Cognitive and histopathological effects of olive leaf extract in colchicine-induced hippocampal neurodegeneration in rats

: OLE seems to have a considerable neuroprotective effect on hippocampal pyramidal cells improving memory improvement potential. More extensive evidence-based research is required to authenticate and screen its medicinal benefits and compare the biological activities of its active ingredients for the prevention and treatment of various diseases, including age-related neurodegeneration


Introduction
Olive tree (Olea europaea L.) is one of the oldest agricultural tree crops and has been a primary source of oil for centuries.It has been cultivated widely since prehistoric times in the Mediterranean region, the Indian subcontinent, and Asia.Because of its close association with human religion, culture, medicine, and nutrition, the leaves of the olive tree have been used as a traditional herbal tea for its beneficial effects among people around the Mediterranean basin (1).Olive leaf extract (OLE) has been the subject of several recent studies due to its potential beneficial effects on human health as it bears anti-oxidant, anti-atherosclerotic, cardioprotective, and hypoglycaemic effects.The consumption of OLE was reported to be associated with a lower incidence of cardiovascular diseases, such as hypertension and metabolic syndrome, including diabetes and hyperlipidaemias (2).Moreover, memory-enhancing effects of OLE have been claimed in animal experiments (3).
Oxidative stress is a condition created by the imbalance between oxidants and antioxidants in a biological system.The imbalance occurs due to the excess level of reactive oxygen species (ROS) or improper functioning of the antioxidant system.Elevated levels of ROS and a decrease in antioxidants disturb cellular homeostasis, leading to harmful effects on biomolecules and causing various age-related pathologies and diseases such as cancer, cardiovascular, and neurodegenerative diseases (4).
Colchicine (COL) is an alkaloid extracted from the plants of the genus Colchicum (Autumn crocus).COL is a microtubule-disrupting agent that produces marked destruction of hippocampal granule cells, mossy fibres, and septohippocampal pathways.It induces neurofibrillary degeneration by binding to tubulin, the structural protein of the microtubule, which is associated with the loss of cholinergic neurons and a decrease in acetylcholinetransferase resulting in impairment of learning and memory.
COL-induced cognitive impairment has been established as an animal model of sporadic dementia of Alzheimer's type (5).It causes instability and depolymerization of the microtubules and inhibits axonal conduction, eventually leading to the death of the nervous cells (6).Alzheimer's disease is associated with microtubule dysfunction and is characterized by the appearance of specific cytoskeletal cellular abnormalities, which are associated with cognitive impairment (5).
Since the Morris water maze (MWM) is an established paradigm for assessing the cognitive memory performances in rats with chronic cerebral neurodegeneration (7,8), therefore, this study aimed to study the neuroprotective and therapeutic effects of OLE on COL-induced neurodegeneration by assessing the short-term memory (STM) levels of animals using MWM test.

Materials and Methods
Methanolic extraction of OLE One kilogram of olive leaves was morphologically authenticated at the pharmacognosy lab by Dr. Norazian Mohd Hassan, Faculty of Pharmacy, IIUM.A specimen was archived at the faculty herbarium with the specimen number (MH-1344-OL/KOP).The overnight air-dried olive leaves were ground into fine powder and added to 5 L of 80% methanol.The mixture was then filtered after 24 hours.The filtrate was then concentrated to yield a solid extract under a reduced-pressure rotary evaporator.After freeze-drying, the OLE was stored in a fridge at 7oC until it was used for forced oral treatment.The yield of OLE was 39.64 grams.The solid extract was dissolved in deionized water before oral treatment (9).

