[1] 15w Determination of the Best Concentration of Streptozotocin to Create a Diabetic Brain Using Histological Techniques

[1] 200w Alzheimer's disease (AD) is one of the most important disorders among neurodegenerative diseases which is characterized by neurofibrillary tangles and senile plagues. Intercerebroventricular (ICV) streptozotocin administration is a form of sAD which was applied to examine different factors following AD. Previous reports used different doses of streptozotocin (STZ) to create Alzheimer's model, but no standard dose has been introduced. Therefore, we decided to investigate the best concentration of STZ to induce a diabetic brain with lowest mortality rate and high severity of destruction. We treated rats with three different doses of STZ (STZ 1.5, 2.25, and 3 mg/kg, ICV). Spatial memory for treated rats was evaluated by Morris water maze (MWM). Locomotor activities of rats were assessed by open field test. Histological observation such as immunohistochemistry, immunofluorescence, and Nissl staining were performed on the brain especially in CA1, CA3, and DG regions of hippocampal neurons at residues P-ser396 and P-ser404. Our data suggest that although the percentage hyperphosphorylation of tau protein by injection of STZ 3 mg/kg was about 10 % more than STZ 2.25 mg/kg compared to the control group, we considered the latter doses due to no effect on motor activities and enhance the number of glial cells.

[1] 90w Alzheimer's disease (AD) is the most common form of neurodegenerative disorders that affects several parts of the brain such as the hippocampus, olfactory bulb, cortical regions, cerebellum, and hypothalamus (El Khoury et al. 2014). Some etiological factors have been identified responsible for the occurrence of AD including genetics and environment (Huang and Mucke 2012). Two proteinopathies play a pivotal role to create the mentioned disease including amyloidopathy which leads to the accumulation of Aβ peptide in amyloid plaques and tauopathy by hyperphosphorylated and aggregated tau (van Eersel et al. 2010).

[2] 110w Tau protein is a random coiled and asymmetric protein which contains special characteristics such as natively unfolded conformation, posttranslational modifications, and thermal and acidic stability (Hirokawa et al. 1988;Ren and Sahara 2013). Tau protein has been recognized as a major neuronal microtubule-associated protein (MAP) which promotes microtubule (MT) polymerization and stabilizes MT polymer structure (Gong and Iqbal 2008). MTs are composed of two subunits, α and β tubulin, with high negative charges at the C-terminal end (Howard and Hyman 2003). The interaction between MT and tau is regulated through phosphorylation and dephosphorylation on tau protein by several enzymes such as kinases and phosphatases (Cho and Johnson 2004;Gong, et al. 2010).

[3] 82w Glycogen synthase kinase 3 (GSK-3β), cyclin-dependent kinase 5 (CDK-5), and cAMP-dependent protein kinase (PKA) play a key role in tau phosphorylation. However, protein phosphatase 2A (PP2A), the major tau phosphatase in the human brain, contributes to tau dephosphorylation (Xu et al. 2014). Several studies have shown GSK-3β phosphorylates protein in Ser199, Thr212, Thr217, Thr231, Ser181, Ser262, Ser396, Ser400, Ser404, and Ser413 sites. Also, CDK-5 phosphorylates tau protein in pathological sites including Ser202, Ser235, and Ser404 (Evans et al. 2000;Li and Paudel 2006).

[4] 61w Memory dysfunction is the main symptom of central nervous system complication in type I diabetes mellitus which has been characterized in AD patients. Impairment in insulin signaling pathway and glucose uptake into cell increases the activity of GSK-3β. The previous studies have indicated that decrement in the expression of GSK-3β leads to exacerbate hyperphosphorylation in tau protein (Schaffer et al. 2008).

