The protective effects of Cyperus rotundus on behavior and cognitive function in a rat model of hypoxia injury

Abstract

Context: Hypoxia injury (HI) with its long-term neurological complications is one of the leading causes of morbidity and mortality in the world. Currently, the treatment regimens for hypoxia are aimed only at ameliorating the damage without complete cure. The need, therefore, for novel therapeutic drugs to treat HI continues.

Objective: This study investigates the protective effects of the ethanol extract of Cyperus rotundus L. (Cyperaceae) (EECR), a medicinal plant used in Ayurvedic traditional medicine against sodium nitrite-induced hypoxia in rats.

Materials and methods: We have evaluated the protective effect of 200 and 400 mg/kg of EECR against sodium nitrite-induced hypoxia injury in rats by assessing the cognitive functions, motor, and behavioral effects of EECR treatment along with the histological changes in the brain. By comparing the protective effects of standard drugs galantamine, a reversible cholinesterase inhibitor and pyritinol, an antioxidant nootropic drug against sodium nitrite-induced hypoxia in rats, we have tested the protective ability of EECR.

Results: EECR at doses of 200 and 400 mg/kg was able to protect against the cognitive impairments, and the locomotor activity and muscular coordination defects, which are affected by sodium nitrite-induced hypoxia injury in rats.

Conclusion: Based on our results, we suggest that the medicinal herb C. rotundus possesses a protective effect against sodium nitrite-induced hypoxia in rats. Further studies on these protective effects of EECR may help in designing better therapeutic regimes for hypoxia injury.

• EECR
• galantamine
• neuroprotection
• pyritinol
• sodium nitrite

Introduction

Hypoxic injury (HI) is a life threatening condition in which oxygen delivery is inadequate to meet the metabolic demands of tissues. HI, with its long-term neurological complications, is one of the leading causes of morbidity and mortality in the world (Lawn et al., 2005; Rees et al., 2008). It is the third most common cause of death next to coronary heart disease and cancer worldwide. According to the World Heart Federation, every year 6 million people die from stroke (http://www.world-heart-federation.org/cardiovascular-health/stroke). Death from hypoxia injury is projected to rise to 6.5 million by 2015 in the world (Strong et al., 2007). It was suggested that the cause of death in more than 87% of patients with hypoxia injury is due to cerebral ischemia (Rosamond et al., 2008), which also leads to delayed neuronal death resulting in significant morbidity with problems of cognition, memory, and behavioral deficits (Volpe & Petito, 1985).

Although our understanding of the cellular and biochemical changes that occur after acute and chronic hypoxia has increased significantly, the various categories of drugs currently used to treat hypoxic brain injury including calcium channel blockers (nifedipine), cholinesterase enzyme inhibitors (galantamine and donepezil), nootropic agents (piracetam), anti-epileptic agents (felbamate), and antidepressants (fluoxetine) are aimed at slowing the progression without cure. Hence, the search for an ideal drug to cure HI continues and has also been extended to herbal drugs as a better alternative to synthetic drugs.

Cyperus rotundus L. (Cyperaceae) is a well-known Ayurvedic plant drug which has been shown to have anti-inflammatory and wound healing (Puratchikody et al., 2006), hepatoprotective (Kumar & Mishra, 2005), antidiarrheal (Uddin et al., 2006), and antioxidative (Yazdanparast & Ardestani, 2007) activities. Studies have also shown that it possesses antimalarial and antihyperlipidemic effects (Mengi & Patel, 2008). Cyperus rotundus is also used to treat central nervous system (CNS) disorders like loss of memory, depression, Parkinson disease, and epilepsy (Lee et al., 2010; Sharma et al., 2001). Although some of these properties have been scientifically evaluated, the protective effect of C. rotundus against HI is not well understood.

In a recent study, we have evaluated the physiochemical properties and toxicological effects of the ethanol extract (EECR) of C. rotundus. It was found to have phenols, tannins, glycosides, and flavonoids and safe up to 2000 mg/kg body weight in Wistar rats (Jebasingh et al., 2012). The present study evaluated the protective effect of C. rotundus against sodium nitrite-induced hypoxia using EECR. By comparing EECR with galantamine (a reversible cholinesterase inhibitor) and pyritinol (an antioxidant nootropic), the drugs presently used for the treatment of HI, we have assessed the learning, memory, and behavioral deficits produced by sodium nitrite.