Animals
Eighteen male Sprague Dawley rats weighing 255-360 g were equally divided into three groups; control group (injected with artificial CSF only), colchicine-injected (COL) group, and 100 mg/kg OLE-treated-colchicineinjected (COL+OLE) group.The rats were housed in standard cages in a controlled colony room (21 ± 3°C).They were maintained under a 12:12 hours light/dark cycle with access to tap water and food pellets ad libitum.All procedures complied with the good laboratory practice guidelines of animal handling and were approved by UniSZA pre-clinical animal research ethical committee with the reference number (EA-20-FP-122/L-03).The COL and COL+OLE groups were injected with 15 µg COL dissolved in 5 µL artificial CSF via an intracerebral hippocampal single injection.The treatment with OLE started a week after the injection and continued for 15 days via forced oral feeding.Distilled water was forced orally into the control and COL groups.The experimental design of the study is shown in Figure 1.Apparatus A 2-meter diameter black fiberglass circular tank, 60 cm in height, filled with water up to 30 cm, was used (Figure 2).The escape platform (EP) was a black 10 cm diameter cylinder.Water temperature was maintained at 26°C throughout the test period.Colored posters were pasted on the surrounding walls near the tank for the rats to develop spatial memory.ANY-maze video tracking software (Stoelting Co., USA) was used to record the swimming time of the rats.Four hypothetical points (N, S, E, and W) depicted the swim starting point of the rats for the four daily tests.The starting point was not repeated for subsequent days of trial.The pool was divided into four virtual quadrants: NE, SE, SW, and NW, as shown in Figure 2.
The habituation training session was done on the 11 th day of treatment.The STM test paralleled the last three days of OLE treatment according to the protocol previously described by Azzubaidi et al (10).Each animal attempted four daily swimming trials with visible EP for three successive days.A probe test was done on day 15 with the EP removed from the pool; each animal was given a maximum time of 60 seconds starting from the SE pole.The number of annulus crossings was recorded.
Colchicine-injection procedure The procedure was done under aseptic conditions.Intraperitoneal sodium pentobarbital 30 mg/kg was injected.A 2 cm longitudinal midline skin incision was made on the rat's scalp.Fifteen micrograms COL dissolved in 5 µL artificial CSF (11) was slowly injected into left and right hippocampi using an insulin syringe for the COL and COL+OLE groups.Artificial CSF was injected into the rat's hippocampus of the control group.The cranial landmark for intrahippocampal injection was 2 mm anterior to the lambdoid suture along the sagittal suture and then 2.25 mm lateral to the sagittal suture on both sides (Figure 3).The skin incision was sutured with a nonabsorbable suture.Postoperative diclofenac sodium 10 mg/kg analgesic was injected intraperitoneally to reduce the post-operative pain.

Histological study
The animals were euthanized with an overdose of inhaled diethyl ether solution after four weeks of COL intracranial injection.Rat brains were quickly excised and dissected on an ice-cold metal plate.The right hemispheres were grossly dissected 3 mm posterior to the lower end of the fornix, as previously described previously (12).After a 72hour fixation in 10% formaldehyde solution, staining with cresyl violet was done on 5 mm thickness sectioned slides for viewing under light microscopy.

Statistical analysis
Results were expressed as mean ± standard error of the mean (SEM).Two-way analysis of variance (ANOVA) with repeated measures was used to analyse the significant difference in escape latency and body weight of rats per week.Post hoc comparison (Tukey HSD's test) was utilized to determine the overall significance of treatment as compared to the control and COL groups.One-way ANOVA was employed to analyse the probe memory test.P value less than 0.05 (P < 0.05) was considered statistically significant.

Results
Short-term memory test findings STM test results on days 12-14 of the treatment with OLE indicated a significant difference (P < 0.05) of the control and COL+OLE groups compared to the COL group.On day "14", the control and COL+OLE groups demonstrated a significantly faster (P < 0.05) escape latency than the COL group.The escape latency values on day "14" for control, COL, and COL+OLE were 11.38 ± 0.17, 60.00 ± 0.19, and 29.25 ± 0.19 seconds, respectively (Figure 4).