[5] 113w The most common diabetogenic chemical agent used to create diabetic model in experimental conditions is streptozotocin (STZ). It is a well-known diabetogenic drug (i.p. injection) that mainly targets pancreatic beta cells (Song et al. 2014). The mentioned molecule is a hydrophilic compound due to hexose group in its structure which plays an important role to be taken up by cells (Chen et al. 2013). STZ can enter into cell via the low-affinity glucose 2 transporter in plasma membrane. The cell which contains GLUT 2 is sensitive to STZ entrance, because STZ has a cytotoxic effect on cells due to DNA and chromosomal fragmentation by mechanisms involving free radical generation (Ngarmukos et al. 2001).

[6] 62w Although several researchers have applied streptozotocin to induce an Alzheimer's model (ICV injection), the exact mechanism of STZ on brain cells has not been recognized (Grünblatt et al. 2007;Zamani et al. 2012;Zhou et al. 2013). Moreover, STZ inhibits the activity of tau protein in the brain, so it makes prolonged impairment of memory which is a character of Alzheimer's patients (Kamat 2015).

[7] 56w Numerous studies have reported that tau protein undergoes a specific modification in the normal brain by attaching hydroxyl group of serine/threonine residues to β-N-acetyl-glucosamine in the presence of glucoside hydrolase-O-GluNAcase. Tau O-GluNAcylation is regulated by the entrance of glucose into cell, so if glucose uptake/metabolism is disturbed, tau O-GluNAcylation may be diminished (Konrad et al. 2001).

[8] 134w STZ possibly affect two different mechanisms in the brain; on one hand, the activity of insulin signaling is rebated by interruption of phosphorylation of PI3K and the downstream GSK-3β and thereby provokes hyperphosphorylation of tau protein (Barilar et al. 2014). On the other hand, STZ is a crucial component able to inhibit the activity of glucoside hydrolase-O-GluNAcase. Since phosphorylation and glycosylation of tau protein is a reciprocal process, therefore, depletion of brain glucose uptake/metabolism resulting from the effect of STZ leads to the decline of the rate of glycosylation reactions. Finally, the phosphorylation of tau is elevated in the STZ-injected rat brains (Lenzen 2007;Hart et al. 2011). In pathological conditions, hyperphosphorylation and aggregation of tau in tauopathies play an important impact on memory formation and long-term potentiation in Alzheimer's brain (Lindwall and Cole 1984).

[9] 41w Previous reports used different doses of STZ to create Alzheimer's model, but no standard dose is introduced. Therefore, we decided to investigate the best concentration of streptozotocin to induce a diabetic brain with lowest mortality rate and high severity of destruction.

[1] 115w Escape latency was significantly increased in STZ treated with 3 and 2.25 mg/kg (F 3,26 = 41.89, P < 0.001, one-way repeated measure ANOVA). Average path length for locating the platform below the water surface on 5 days of acquisition trials was significantly prolonged in STZ 2.25 and 3 mg/kg as compared with aCSF group (F 3,26 = 34.255, P < 0.001). No statistically significant differences in swimming speed was observed between STZ-treated (2.25 and 1.5 mg/kg) and aCSF groups (F 3,26 = 6.237, P < 0.05), but there was a significant difference (F 3,26 = 6.237, P < 0.001) between STZ 3 mg/kg and the control group in terms of swimming speed (Fig. 3a-c).

[2] 208w All rats treated with STZ and aCSF showed a significant reduction in escape latency and path length during 5 days of training trial test which were evaluated according to repeated one-way ANOVA (P < 0.001). The effect of different doses of STZ treatment vs. training trial days was surveyed. The observed results of path length treated with STZ 3 and 2.25 mg/ kg had the most effectivity on spatial acquisition in days 1, 2, and 5 of the training test (F 3,28 = 19.469, P < 0.001; F 3, 28 = 4.218, P < 0.01; F 3,28 = 5.041, P < 0.01, respectively). The effect of STZ treatment in day 4 showed no significant difference (F 3,28 = 0.709, P > 0.05) on the training test. All days of training test except day 4 showed a significant difference in the swimming duration between STZ-treated animals and the control group (F 3,28 = 5.131, P < 0.01; F 3,28 = 3.235, P < 0.05; F 3,28 = 7.078, P < 0.001; F 3,28 = 3.045, P < 0.05, respectively). No significant difference was observed in swimming speed during 5 days except day 3 for STZ 3 mg/kg treatment (F 3,28 = 3.999, P < 0.001) (Fig. 4a-c). Probe Test