Materials and methods

Plant materials

The fresh tubers of C. rotundus were collected from Kanyakumari district of Tamil Nadu during the months of November and December 2008. The plant was identified and authenticated by Prof. P. Jayaraman, Director, Plant Anatomy Research Centre, Tambaram, Chennai, India, and a voucher specimen (PARC/2008/140) was deposited at the Department of Pharmacology, CL Baid Metha Research Foundation, Chennai, for further reference.

Extraction and preparation of test sample

Freshly collected tubers of C. rotundus, were washed, shade-dried, powdered, and soaked in chloroform for 48 h. The resulting extract was filtered, distilled, and extracted with chloroform. The resulting marc was extracted with alcohol exhaustively and the EECR was prepared as a suspension in 1% sodium carboxy methyl cellulose (SCMC). It was analyzed by HPLC and found to have 13 peaks (Jebasingh et al., 2012).

Pharmacological study: animals

Inbred male Wistar rats weighing between 150 and 180 g were received from the Committee for the Purpose of Control and Supervision on Experimentation on Animals (CPCSEA) approved animal house of Mohammed Sathak A. J. College of Pharmacy, Chennai, India. All the experimental protocols were approved by the Institutional Animal Ethical Committee (IAEC) (Ref no. AJ/IAEC/10/05). Rats were housed at 25 ± 1 °C with a relative humidity of 55 ± 5% and were fed with a standard pellet diet and water ad libitum. They were maintained under a 12 h light/dark cycle. The animals were acclimatized to laboratory conditions for 1 week prior to the initiation of the study.

Grouping

The animals were weighed, numbered, and divided into five groups of six. Sodium nitrite, which reduces the oxygen carrying capacity of the blood by changing normal hemoglobin to methemoglobin, was used to induce hypoxia. It was given intraperitoneally (i.p.) daily for 30  d, at 60 mg/kg with or without other drugs (Abdel-Baky et al., 2010). The standard drugs galantamine, a reversible cholinesterase inhibitor and pyritinol, an antioxidant nootropic drug, were used as a positive control. Drugs were administered per os (p.o.).

The animals were grouped as follows:

• Group I animals received 1% SCMC at a dose of 10 ml/kg, p.o. (vehicle control).

• Group II animals received sodium nitrite 60 mg/kg, i.p. (negative control).

• Group III animals received pyritinol 100 mg/kg, p.o. + galantamine 0.5 mg/kg, p.o. + sodium nitrite 60 mg/kg, i.p. (Dimitrova & Getova-Spassova, 2006) (positive control).

• Group IV animals received EECR 200 mg/kg, p.o. + sodium nitrite 60 mg/kg, i.p. (Jebasingh et al., 2012).

• Group V animals received EECR 400 mg/kg, p.o. + sodium nitrite 60 mg/kg, i.p. (Jebasingh et al., 2012).

We also tested the effect of EECR 200 mg/kg, p.o.; EECR 400 mg/kg; pyritinol 100 mg/kg, p.o, and galantamine 0.5 mg/kg alone and did not see any significant change in brain morphology, toxicity or behavior of rats (Jebasingh et al., 2012).

The cognitive, behavioral, and physical effects of sodium nitrite-induced hypoxia and the ameliorating effects of test and standard drugs were evaluated in rats using the Cooks pole climbing apparatus, Morris water maze, actophotometer, rotarod, elevated plus maze, and two compartment passive avoidance apparatus. The responses of the animals were recorded on days 1, 10, 20, and 30 of the experiment unless otherwise explained.

Assessment of learning and memory using Cook’s pole climbing apparatus

The learning and memory of the animals were evaluated by assessing the conditioned avoidance response using the Cooks pole climbing apparatus (Cook & Weidley, 1957). Male Wistar rats were trained in such a way that the animal had to climb the pole (shock free zone) within 30 s to avoid a shock. The shock was preceded by a buzzer that lasted for 15 s. The animals were trained to climb the pole at the sound of the buzzer (conditioned avoidance response). At particular intervals, 20 trials were given for each animal and the shock avoidance and mistakes were recorded. Trained animals were then treated with the SCMC, test drugs, or standard drugs and the conditioned avoidance responses were assessed.

Assessment of retention of learned behavior using the Two Compartment Passive Avoidance test

The retention of learned behavior was assessed using the Two Compartment Passive Avoidance test (Elrod & Buccafusco, 1988). The apparatus consists of a square box with a floor grid of 50 × 50 cm and wooden walls of 35 cm height. This box was illuminated with 100 watts bulb. In the center wall, there was an opening of 6 × 6 cm, which leads to a small (15 × 15 cm) dark compartment provided with an electrifiable floor (Hugo Sachs Electronics, Baden-Württemberg, Germany). The animals were trained by placing them in the illuminated chamber facing away from the entrance to the dark compartment. The latency to enter the dark compartment was recorded and a 1 mA foot shock was given for a period of 2 s when the rat stayed for more than 5 s in the dark chamber. Then the animal was returned to the cage. All the animals were trained for a week before testing with the drug. About 24 h after the trial period, each animal was placed again in the illuminated chamber as before for a maximum period of 180 s. The transfer latency of the animals (in seconds) to re-enter the dark compartment was assessed.

