1. Introduction

njection of STZ in ICV form in rats cause a long-term and progressive deficits in learning, memory, and cognitive performance in rats that is similar to Alzhimer’s disease (AD) (1). AD has been known as a chronic neurodegenerative disorder characterized by progressive cognitive impairment, memory loss, and behavioral disturbances (2) and is considered as the most common cause of dementia in elderly patients (3). Interventions that could delay SAD onset would have a major public health impact (2). Free radical generation has been associated with cognitive impairment in ICV STZ model of SAD in rats (1, 4) and ICV STZ has also been known to impair cholinergic neurotransmission (5).

There is also experimental evidence that saffron constituents are involved in cognition. However, chemical analysis of this plant has shown the presence of water-soluble carotenoids, and its glucoside (safranal and picrocrocin) and flavonoids (quercetin and kaempferol) (6, 7). Its crude extract and purified chemicals have been demonstrated to prevent tumour formation (8-10), atherosclerosis or hepatic damage (11, 12). It has been shown that administration of saffron and its constituents crocins could antagonized ethanol-induced memory impairment in the passive

avoidance in mouse (13, 14). Also, recently, it has demonstrated that saffron extracts  counteracted recognition memory deficits and persist against scopolamine-induced,  performance impairments in the passive avoidance task in rat (15). Rgarding to mentioned reports the aim of the present study was to investigate the role of a main saffron constituent safranal in learning and memory processes. For this purpose, So, there was evaluated the effect of safranal in antagonizing extinction of recognition memory in STZ–induced model of AD in the rats using Passive avoidance and Y maze tasks.

2. Materials and Methods

2.1. Materials

Streptozotocin (STZ) was purchased from Sigma Chemicals Co (St. Louis, MO, USA). Ketamone (10 %) and xylazine (2 %) were purchased from Alfasan Co (Holland). Chemical constitutes ACSF to make ready from Merck Co (Germany). Saffron were provided from local market and then scientifically identified by the department of botany of Shaheed Beheshti University (SBU).

2.2. Methods

2.2.1. Animals

Male adult Wistar rats (310-350 g) (Pasteur’s Institute, Tehran), at the start of the experiment were housed three to four per cage in a temperature-controlled colony room under 12 h light/dark cycle. Animals were given free access to water and kept at 80–85% of their free feeding body weight throughout the experiment. All behavioral experiments were carried out between 11 a.m. and 4 p.m. This study was carried out in accordance with the policies set forth in the Guide for the Care and Use of Laboratory Animals (NIH).

2.2.2. Experimental procedure

Rats (n = 60) were randomly divided into the following groups: 1. Control group, 2. Sham-operated group (SH ) that received bilateral ICV injection of artificial CSF (ACSF) (10 µl on each side) as the solvent of STZ. 3. Safranal-treated control group (safranal), 4.STZ-injected group (STZ) which received ICV injection of STZ. and 5. safranal-treated STZ group (STZ + safranal), which also received safranal (60 mg/Kg; i.p.) once for 2 day. STZ and safranal-treated STZ groups were given a bilateral ICV injection of STZ (Sigma, St. Louis, USA) (3 mg/kg). STZ was freshly dissolved in cold artificial CSF and at a volume of 10 µl on each side. The injection was repeated on day 3. In the sham group, only artificial CSF (120 mM NaCl; 3 mM KCl; 1.15 mM CaCl2; 0.8 mM MgCl2; 27 mM NaHCO3; and 0.33 mM NaH2PO4 adjusted to pH 7.2) (Merck Chemical, Germany) was ICV injected. For stereotaxic surgery, rats were anesthetized with a combination of ketamin (100 mg/Kg, i.p.) and xylazine (5mg/Kg, i.p.), placed in a stereotaxic apparatus (Stoelting, USA). The scalp was cleaned with iodine solution, incised on the midline and a burr hole was drilled through the skull (A, -0.8 mm from bregma; L, 1.4 mm; 3.4 mm below the dura) according to the stereotaxic atlas (16).

2.3. Behavioral tests

2.3.1. Spatial Y-maze memory

The experimental apparatus for Y-maze consisted of a black-painted maze made of Plexiglas. Each arm of the Y-maze was 40 cm long, 30 cm high and 15 cm wide (17, 18) and positioned at an equal angle (labeled A, B and C) and converged in an equilateral triangular central area with 15 cm at its longest axis. Each rat, naïve to the maze, was placed at the end of one arm and allowed to move freely through the maze during an 8 min session. The sequence of each arm entry recorded manually (i.e., ACBABACACBCACAC , etc.).

 A spontaneous alternation behavior, which is regarded as a measure of spatial memory, was defined as the entry into all three arms on consecutive choices in overlapping triplet sets (i.e., ACB,ABA,CAC,BCA,CAC,) (18). The percent spontaneous alternation behavior was calculated as the ratio of actual to possible alternations. Percent Alternation = [Actual Alternation (i.e., ACB, BCA = 6)/Maximum Alternation.

*(i.e., ACBABACACBCACAC = 15 – 2 = 13)] ×100 = (6/13) ×100 = 46.15 %.

* Total Number of arms entered minus 2 (18). Each test was done once on each animal.

2.3.2. Single trial passive avoidance test

The apparatus (BPT Co., Tehran) consisted of an illuminated chamber connected to dark chamber by a guillotine door. Electric shocks were delivered to the grid floor by an isolated stimulator. On the first and second days of testing, each rat was placed on the apparatus and left for 5 min to habituate to the apparatus. On the third day, an acquisition trial was performed. Rats were individually placed in the illuminated chamber. After a habituation period (2 min), the guillotine door was opened and after the rat entering the dark chamber, the door was closed and an inescapable scrambled electric shock (1 mA, 1 s once) was delivered. In this trial, the initial latency (IL) of entrance into the dark chamber was recorded and rats with ILs greater than 60 s were excluded form the study. Twenty-four hours later, each rat was placed in the illuminated chamber for retention trial. The interval between the placement in the illuminated chamber and the entry into the dark chamber was measured as step-through latency (STL up to a maximum of 150 s). This test was conducted after 3 weeks post-surgery.

2.4. Statistical analysis

All results were expressed as mean S.E.M. For the passive avoidance test, nonparametric Kruskal-Wallis test was used, which if significant, was followed by Mann-Whitney U-test for pair-wise comparisons. Data for the Y maze task were evaluated by Wilcoxon’s rank sum test. In all calculations, a difference at p

3. Results

3.1. Effect of safranal on memory retention deficit in passive avoidance test

The initial latency was 33.11, 29.89, 30.25, 37.57 and 26.66 s in Control, SH, Safranal, STZ, and STZ+Safranal groups respectively. There was no significant difference between experimental groups. Step through latency in STZ (20.28) and STZ+CSE (33.95) groups reduced markedly in comparison to control (53.18) animals. On the other hand, the STZ+Safranal group exhibited significant reversal of STL ( P

Fig. 1: The effect of Safranal treatment (60 mg/Kg) on passive avoidance performance after ICV injection of STZ in rats as indicated by initial and step-through latencies after 3 weeks. Values are expressed as means ± S.E.M. * P

3.2. Effect of Safranal on spatial cognition deficit in Y maze task

As shown in Fig. 2, the mean scores of alternation behavior for control, SH, Safranal, STZ and STZ+Safranal were 79.23, 75.89, 85.23, 39.91 and 65.68 % respectively. There was found a marked reduction in alternation score in STZ and STZ+Safranal groups regarding to control animals. However in STZ+Safranal group the treatment could significantly modified the STZ memory impairment effect (P < 0.05).