[3] 261w The results of probe trial were evaluated based on two parameters, the time spent (%) and crossing numbers (s) in the target quadrant. The results of the first factor showed there was a significant difference between STZ-treated animals (3 and ) % ( t n a r d a u q t e g r a t n i e m i T a C S F S T Z 1 . 5 m g / k g S T Z 2 . 2 5 m g / k g S T Z 3 m g / k g 0 5 10 15 20 *** ** A ) s e m i t ( s r e b m u n g n i s s o r C a C S F S T Z 1 . 5 m g / k g S T Z 2 . 2 5 m g / k g S T Z 3 m g / k g 0 2 4 6 8 ** *** B C Fig. 5 Effect of different doses of STZ treatment in target quadrant. a The time spent in the target quadrant (%). b The numbers of crossing in the target quadrant. c The swimming tracks. Data were presented as mean ± SD. (a **P<0.01 significant different between STZ 3 mg/kg and control group (aCSF); ***P<0.001 significant different between STZ 2.25 mg/kg and control group (aCSF). b **P<0.01 significant different between STZ 2.25 mg/kg and control group (aCSF); ***P<0.001 significant different between STZ 3 mg/kg and control group (aCSF))

[4] 13w 2.25 mg/kg) and the control group (F 3,26 = 4.937, P < 0.005).

[5] 38w On the other hand, a remarkable difference of the crossing numbers in the target zone was observed between STZ treatment (3 mg/kg and 2.25 mg/kg) and aCSF groups (F 3, 26 = 5.807, P < 0.005) (Fig. 5a-c).

[6] 47w Total duration of highly walk of STZ treated with 3 mg/kg showed a significant increment during 5 min (F 3,25 = 13.18, P < 0.001). The percentage time of highly walk was exacerbated (F 3,23 = 26.35, P < 0.001) in STZ (3 mg/kg) (Fig. 6a, b).

[7] 13w Tau Hyperphosphorylation at Residues P-ser396 and P-ser404 in the Hippocampal Regions of Rats

[8] 27w In order to confirm cognitive and spatial memory impairment resulting from injection of STZ reagent, the formation of phosphorylated tau was investigated through IHC on specific residues.

[9] 34w Tau phosphorylation level at the ser396 site was significantly increased in the hippocampus at CA1 and CA3 regions (F 3, 14 = 21.165, P < 0.001; F 3,14 = 15.789, P < 0.001, respectively).

[10] 98w Our results showed that tau was significantly phosphorylated at residue 404 in CA1 and CA3 regions of the hippocampus (F 3, 14 = 22.491, P < 0.001; F 3,14 = 9.801, P < 0.001). Since CA4 region of the hippocampus is the prominent location in the brain which is susceptible to posttranslational modification such as phosphorylation, comparison between different doses, i.e., 2.25 and 3 mg/kg of STZ, showed CA3 and CA4 regions are phosphorylated in both residues more than CA1 region (F 1,26 = 21.541, P < 0.05; F 1,24 = 44.917, P < 0.05, respectively) (Fig. 7a-c).

[11] 103w A significant difference in the treated animals by STZ in CA1 and CA3 of hippocampal regions from different groups was observed. Induction of STZ resulted in a significant increase in GFAP immunoreactive astrocytes in the CA1 and CA3 regions. Comparison between different doses of STZ showed astrocyte cell counts in CA3 region was more than those in CA1 region (F 3,28 = 4.003, P < 0.01, and F 3,23 = 7.787, P < 0.000, respectively). The percentage increment of cell number in STZ 2.25 mg/kg was about 4 and 24 % relative to STZ 3 mg/kg in CA1 and CA3, respectively (Fig. 9a-c).