Assessment of anxiety like behavior using elevated plus maze

Elevated plus-maze (Pellow et al., 1985) was used to assess the anxiolytic effect of EECR. The elevated plus maze apparatus consisted of a central platform (10 cm × 10 cm) connected to two open arms (50 cm × 10 cm) and two covered (enclosed) arms (50 cm × 40 cm × 10 cm) and the maze was elevated to a height of 50 cm from the floor. During the experiment, each rat was placed at the end of an open arm, facing away from the central platform and the time (in seconds) spend in the open or closed arms and the number of entries into the open or closed arms with all its four legs were noted for the 5 min observation period.

Assessment of spatial learning using Morris water maze

The water maze test measures the spatial learning and memory of previously trained animals, which have learned to find a platform (Morris, 1984). It consists of a circular tank (100 cm diameter and 20 cm depth) with a circular white platform of 9 cm diameter hidden 2 cm below the water level. The water at 23 °C was made opaque by powdered milk during the experiment. The animal was left at one of the four assigned pole positions and the time taken for the animal to reach the platform was noted. Each animal received four consecutive trials per day with an inter-trial interval of 6–10  min for 3 d. After the trial period, the platform was removed and the experiment was repeated. On the day of experiment, the animal was placed in one of the four assigned polar positions and the time taken by the animal to reach the platform in the first 60 s was recorded.

Assessment of motor skill learning using rotarod

The motor coordination was evaluated using a rotarod apparatus (Caston et al., 1995; Lalonde et al., 1995). The apparatus consists of a horizontal metal rod with grip attached to a motor, whose speed can be adjusted. The rod is at a height of 50 cm above the table in order to discourage the animals from jumping off the roller. Before the experiment, all animals in each group were habituated to balance for 180 s. The rotarod speed of 20 revolutions per min (rpm) was used for the experiment. The time each animal was able to balance in the rotating rod with the prescribed speed up to 180 s in each experiment was recorded. The experiments were repeated five times in one session.

Assessment of locomotor activity using actophotometer

Actophotometer records the locomotor activity of the animal (Turner, 1965). The actophotometer operates on photoelectric cells which are connected to a counter and a count is recorded when the beam of light falling on the photocell is cut off by the movement of an animal. The animals were kept individually in the cage for 5 min and activity scores of each group of animals were recorded on days 1, 10, 20, and 30.

Assessment of brain histology

After the stipulated period of exposure to the drugs or the vehicle, the animals were sacrificed with an overdose of sodium pentobarbital and the brains were dissected out and fixed with 4% para-formaldehyde. They were dehydrated using graded alcohol and embedded in wax. About 5 µm coronal section was taken at bregma-4.16 (Paxinos & Watson, 1986). Sections were stained with hematoxylin and eosin and the morphology of the hippocampus, cortex, thalamus and the cerebellum was analyzed by light microscopy. The extent of neuronal damage was scored blindly in six different regions within the hippocampus, cortex, thalamus, and the cerebellum according to a previously described method (van den Tweel et al., 2005). Scores were from 0 to 3: 0 = 91–100% of neurons damaged, 1 = 51–90% of neurons damaged, 2 = 11–50% of the neurons damaged, 3 = less than 10% neuronal damage. Six sections/rat were scored, averaged, and the scores of six rats/treatment group were added to obtain the final score.

Statistical analysis

All experimental data were expressed as mean ± S.E.M of six animals in each group. The statistical analysis was carried out using a one-way ANOVA with the Bonferroni correction post hoc. Difference in the values at p < 0.05 was considered as statistically significant.

Results

Evaluation of learning and memory using Cook’s pole climbing apparatus

Learning and memory were evaluated on days 1, 10, 20, and 30 using Cook’s pole climbing apparatus. On day 1, there was no significant difference between the sodium nitrite-administered Group II animals compared with those of the vehicle control Group I animals or with those of any drug-treated animal groups (III, IV, and V) in terms of the conditioned avoidance responses.

