported that Li-Pc disrupts the OS-related indices in brain regions [2,3,64]. Thus, it is likely that similar pathomechanism might be contributing at least in part to the pathophysiology of the seizure activity herein.

The present study suggests at a preliminary level that the natural food products CUR, NS, and CIN have promising anticonvulsant and antioxidant activity against SE in rats in the order NS<CIN<CUR. However, further studies on these lines might be encouraging in developing neurotherapeutic interest in these natural food products for neurodegenerative disorders.

The authors are thankful to the Deanship of Scientific Research, College of Nursing, Research Centre at King Saud University for supporting this research.

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