[1] 171w Learning and memory are two important events involving several neurodegenerative diseases such as Alzheimer's diseases (AD) (Slutsky et al. 2010). Almost all researchers have used several different compounds to induce Alzheimer's disorder in experimental level (Wang et al. 2001;Montilla-López et al. 2002;Song et al. 2014). To obtain better insight of neuronal impairments and their effect on memory function, STZ is the most m m 0 0 1 r e p s r e b m u N 2 P S 3 9 6 P S 4 0 4 0 10 20 30 40 STZ (3mg/kg) control STZ (2.25 mg/kg) STZ (1.5 mg/kg) # # # # # *** * B A m m 0 0 1 r e p s r e b m u N 2 P S 3 9 6 P S 4 0 4 0 10 20 30 40 50 STZ (3mg/kg) control STZ (2.25 mg/kg) STZ (1.5 mg/kg) # # # # # *** * C potent reagent used to create a diabetic model and Alzheimer's disease.

[2] 95w Previous reports have pointed out that ICV-STZ causes learning and memory impairments in rats with the appearance of neurodegenerative effects, oxidative stress, and tau protein phosphorylation (Perry et al. 1998;Raza and John 2012). A part of structure of STZ includes glucose, so cells uptake STZ by GLUT2. The exact mechanism of STZ in the brain has not been cleared, but its effects are comparable with events taking place in beta-cell of pancreas. STZ facilitates the hyperphosphorylation of tau and accelerates the aggregation of neurofibrillary tangles (NFTs) which are hallmarks in AD (Sadigh-Eteghad et al. 2015).

[3] 90w Several studies have reported different doses of STZ induce a diabetic brain (Chu and Qian 2005;Salkovic-Petrisic et al. 2006;Grünblatt et al. 2007;Deng et al. 2009); therefore, we investigated the best concentration of STZ to create sAD with cognitive impairment and neurophatological changes. Accordingly, it is worthwhile to assess the best potential of STZ to function as a neurodegeneration inducer in an Alzheimer's model. In this study, rats were treated with different doses of STZ treatment (1.5, 2.25, and 3 mg/kg) to induce sporadic dementia without their effect on motor activity.

[4] 303w Fig. 7 Effect of STZ in tau hyperphosphorylation in the hippocampus at residues Ser396 and Ser404. a The accumulation of P-tau 396 and 404 in the CA1 and CA3 regions of hippocampal neurons by immunohistochemistry. The image was shown at the magnification of ×400. b Quantitative analysis of tau hyperphosphorylation in the CA1 area of the hippocampus. c Quantitative analysis of tau hyperphosphorylation in the CA3 area of the hippocampus. Data were presented as mean ± SD. (b *P<0.05 significant different between STZ 2.25 mg/kg and control group (aCSF) (Ps396); ***P<0.001 significant different between STZ 3 mg/kg and control group (aCSF) (Ps396); ## P<0.01 significant different between STZ 2.25 mg/kg and control group (aCSF) (Ps404); ### P<0.001 significant different between STZ 3 mg/kg and control group (aCSF) (Ps404). c *P<0.05 significant different between STZ 2.25 mg/kg and control group (aCSF) (Ps396); ***P<0.001 significant different between STZ 3 mg/kg and control group (aCSF) (Ps396); ## P<0.01 significant different between STZ 2.25 mg/kg and control group (aCSF) (Ps404); ### P<0.001 significant different between STZ 3 mg/kg and control group (aCSF) (Ps404)). Scale Bar: 20 μm STZ leads to induce tau hyperphosphorylation at many residues such as Ser199, Thr181, Thr205, Ser404, and Ser396 which is created by activating several kinases like GSK-3β, CDK-5, and MAPK (Sjolander et al. 2009;Ackermann et al. 2010). On the other hand, inhibition of some phosphatases, i.e., PP2A and PP2B, in the presence of STZ is an important process in the hyperphosphorylation tau protein pathway. Ser396 and Ser404 are two important residues involving interaction between tau protein and the microtubule. Phosphorylated tau at residues Ser396 and Ser404 is separated from the microtubule, so several intracellular cascades would be followed such as disturbance in the synaptic plasticity, instability of microtubules, formation NFT, and eventually, cognitive impairment (Evans et al. 2000;Regan et al. 2015).