On day 10 onwards, there was a significant difference between the sodium nitrite-administered Group II animals compared with those of the vehicle control Group I animals in terms of the number of conditioned avoidance responses that decreased significantly (Figure 1A). The positive control Group III animals, which received the combination of pyritinol and galantamine, showed a significant improvement in learning and memory when compared with Group II animals (p < 0.001). No significant differences in conditioned avoidance responses were observed between 200 and 400 mg/kg of EECR treatment groups on days 10, 20, and 30 in conditioned avoidance responses (Figure 1A). Interestingly both 200 and 400 mg/kg of EECR treatment Groups IV and V also showed significant improvement of learning and memory when compared with Group II animals (p < 0.001), which was comparable with those of the positive control Group III animals (Figure 1A).

Figure 1. Effect of EECR on learning and memory using Cook’s pole climbing apparatus. (A) Comparison among day 1, day 10, day 20, and day 30. (A(a)) Group I versus Group II, (A(b)) Group II versus Groups III, IV, and V and (B) within each group. * Significant difference, *p < 0.05, **p < 0.01, ***p < 0.001. Group I, vehicle control; Group II, sodium nitrite-treated animals (negative control); Group III, pyritinol, galantamine, and sodium nitrite (positive control); Group IV, EECR 200 mg/kg and sodium nitrite; Group V, EECR 400 mg/kg and sodium nitrite. Values are expressed as mean ± SEM from six male animals in each group.

Compared with day 1, all the groups except the sodium nitrite-treated Group II animals showed an increase in their conditioned avoidance responses at days 10, 20, and 30 (Figure 1B). Only the sodium nitrite-treated Group II animals showed a non-significant decrease in their conditioned avoidance responses (Figure 1B).

Evaluation of retention of learned behavior using the Two Compartment Passive Avoidance (TCPA) test

The effects of EECR on retention of learned behavior were assessed using the TCPA test and the results are depicted in Figure 2. On day 1, the sodium nitrite-treated Group II animals had a lower response (dark to bright chamber) when compared with the vehicle-treated control Group I animals (p < 0.01; Figure 2A).

Figure 2. Effect of EECR on retention of learned behavior using Two Compartment Passive Avoidance (TCPA) test. (A) Comparison among day 1, day 10, day 20, and day 30. (A(a)) Group I versus Group II, (A(b)) Group II versus Groups III, IV, and V, and (B) within each group. *Significant difference, *p < 0.05, **p < 0.01, ***p < 0.001. Group I, vehicle control; Group II, sodium nitrite-treated animals (negative control); Group III, pyritinol, galantamine, and sodium nitrite (positive control); Group IV, EECR 200 mg/kg and sodium nitrite; Group V, EECR 400 mg/kg and sodium nitrite. Values are expressed as mean ± SEM from six male animals in each group.

The positive control Group III animals, which received the combination of pyritinol and galantamine, showed a significant improvement in their transfer latency when compared with the sodium nitrite-treated Group II animals from day 1 to day 30 (p < 0.05 on day 1 and p < 0.001 from day 10 onwards; Figure 2A). The EECR-treated Groups (IV and V animals) also showed a faster response when compared with the sodium nitrite-treated Group II animals with no significance variation in transfer latency between them from day 10 onwards (p < 0.001; Figure 2A).

Compared with day 1, all the groups except the sodium nitrite-treated Group II animals showed a faster response at days 10, 20, and 30 (p < 0.001; Figure 2B). The sodium nitrite-treated Group II animals showed either no change or a significant decrease in their retention of learned behavior as seen by the increased latency at day 20 (p < 0.05; Figure 2B).

Evaluation of animal anxiety like behavior using elevated plus maze

Elevated plus maze was used to evaluate the behavioral parameters such as anxiety and exploratory activity and the results are shown in Figure 3. On day 1, the sodium nitrite-treated Group II animals did not show any changes in the anxiety level or exploratory activity when compared with all other groups (Figure 3A). There was no significant difference in the number of entries into the open or closed arms between the sodium nitrite-treated Group II animals and the drug-treated Groups III, IV, and V on day 1 (Figure 3C).

Figure 3. Effect of EECR on anxiety like behavior using elevated plus maze (A–D). The results on time spent in open and closed arms are shown in (A) and (B), while the results of open and closed arm entries are shown in (C) and (D). Values are expressed as mean ± SEM from six male animals in each group. (A) and (C) Comparison among day 1, day 10, day 20, and day 30. (A(a)) Group I versus Group II, (A(b)) Group II versus Groups III, IV, and V, and (B) within each group, day 1 versus 10, 20, and 30 d. (C(a)) Group I versus Group II, (C(b)) Group II versus Groups III, IV, and V. (D) Within each group, day 1 versus 10, 20, and 30th days. *Significant difference, *p < 0.05, **p < 0.01, ***p < 0.001. Group I, vehicle control; Group II, sodium nitrite-treated animals (negative control); Group III, pyritinol, galantamine, and sodium nitrite (positive control); Group IV, EECR 200 mg/kg and sodium nitrite; Group V, EECR 400 mg/kg and sodium nitrite.