Probe memory test findings
The number of annuli crossing the EP zone for the control, COL, and COL+OLE groups was 4.00 ± 0.447, 1.00 ± 0.00, and 2.00 ± 0.00, respectively.The COL+OLE group revealed a statistically significant difference (P < 0.05) when compared with the control group (Figure 5).control group showed typical pyramidal cells with welldemarcated cell membranes, clear cytoplasm, and a welldelineated nucleus.These were normal findings for the viable neurons in the control group (Figure 6a).However, in the COL-injected animal group, the toxic effect of the COL on the pyramidal cells manifested itself histopathologically as distorted shrunken pyramidal (non-viable) cells with irregular boundaries.The presence of dark cytoplasm and indistinct pyknotic nuclei of the pyramidal neurons of the CA1 layer in the COL-injected group proves that   a neurodegenerative process has taken place as a result of COL toxicity (Figure 6b).Moreover, treatment with OLE improved the pyramidal cell morphology in COL+OLEtreated rat hippocampal sections with apparent and welldemarcated nuclei (Figure 6c).

Discussion
This study was designed to investigate potential neuroprotective effect of olive leaf methanolic extract on the COL-induced neurodegeneration of the rat brain.This work's findings confirmed COL's detrimental effect on the pyramidal cell layers in the CA1 area of the hippocampus, thus supporting the conclusion of previous studies that COL neuronal toxicity irreversibly damages dendrites by disrupting their microtubules supporting network (6).COL toxicity mimics Alzheimer's disease pathogenesis in several mechanisms, such as disrupting the neuronal microtubule structure causing it to be unstable and depolymerized, eventually leading to the necrosis of the neurons.Besides the CA1 hippocampal neurodegeneration, the granular cell layer in the dentate gyrus is lost (13).The intrahippocampal COL injection into the rats was found to cause cognitive deficits similar to those of Alzheimer's disease.These features are manifested by learning defects and impairment of recent memories, which have been consolidated (11).Furthermore, COL is reported to increase the level of free radicals, thereby aggravating the oxidative stress status and simultaneously decreasing the glutathione level, which is an essential antioxidant in the body.Consequently, necrosis of the pyramidal cells in the hippocampus is inevitable (5).Moreover, other studies have affirmed that the intracranial COL injection would significantly weaken memory secondary to a reduction of neurotransmitters levels, such as norepinephrine, dopamine, and serotonin in the cerebral cortex, hippocampus, and the caudate nucleus, which ultimately leads to learning difficulties (11).
The daily oral treatment of 15 mg/kg OLE markedly ameliorated the histopathological changes in the hippocampal pyramidal cells and showed less overall neuronal cell loss.This might be attributed to the potent antioxidant, free radical scavenging, and antiinflammatory properties of OLE, which play an essential role in preventing neuronal damage from toxic free radicals' effect and oxidative stress caused by COL.Previous studies have reported that free radicals' deleterious effects can mediate necrosis and neuronal degeneration in brain regions abundant with such ROS (14).It is well-known that oxidative stress is involved in neuronal cell death, which is associated with neurodegenerative disorders such as AD.Researchers have shown that COL derivatives could augment ROS to the level that can activate apoptosis in cancerous lymphocytes (15).
The retention probe memory test on the following day confirmed the considerable deterioration in memory water maze performance by the COL group between the 12 th and the 14 th days of the study.Animals of the COL group swam by circling the edge of the pool, and there was a time when rats failed to find the platform.Therefore, the number of annulus crossings in the EP zone was significantly worse than the control group.Although it is observed that the level of improvement in escape latency pattern with the OLE-treated group was not as robust as it was reported in the previous study with the use of Nigella sativa treatment on spatial cognitive functions in the study done by Azzubaidi and colleagues ( 10), yet the escape latency time for the three trials was significantly faster than COL group.Nevertheless, the preservation of cognitive function by OLE treatment was proved as it performed well on the day of the probe memory test.The animal learning behavior was observed in the COL+OLE-treated group; i.e., the rats in the OLE-treated group showed a rearing behavior after climbing the platform.Animals were noticed to keep looking around at the surrounding cues, which indicates the learning process.Unlike the OLE-treated group, the COL group was not observed to swim to the EP zone group, only after spending significant time swimming away from the EP quadrant.These improvements in the OLE-treated rats may be related to the neuroprotective effect of OLE and its antioxidant properties.The histological observation of the CA1 area proved that there was a neurodegenerative process affecting the pyramidal neurons of the hippocampus in COL group rats.Electron microscopic assessment is in progress to study the detailed ultrastructural changes that occurred in the COL and COL+OLE groups.
The phenolic compounds that form the active constituents of the olive leaf, such as oleuropein, hydroxytyrosol, flavones, flavonols, and flavan-3-ols, contribute to OLE's robust antioxidant activity (16).Oleuropein was reported to reduce lipid peroxidation activity in the oleuropein-treated group (2).Oleuropein in olive leaf might exhibit its antioxidant activity by increasing superoxide dismutase activity and glutathione peroxidase activity in the oleuropein-treated group (17).Other in vivo studies have demonstrated that hydroxytyrosol, a phenolic compound extracted from the olive leaf and oil, possesses antioxidant and anti-inflammatory properties (18), besides reducing the levels of Aβ plaques in an AD mice model (19).Moreover, OLE was observed to relieve oxidative stress and hippocampal neuronal damage in transient cerebral ischemia (16).Additionally, OLE at doses ranging between 50 and 200 mg/kg has proved to possess anti-inflammatory and analgesic activities in a dose-dependent manner (20).OLE may exert its neuroprotective effect against amyloid toxicity by directly interfering with the aggregation process, thus inhibiting the formation of toxic soluble oligomers and amyloid fibrils.Cell viability is seen to be preserved with OLE since it interferes with the initiation of the amyloid aggregation process.As a result of decreasing the level of the toxic oligomer, the noxious effect of the amyloid fibrils on the brain is alleviated (21).