[5] 208w The Morris water maze test was employed to survey spatial learning and memory based on time taken by rats to reach the hidden platform. Escape latencies, time spent (%), and frequency in the target quadrants were evaluated. Rats attempted to escape from water by locating the hidden platform in a target quadrant. The time required in finding the hidden platform gradually decreased in training trials; rats which were treated with 1.5 mg/kg spent lesser time to locate the platform compared to other groups (2.25 and 3 mg/kg). The level of impairment for the other doses was more than that of the control group during the training trial test. Rats which were treated with 2.25 and 3 mg/kg showed a fluctuation in spatial memory during 5 days. The length that animals swam before finding the hidden platform was longer for the two latter doses. In order to assess the effect of the three doses on motor activity, we evaluated swimming speed of rats. Surprisingly, treated rats by 3 mg/kg had the highest velocity to find the platform. It seems that the mentioned dose leads to impair learning and memory during the training trials, but it is harmful for motor activity and change in cognitive process (memory, attention, and performance).

[6] 88w The step-down open filed test was applied to evaluate the exact effect on the two latter doses (2.25 and 3 mg/kg) on motor activity. In our study, 3 mg/kg of STZ showed an improvement in velocity of rats treated in ICV-STZ relative to that of the control group. It revealed the time spent and frequency of mobility during 5 min of open filed test; STZ 2.25 mg/kg is the more effective than STZ 3 mg/kg in terms of memory impairment and also without any effect on motor activities.

[7] 248w In order to confirm spatial destruction in MWM, we evaluated one of the risk factors involving creation Alzheimer's disease, tau phosphorylation, at two residues Ser404 and 9 The results of immunofluorescence analysis of the effect of STZ administration on innate immunity. a Representative fluorescence microscopy images of aCSF and STZ 1.5 mg/kg treatments in CA1 and CA3 regions of the hippocampus. b STZ 2.25 mg/kg and 3 mg/kg. c The percentage number of glial cells. Values are presented as mean ± SD. (a **P < 0.01 significant different between STZ 1.5 mg/kg and control group (aCSF) (CA1); ***P < 0.001 significant different between STZ 2.25 mg/kg and control group (aCSF) (CA1); ***P < 0.001 significant different between STZ 3 mg/kg and control group (aCSF) (CA1); ##P < 0.01 significant different between STZ 2.25 mg/kg and control group (aCSF) (CA3)). Scale bar, 50 μm Ser396. ICV-STZ 2.25 mg/kg and STZ 3 mg/kg lead to induce tau hyperphosphorylation at two important residues. Our results showed the effect of STZ 2.25 mg/kg is approximately the same as that of STZ 3 mg/kg which is demonstrated by IHC. The process of memory impairment and tau phosphorylation were exacerbated in CA3 and CA4 regions relative to the control group. It seems that pyramidal cells of the mentioned sites are more susceptible to destruction compared to CA1 region. Enhanced phosphorylated tau in these regions leads to affinity decrement to the microtubule in the pathological conditions, so the disability of the microtubule disrupts intracellular events.

[8] 231w To assess the effect of hyperphosphorylated tau deposits in neuronal cells and theirs damages, we demonstrated the presence of dark neurons by Nissl staining. Dark neurons are a kind of neuron resulting from the aggregation of basophilic proteins and condensation of cytoplasm (Garman 2011). Neurofibrillary tangles are basophilic components composed of hyperphosphorylated tau protein. Histological examination of the hippocampus using Nissl staining in treated rats with different doses of STZ showed a significant large number of death neurons compared to the control group. Our data have demonstrated that the extent of neuronal impairment arising from administration of STZ 2.25 mg/kg is similar to STZ 3 mg/kg. Detected dark neuron using cresyl violet dye was in CA3 and DG regions more than other parts of the hippocampus. DG region was at a higher rate than those observed in the CA3 part. Our results showed some of neurons died after injection of different doses of STZ treatment. On the other hand, hippocampal neurons are the most effective cells in the treated rat brain which undergoes a higher damage. It seems that these cells are more vulnerable among the neurons of the other parts of the brain. The fate of DG following administration of STZ 3 mg/kg treatment in the hippocampus showed about 5 % more than that of the 2.25 mg/kg group compared to aCSF group in which there was no significant difference.