Compared with the sodium nitrite-treated Group II animals, all the other groups showed significant decreases in their anxiety and an increase in their exploratory activity from day 10 onwards (p < 0.001; Figure 3A). The number of entries into open arm increased non-significantly in Group III animals on day 10 while it increased significantly on days 20 and 30 (Figure 3C). Compared with the sodium nitrite-treated Group II animals the EECR-treated Groups IV and V animals showed significant increases in the number of entries from day 10 onwards (p < 0.05, Group V, on day 10 to p < 0.001 on day 30; Figure 3C).

Compared with day 1, the sodium nitrite-treated Group II animals showed a significant reduction in the amount of time spent in the open arm at days 10, 20, and 30 (p < 0.001; Figure 3B).

A significant reduction in the number of entries into the open arm from day 1 to day 30 was also seen (p < 0.01; Figure 3D). The positive control Group III animals, which received the combination of pyritinol and galantamine showed a non-significant increase in the number of open arm entries (Figure 3D).

The 400 mg/kg EECR-treated animals also showed a non-significant increase in the number of open arm entries from day 1 to day 30 (Figures 3B and D). The 200 mg/kg of the EECR-treated Group IV animals showed a significant increase in the number of open arm entries on days 10 and 20, which decreased on day 30 (p < 0.01 on day 10 to p < 0.001 on day 20; Figure 3B and D).

Animals in the drug-treated Groups (III, IV and V) spent almost the same time in the open arms on all days with a slight decrease in days 20 and 30, although this is higher compared with Group II animals (Figure 3B).

Evaluation of spatial learning using Morris water maze

Using the water maze, we have evaluated the effect of EECR in spatial learning and the results are given in Figure 4. On day 1, there was no significant difference between the sodium nitrite-administered Group II animals compared with those of the vehicle control Group I animals or with those of any drug-treated animal Groups (III, IV, and V) in terms of the time taken by the animals to identify the platform (Figure 4A). From day 10 onwards, the time spend in identifying the platform to rest increased significantly in the sodium nitrite-administered Group II animals compared with those of the vehicle control Group I animals (p < 0.001; Figure 4A). Compared with the sodium nitrite-administered Group II animals, animals in Groups IV and V treatment with 200 and 400 mg/kg of EECR had a faster response time from day 10 onwards in identifying a platform to rest (p < 0.001), which was comparable with the positive control Group III animals which received a combination of pyritinol and galantamine (Figure 4A).

Figure 4. Effect of EECR on spatial learning using Morris water maze. (A) Comparison among day 1, day 10, day 20, and day 30. (A(a)) Group I versus Group II, (A(b)) Group II versus Groups III, IV, and V, and (B) within each group. *Significant difference, *p < 0.05, **p < 0.01, ***p < 0.001. Group I, vehicle control; Group II, sodium nitrite-treated animals (negative control); Group III, pyritinol, galantamine, and sodium nitrite (positive control); Group IV, EECR 200 mg/kg and sodium nitrite; Group V, EECR 400 mg/kg and sodium nitrite. Values are expressed as mean ± SEM from six male animals in each group.

Except for the sodium nitrite-treated Group II animals, spatial learning ability also improved in all the animal groups from day 1 to day 30 (p < 0.001; Figure 4B). In contrast, the sodium nitrite-treated Group II animals took more time to identify a platform from day 10 onwards, which increased significantly at days 20 and 30 (p < 0.001) suggesting a reduction in spatial learning ability (Figure 4B).

Evaluation of motor coordination by rotarod performance test

We have evaluated the motor coordination ability of the animals by assessing their ability to balance, using a rotarod, which in turn depends on the skeletal muscle contraction/relaxation state and the results are given in Figure 5. There was no significant difference between the sodium nitrite-administered Group II animals with those of the vehicle control Group I animals or with those of any drug-treated animal Groups (III, IV, and V) on day 1 (Figure 5A).