Conclusion
OLE treatment led to a considerable preservation in the STM function of rats challenged with intrahippocampal COL injection.This memory improvement of the OLE might be attributed to its promising neuroprotective potential on hippocampal pyramidal cells.

Figure 1 .
Figure 1.Experimental design of the study groups.OLE treatment was started a week after colchicine injection into the hippocampus.Habituation training was performed on day "11".The STM test was started on day "12" for three successive days, followed by the # probe retention test on day "15".

Figure 2 .
Figure 2. Image depicting Morris water maze pool as viewed from above.The four starting points: N, S, E, and W, were randomly chosen for the training and trials.EP was the target rescue area.The probe memory test started from the SE quadrant (arrowhead).N= north, S= south, E= east, W= west, EP= escape platform, NE= northeast, SE= southeast, SW= south west, and NW= northwest.

Figure 4 .
Figure 4. Line graph depicting significant intergroup differences in escape latency for three successive days(12, 13, and 14).COL+OLE and control groups demonstrated a significantly faster escape latency than the COL-injected group.*P<0.05 as compared to the COL group.OLE: Olive leaf extract; COL: Colchicine.

Figure 6 .
Figure 6.Cresyl violet stained light microscopy photomicrographs defining the CA1 hippocampal region showing the pyramidal neurons (arrows) of (a) the control group, depicting the typical shape of pyramidal neurons and a distinct nucleus and a clear cytoplasm (b) Colchicine (COL)-injected group, depicting shrunken distorted cell with an indistinct nucleus that cannot be differentiating from the cytoplasm (c) COL+OLE treated group, depicting pyramidal neurons that are partially contracted with an overall preserved nucleus indicating cellular viability.OLE: Olive leaf extract.

Figure 5 .
Figure 5. Histogram depicting the (day 15) probe memory test findings represented by the number of annuli crossings the escape platform zone within 60 seconds.*P < 0.05 versus the control group.OLE: Olive leaf extract; COL: Colchicine.