[9] 118w In order to assess the effect of different doses of STZ on the innate immunity such as astrocyte and microglial population, glial fibrillary acidic protein (GFAP) was evaluated as a marker of astrocytes in the hippocampus. Immunofluorescence demonstrated a significant increase in the number of glial cells in the CA1 and CA3 regions of treated rat by STZ 2.25 and 3 mg/kg compared to the control group. Glial alteration in STZ 2.25 mg/kg in CA3 was higher than that in STZ 3 mg/ kg. It confirmed that STZ administration disturbs neuron cells in different regions of the hippocampus especially in CA3 area, so the number of glial cells was increased to play a protective role in the brain.

[10] 115w We concluded that based on our results, the effect of different doses of STZ treatment showed ICV injection of STZ 3 mg/kg had a negative effect on locomotor activities of rats. STZ 2.25 mg/kg seems to be the most effective dose on memory impairment without alteration of motor activities. On the other hand, the proliferation of astrocytic cells in the damaged areas of the hippocampus in STZ 2.25 mg/kg is a common end result of damage to neurons in the CNS. We, as a consequence, suggest that STZ 2.25 mg/kg causes to hyperphosphorylate tau protein on the brain especially in the hippocampal neurons of rats and neuronal damage and increases the number of glial cells.

[1] 95w Adult male Wistar rats weighting 250-300 g were purchased from Pasteur Institute of Iran, and housed ten per cage in large cages before surgery, handled daily, and were maintained at room temperature (25 ± 2 °C) under standard 12-h light cycle and 12-h dark cycle with lights on at 7.00 A.M.; food and water were available. These animals' experiments were carried out in accordance with recommendations from the Declaration of Helsinki and the internationally accepted principles for the use of experimental animals. All efforts were made to minimize animal suffering (Care, Animals and Resources 1985).

[2] 145w Different concentrations of streptozotocin (STZ) treatment (1.5, 2.25, and 3 mg/kg of body weight) from Sigma-Aldrich Chemical Co., St. Louis, USA, were dissolved in artificial cerebrospinal fluid (aCSF-124 mM NaCl, 3 mM KCl, 2 mM CaCl 2 , 1.25 mM KH 2 PO 4 , 36 mM NaHCO 3 , 10 mM D-Glucose, and 1 mM MgSO 4 , pH adjusted to 7.2 by 5 % CO 2 insufflation, filtered through disposable sterile 0.2-μm filters and adjusted to body temperature). These solutions were prepared immediately prior to injection. Pser-404 tau (ab92676; 1:800 v/v), Pser-396 tau (ab109390; 1:1000 v/ v), anti-glial fibrillary acidic protein (GFAP; z0334; 1:200 v/ v), secondary antibody anti-GFAP (ab97050; 1:300 v/v), propidium iodide (Pi) (P4864, 1:1000 v/v), and mouse-and rabbit-specific HRP/3,3′-diaminobenzidine (DAB) (ABC) detection immunohistochemistry (IHC) kit were purchased from Abcam Biotechnology Inc., USA. All other chemicals were of analytical reagent grade.

[3] 32w Rats were anesthetized with an i.p. combination of ketamine (5 mg/100 g of body weight (b.w.)) and xylazine (1 mg/100 g b.w.) and positioned in a stereotaxic frame (Naghdi et al. 2003).

[4] 61w After a week of adaptation to colony room conditions, the rats were randomly divided into four groups (n = 10): group 1control (vehicle): control-operated rats were infused with aCSF in each ventricle on days 1 and 3. Group 2-lesion (1.5, 2.25, and 3 mg/kg of body weight): rats were performed with bilaterally ICV infusion of STZ on days 1 and 3.