Figure 5. Effect of EECR on motor coordination using a rotarod. (A) Comparison among day 1, day 10, day 20, and day 30. (A(a)) Group I versus Group II, (A(b)) Group II versus Groups III, IV, and V, and (B) within each group, day 1 versus 10, 20, and 30 d. *Significant difference, *p < 0.05, **p < 0.01, ***p < 0.001. Group I, vehicle control; Group II, sodium nitrite-treated animals (negative control); Group III, pyritinol, galantamine, and sodium nitrite (positive control); Group IV, EECR 200 mg/kg and sodium nitrite; Group V, EECR 400 mg/kg and sodium nitrite. Values are expressed as mean ± SEM from six male animals in each group.

From day 10 onwards, there was a significant decrease in the balancing time in the sodium nitrite-administered Group II animals compared with those of the vehicle control Group I animals (Figure 5A; p < 0.001) or the positive control Group III animals, which received a combination of pyritinol and galantamine or the 200 and 400 mg/kg of EECR treatment Groups IV and V animals (p < 0.001; Figure 5A).

In the case of sodium nitrite-treated Group II animals, there was a gradual decrease in the balancing time from day 1 onwards. There was a significant reduction in the balancing time towards the end of day 30 (p < 0.001; Figure 5B). The balancing time was not significantly different between days 1 and 30 in the control Group I animals, in the positive control Group III, and in the EECR-treated Groups IV and V animals, although a non-significant reduction in balancing time was seen towards day 30 (Figure 5B).

Evaluation of locomotor activity by actophotometer

The locomotor activity was assessed using an actophotometer and the results are shown in Figure 6. There was no significant difference between the sodium nitrite-administered Group II animals compared with those of the vehicle control Group I animals or with those of the 200 and 400  mg/kg of EECR treatment Groups IV and V on day 1 in their locomotor activity. The positive control Group III animals, which received a combination of pyritinol and galantamine showed an increase in their locomotor activity on day 1 compared with the sodium nitrite-administered Group II animals (p < 0.01; Figure 6A).

Figure 6. Effect of EECR on the locomotor activity using an actophtometer. (A) Comparison among day 1, day 10, day 20, and day 30. (A(a)) Group I versus Group II, (A(b)) Group II versus Groups III, IV, and V, and (B) within each group, day 1 versus 10, 20, and 30 d. *Significant difference, *p < 0.05, **p < 0.01, ***p < 0.001. Group I, vehicle control; Group II, sodium nitrite-treated animals (negative control); Group III, pyritinol, galantamine, and sodium nitrite (positive control); Group IV, EECR 200 mg/kg and sodium nitrite; Group V, EECR 400 mg/kg and sodium nitrite. Values are expressed as mean ± SEM from six male animals in each group.

Compared with the sodium nitrite-administered Group II animals, the drug-treated Group III, IV, and V animals showed a significant increase in their locomotor activity on days 20 and 30 (p < 0.001; Figure 6A).

The sodium nitrite-administered Group II animals showed a significant reduction in the locomotor activity, at days 20 and 30 (p < 0.001) compared with day 1, although there was an increase at day 10 (p < 0.01). The locomotor activity was unchanged in the control Group I between day 1 and day 30. The locomotor activities were comparable at days 1 and 30 in the positive control Group III animals as well as in the 400 mg/kg of the EECR-treated Group V animals. The 200 mg/kg dose of EECR Group IV animals showed a slight decrease in their locomotor activity at day 30 when compared with day 1 (p < 0.05; Figure 6B).

Morphological evidence of the protective effect of EECR in the brain

We have assessed the morphological changes in the cortex, hippocampus, thalamus, and cerebellum of the brain of sodium nitrite-administered Group II animals compared with those of the vehicle control Group I, drug-treated positive control Group III, and EECR-treated Groups IV, and V animals (Figures 7 and 8). The cortex, hippocampus, thalamus, and cerebellum of the brain from the sodium nitrite-administered Group II animals showed significant morphological changes with pyknotic and rounded nuclei and with fragmented dead neurons (Figures 7B, G, L, Q, and 8). Vacuolization was also seen especially in the cortex, thalamus, and the hippocampus (Figure 7B, G, and L). The neuronal layer also shrank considerably in the cortex and hippocampal regions (Figure 7B and G). The Purkinje cells in the cerebellum were replaced with vacuoles (Figure 7Q). There was a significant protection against these changes in the pyritinol and galantamine-treated positive control Group III animals (Figure 7C, H, M, and R). EECR-treated Groups IV and V animals also showed protection against these morphological changes in all examined (cortex, thalamus, hippocampus, and cerebellum) brain regions (Figure 7D, E, I, J, N, O, S, and T). Qualitatively, there was more protection against the morphological changes in the 400 mg/kg EECR-treated Group V animals (Figure 7E, J, O, and T) compared with the sodium nitrite-treated animals (Figure 8). This was comparable with the positive control Group III animals (Figure 7C, H, M, and R). The cortex and the hippocampus were even comparable with those of the vehicle control group (Figures 7A and F and 8).

