1. Faezeh Tashakori Sabzevar
Crocetin attenuates spatial learning dysfunction and brain damage after chronic cerebral hypoperfusion in rats
Faezeh Tashakori Sabzevar

2. Introduction
Cerebral hypoperfusion is a major cause of cognitive deficits and memory impairment, in aging and in age-related neurological disorders.
Alzheimer's disease (AD), vascular dementia and post-stroke hypoperfusion are the most important disorders, especially in elderly people after the age 65.
In people with AD the increasing impairment of learning and memory eventually leads to a definitive diagnosis.
The reduced cerebral blood flow (CBF) is an important factor in progression of AD.
AD is the most common neurodegenerative disorder.

3. So In the search for effective treatments, investigators employ animal models (rat models)of disease to test potential therapeutic agents.
Vascular dementia is a common form of dementia after Alzheimer's disease (AD).
Bilateral permanent occlusion of the common carotid arteries (2-VO) in rats has been widely used as a suitable model to investigate the chronic cerebral hypoperfusion.
2VO used to evaluate the different managements and chemicals in delaying the progression of vascular dementia associated with reduced CBF.

4. Crocetin
Crocetin could be obtained from natural sources e.g. saffron or Gardenia jasminoides Ellis by acidic hydrolysis.
Crocetin, as a traditional herbal medicine, has multiple pharmacological properties such as: neuroprotective effects, anti-inflammatory effects, excellent antioxidant, anti fatigue.
Crocetin is a carotenoid with a 20-carbon chain, six double bonds and two carboxylic acid groups at the ends of the carbon chain .

6. Methods Preparation of crocetin
The crocetin was prepared from crocin by acidic hydrolization
2. Analysis of crocetin(for purity evaluation)
UV-Visible spectrophotometry analysis
High performance liquid chromatography(HPLC)
Thin layer chromatography (TLC)
3. Animals
30 Male Wistar rats ( g) divided into five groups; Sham, negative control and treatment groups 2,4,8 mg/kg.
Methods

7. Surgery and experimental procedure
The rats were anesthetized with a mixture of ketamine and xylazine
In ischemia rats, through a midline neck incision, left common carotid was ligated with surgical silk.
After several days, the right common carotid artery was ligated in the same way.
The animals received drugs or vehicle intraperitoneally 1 h after the second step of operation.
The non-ischemic rats received the same two-step operation procedure without ligatures.

9. Behavioral experiments
The performances of the spatial memory were investigated using a Morris water maze.
The water maze was a black circular tank with a 136cm diameter and a 60cm height. The tank was filled with water (20±1°C) to a depth of 35cm. It has four geographical quadrants.
The hidden platform was located in the target quadrant under water.
A video camera was applied above the water maze to record the rats’ swimming.

10. The escape latency time, the traveled distance, the swimming speed of each rat and the swimming time in the target quadrant were measured by the tracking system software.
The rats were given four training trials each day on 5 consecutive days.
they were allowed to rest on the platform for 20s before the next trial.
In the final day ,the platform was removed and the final test trial was performed to assess statistically the memory of the correct platform location.

13. After final test terial,2-3 rats from each group were randomly selected and euthanatized in CO2 chamber.
Tissue samples were taken from the brain.
After preparation , all samples being viewed under a light microscope.
Cerebral cortex and hippocampus of each brain were studied at a total magnification of 10×, 20× and 40×.
Pathology

14. Results

15. The escape latency time

16. Time target quadrant

17. Percentages of traveled distance

18. Swimming speed

19. Neuronal damage in the CA1 and CA3 regions of the hippocampus and cerebral cortex was extensively widespread in control group.
Neuronal cells were shrunken with deeply eosinophilic cytoplasm and pyknotic nuclei and frequently angular to triangular in shape especially in control ischemic group and crocetin 2mg/kg group.
Intensity of neuronal changes was less in crocetin 4mg/kg group, but not as neurons of the sham operated group and crocetin 8mg/kg group.
Crocetin 8mg/kg group had the least intensity of neuronal changes in the CA1 and CA3 regions of the hippocampus and cerebral cortex.

20. Hippocampus of a normal sham
rat
CA1 region of hippocampus of a normal sham rat with normal neurons (arrows), H and E, ×400(b)

21. Cerebral cortex of a normal sham rat with normal neurons (arrows)
H and E, ×400
Hippocampus of an untreated control rat showing widespread ischemic cell change in affected neurons (arrows), H and E, ×200

22. CA3 region of hippocampus of an untreated control rat
CA3 region of hippocampus of an untreated control rat. There are many triangular neurons with dark basophilic nuclei (arrows), H and E, ×200
Cerebral cortex of an untreated control rat showing laminar pattern of necrotic neurons associated (arrow), H and E, ×200

23. CA1 region of hippocampus of 8mg/kg crocetin treated rat with a few ischemic neurons (arrows), H and E, ×200
Higher magnification of the same region in figure 8g with a few ischemic neurons (arrows), H and E, ×400

24. Cerebral cortex of 8mg/kg crocetin treated rat showing a few ischemic neurons (arrows), H and E, ×200

25. These results suggest that crocetin (as potent antioxidant) has protective effects into ischemia damages and it would be beneficial to the improvement in impaired spatial learning skills. Also it could be applied and considered in the future human studies specially in the therapy of brain neurodegenerative disorders such as vascular dementia.
Conclusion

26. Many thanks from Dr. Seyed Ahmad Mohajeri for his supports.
Mashhad University of Medical Sciences for financial support through the grant number
Acknowledgment

27. Thank you for your considration.