[5] 40w All behavioral tests were carried out to evaluate the capacity of learning and memory for each group between day 15 and day 21. On day 21, the rats were sacrificed for assay of phosphorylated tau by histological tests (Fig. 1).

[6] 128w Rats were anesthetized with an i.p. combination of ketamine (5 mg/100 g of body weight (b.w.)) and xylazine (1 mg/100 g b.w.). The head was poised in a stereotaxic apparatus (Stoelting, USA) and skull exposed. Then a midline sagittal incision was made in the scalp. Two holes were drilled in the skull for the placement of the injection cannula into both of the lateral cerebral ventricles. Coordinate s for the intercerebroventricular (ICV) cannula implantation were 0.9 mm posterior to the bregma, 1.5 mm lateral to the sagittal, and 3.6 mm beneath the cortical surface of the rat brain on the Paxinos and Watson's atlas (Swanson 2004). Two cannulas were inserted into the skull and then were fixed with dental cement. The cannulas were then closed with a steeled.

[7] 100w Animals were housed in a cage during the postoperative period to provide food and water. Rats were infused ICV with either aCSF or STZ. Drug concentration and solution volume were adjusted according to the animal body weight, and a volume of 4 μl/300 g body weight was administrated (2 μl/ ventricle) using a Hamilton microsyringe positioned in the 30gage infusion cannulas 13 mm long, when the subjects were awake and freely moving, then allowing to follow behavioral changes after injection (Grünblatt et al. 2007;Barilar et al. 2014). Control animals received bilaterally an equal volume of vehicle into the lateral ventricles.

[8] 85w During 7 days following surgical methods, for recovery, drug and vehicle were administrated into lateral ventricle bilaterally through guide cannulas (23 gage) using injection needles (30 gage) connected by polyethylene tubing to 10.0-μl Hamilton microsyringe. Four-microliter vehicle or different doses of STZ were injected during 3-4 min. The needle was left in place for another 60 s before it was slowly withdrawn. In all experiments, aCSF is used as vehicle that it has no significant effect on learning and memory in Morris water maze (MWM).

[9] 97w Morris Water Maze and Open Field Apparatus Spatial learning and memory were evaluated using MWM as it is described previously with a minor modification (Brandeis et al. 1989;Zhou et al. 2013). Briefly, it consisted of a dark circular pool (140 cm in diameter and 22 cm high) filled with water (20 ± 1 °C) to a depth of 25 cm. A transparent Plexiglas platform (11 cm diameter) was located 2 cm below the water surface in the center of one of the arbitrarily designed northeast (NE), southeast (SE), southwest (SW), or northwest (NW) orthogonal quadrants (Fig. 2).

[10] 96w The platform provided the only escape from water. Many extra cues such as racks, a window, a door, a book shelve, and pictures on the walls surrounded the room where the water maze was housed. These were kept in fixed positions with respect to the swimming pool to allow the rat to locate the escape platform hidden blow the water surface. The position of the animal was monitored by a camera that was mounted above the center of the pool (Zhou et al. 2013). All behavioral tests were carried out between 9.00 A.M. and 12.00 A.M.

[11] 129w The animals were subjected to a daily session of four training trials for five consecutive days. First of all, the rat holds near the border of the pool (in one of the starting location), facing the wall and allowing it to swim until the platform was found. If the rat did not find the hidden platform a maximal treatments in rats time of 60 s, the animal was then manually guided to the platform, where it remained for a 30-s period before commencement of the next trail (Zhou et al. 2013). For each animal, three factors were evaluated: the amounts of time spent to find the platform (escape latency (duration (s))), the distance animal swam before finding the platform (path length (distance (cm))), and the swimming speed (velocity (cm/s)).

[12] 102w On the following day, a probe test was conducted by removing the platform. Rats were permitted to swim freely in the pool for 60 s. The time spent and frequency in the target quadrant (Q3) in which the platform had previously been located during training phase were recorded over 60 s. These parameters provided an index of the degree of memory consolidation. In the same day which memory consolidation of rats was evaluated, visible test for each rat was done to assess possible deficits in sensory-motor processes, so rats were held in the MWM with a visible platform on a new location.