Figure 7. Morphological evidence of the protective effect of EECR in the brain. Representative photomicrographs of the cortex (A–E), hippocampus (F–J), thalamus (K–O), and cerebellum (P–T) of the brain sections from the different groups. A, F, K, and P are photomicrographs from Group I; B, G, L, and Q are photomicrographs from Group II; C, H, M, and R are photomicrographs from Group III; D, I, N, and S are photomicrographs from Group IV; and E, J, O, and T are photomicrographs from Group V. Note the presence of significant number of intact neurons in the brain sections of EECR-treated Groups IV (200 mg/kg) and V (400 mg/kg) (D, E, I, J, N, O, S, and T). Group I, vehicle control; Group II, sodium nitrite-treated animals (negative control); Group III, pyritinol, galantamine, and sodium nitrite (positive control); Group IV, EECR 200 mg/kg and sodium nitrite; Group V, EECR 400 mg/kg and sodium nitrite. Arrows in Q indicate the Purkinje cells in the cerebellum replaced with vacuoles. Magnification scale bar 20 µM.

Figure 8. Neuronal damage was scored in different groups in the cortex, hippocampus, thalamus, and cerebellum at day 30 in six different areas for each tissue. Scores were from 0 to 3: 0 = 91 to 100% of neurons damaged, 1 = 51–90% of neurons damaged, 2 = 11 to 50% of the neurons damaged, 3 = less than 10% neuronal damage. Six sections/rat were scored, averaged, and the scores of 6 rats/treatment group were added to obtain the final score. Group I, vehicle control; Group II, sodium nitrite-treated animals (negative control); Group III, pyritinol, galantamine, and sodium nitrite (positive control); Group IV, EECR 200 mg/kg and sodium nitrite; Group V, EECR 400 mg/kg and sodium nitrite. (a) Group I versus Group II, (b) Group II versus Groups III, IV, and V. *Significant difference, *p < 0.05, **p < 0.01, ***p < 0.001.

Discussion

An earlier study on the physiochemical characteristics and toxicological effects of the ethanol extract of C. rotundus showed that C. rotundus contains phenols, tannins, glycoside, and flavonoids and was safe even at a dose of 2000 mg/kg body weight in Wistar rats (Jebasingh et al., 2012). In the present study, we have evaluated the protective effect of C. rotundus against the sodium nitrite-induced hypoxia injury in rats. Short-term, long-term, and open field studies in rats have shown that sodium nitrite induces learning, memory, as well as behavioral deficits (Hlinak et al., 1990; Koziar et al., 1994). The sodium nitrite-induced deficits in learning, memory and behavior seen in our study correlated well with earlier reports. We have used standard drugs galantamine and pyritinol as the positive control (Group III) for the comparison of the protective effect of EECR. Pyrinitol is a well-known nootropic agent and was shown to affect the behavior in correlation with the spatial memory in rodents (Valzelli & Tomasikova, 1985). Galantamine is a reversible acetyl cholinesterase inhibitor and has been shown to act as an allosterically potentiating ligand on nicotine α4/β2 subtype acetylcholine receptors (Barnes et al., 2000; Samochocki et al., 2000). Animal studies have shown that galantamine produces significant improvements in learning ability, memory retention, and spatial-learning after ischemia (Dimitrova & Getova-Spasssova, 2006; Iliev et al., 2000). By comparing the protective effects of EECR-treatment with that of standard drugs galantamine and pyritinol (positive control), we were able to make a better correlation about the protective effect of EECR.

The Cooks pole climbing apparatus, using the conditioned avoidance responses, assesses the ability to acquire, retain, and retrieve the learned responses from memory. Accumulation of free radicals is said to affect the conditioned avoidance responses in rodents (Sreemantula et al., 2005). Our results suggest that there is a significant increase in the conditioned avoidance responses in all the groups except the sodium nitrite-administered Group II animals. The EECR-treated animals in Groups IV and V showed an increase in their conditioned avoidance responses, which is comparable with those of the vehicle control animals.

The elevated plus maze is used to assess anxiety responses in rodents (Pellow et al., 1985). The behaviors that are assessed here reflect the animal’s preference for an open area as opposed to a protected place and their motivation to explore a new environment and their anti-anxiety levels (Walf & Frye, 2007). The sodium nitrite-administered Group II animals had an increased anxiety as seen by their significantly reduced time spend in the open arms. The EECR-treated animals in Groups IV and V showed a significant reduction in their anxiety level as seen by the increase in their time in the open arm and the increase in the number of entries. Moreover, the reduction in the anxiety exerted by the doses of EECR was comparable with both the vehicle control and the galantamine and pyritinol-treated positive control animals. The anti-anxiety effects of EECR were further validated by the fact that the number of entries into the open arm decreased significantly after day 10 in the sodium nitrite-administered Group II animals.

The Morris water maze assesses the learning and memory deficits related to the hippocampus, striatum, basal forebrain, cerebellum, and different neocortical areas (D’Hooge & De Deyn, 2001). Different rodent models of ischemia such as the focal, partial, and global cerebral ischemia using the Morris water maze have shown that there is learning and memory deficits and this is comparable with the learning and memory deficit seen in the sodium nitrite-administered Group II animals (Block, 1999; D’Hooge & De Deyn, 2001). Earlier studies have also shown that cholinergic dysfunction induced by choline uptake blockers impairs learning and memory and cholinesterase inhibitors had shown to reverse the effect (Hagan et al., 1989; Socci et al., 1995). The fact that the EECR-treated animals in Groups IV and V showed improvements in the Morris water maze performance suggests that their spatial learning and memory are improved.

It has been shown that the corpus striatum is responsible for controlling a range of motor and cognitive functions, and the rotarod test assesses the motor performance related to neuronal changes in the striatum in rodents (Hikosaka et al., 1999; Lehericy et al., 2005; Poldrack et al., 2005). The motor performance is also connected with movement as neuronal structures innervate muscle fibers and the generated impulses are transmitted to the muscle fibers to coordinate muscle contraction and movement. Thus a reduction in the motor activity is an indication of CNS depression (Rathor & Ram, 2010). Further, lack of muscular coordination is an indication of abnormal muscle relaxation, which in turn leads to loss of muscle grip (Jansen & Low, 1996a,b). In the present study, although the locomotor activity did not change significantly in the beginning in Group II animals, which were treated with sodium nitrite, it started to decrease from day 20 onwards suggesting that there is a motor impairment. The fact that the EECR-treated animals in Groups IV and V showed improvements in their locomotor activity suggested that EECR has the potential to prevent the damaging effects of sodium nitrite-induced hypoxia and restore the cortical and striatal interaction in the brain. Moreover, the ameliorating effect exerted by the higher dose EECR was comparable to the vehicle control as well as the galantamine and pyritinol-treated positive control animals.

The actophotometer assesses the locomotor activity, which is an index of mental alertness (Thakur & Mengi, 2005). Most of the drugs that have an effect on the CNS are also said to influence the locomotor activity (Nehlig et al., 1992; Walker et al., 1996). A similar damage to the brain may reduce the motor activity. Our results have shown that there is a significant reduction in the locomotor activity in the sodium nitrite-administered Group II animals. The EECR-treated animals in Groups IV and V showed no change in their alertness behavior compared with those of the vehicle Group I and positive control Group III animals.

The histological analysis showed that sodium nitrite-induced hypoxia adversely affected the cortex, hippocampus, thalamus, and cerebellum of the brain as there was an increase in the number of pyknotic, shrunken neurons in these regions with increased vacuolization. These brain regions are known to play a significant role in the regulation and coordination of movement and behavioral activities. Earlier studies on hypoxia injury in different animal models reported these changes and suggested that these morphological changes occur because of the apoptosis of neurons or cell degeneration (Cummings et al., 1984; Jensen et al., 1991). It was also proposed that the cortex, hippocampus, and striatum are particularly sensitive to hypoxia-induced damages (Maiti et al., 2007; Nakajima et al., 2000; Ruan et al., 2003). An increase in the oxidative stress is also shown to be a reason for the changes in the morphology and cell death during hypoxia (Maiti et al., 2006). The fact that EECR-treated Groups IV and V animals also showed protection against these changes in all of these brain regions suggested that the medicinal herb, C. rotundus, has a protective effect against sodium nitrite-induced hypoxia injury.

Conclusion

Taken together, our study revealed that the traditionally used medicinal herb C. rotundus has a protective effect against the neurodegenerative changes produced by sodium nitrite-induced hypoxia injury in rats. Further studies using different model systems as well as hypoxic injury induced by different methods will help us to clarify the protective effect of the medicinal herb C. rotundus which may help in designing better intervention strategies for hypoxia injury.

Declaration of interest

The authors declare that there are no conflicts of interests.