[13] 43w Another day after finishing MWM, subjects were introduced in an open field apparatus for 5 min and a number of behaviors scored was carried out using a computer-based video tracking system (Ethovision 1.6 Noldus, Waninggen) to evaluate motor activity (Basso et al. 1995).

[14] 78w During the behavioral testing, rats were deeply anesthetized with ether and perfused transcardially with phosphatebuffered saline (PBS) followed by 4 % paraformaldehyde in 0.2 M phosphate buffer pH 7.4. The whole brain was carefully extracted and kept in 4 % paraformaldehyde overnight. After fixation, the tissue was rehydrated and embedded in paraffin. Fixed frozen sections (5 μm) were prepared and subjected to Nissl (Ooigawa et al. 2006) staining and immunohistochemistry as described in the protocol of the instructions.

[15] 251w To determine the presence of phosphorylated tau protein, immunohistochemistry (IHC) was performed on 5-μm-thick paraffin-embedded sections of treated groups including different doses of STZ regent and its corresponding control group (Clodfelder-Miller et al. 2006;Xu et al. 2014). In brief, sections were deparaffinized in xylene and rehydrated. Endogenous peroxidase activity was inhibited by incubation in 3 % hydrogen peroxide (H 2 O 2 ) in absolute methanol followed by washes in water three times for 5 min. Antigen retrieval was carried out by steaming in citrate buffer (pH value 6) for 40 min. After a 25-min cooling period, sections were incubated for 30 min in protein blocking buffer to inhibit nonspecific antibody binding. The addition of primary antibody was followed by an overnight incubation with phospho-S404 and phospho-S396 tau primary antibody which was diluted in protein blocking buffer at 4 °C. Samples were washed and then incubated in biotinylated goat anti-polyvalent for 30 min at room temperature from IHC kit (Abcam). Sections were treated with streptavidin peroxidase for 30 min at room temperature. A color reaction was developed using 3,3′-diaminobenzidine (DAB) as a chromogen according to the manufacture's protocol to localize site of antibody binding. Slides were counterstained with hematoxylin, dehydrated using graded alcohols and xylene, and preserved with mounting medium and cover glass. All samples were photographed at a magnification of ×400 within the identical areas in the hippocampus by a microscope (Nikon, H600L, Japan) equipped with a digital camera system (Nikon, DS-L3, Japan). A positive reaction appeared as brown color.

[16] 190w GFAP immunohistochemistry staining was used to assay innate immunity changes in hippocampal areas, i.e., CA1 and CA3 regions in the proposed animal model. Briefly, tissue sections of 5 μm were deparaffinized and dipped in citrate buffer (pH 6) for 45 min at 80 °C. The samples were washed twice in PBS containing Tween 20 for 30 min. Sections were immersed in 0.3 % (v/v) triton X-100 for 20 min. They were dipped in a blocking solution at room temperature. The primary antibody rabbit anti-GFAP (cat # Z0334, Dako; Denmark) was diluted 1:200 in PBS containing 0.3 (v/v) Triton X-100 and 1 % (v/v) BSA and incubated for 1 h. Then, the slides were washed in PBS and Tween 20 before adding secondary antibody diluted 1:300 in the solution which was applied for primary antibody. Slides were then washed twice and treated with propidium iodide (Pi) for 5 s. Finally, all tissues were washed in PBS and mounted with glycerol. All samples were photographed at a magnification of ×200 within the identical areas in the hippocampus by a microscope (Nikon, H600L, Japan) equipped with a digital camera system (Nikon, DS-L3, Japan).

[17] 43w Six sections from each animal were used to determine the number of phosphorylated tau and GFAP cells, i.e., astrocytes present in CA1 and CA3 regions of the hippocampus. The mean number of phospho-tau per slice was recorded for each animal in experimental groups.

[18] 86w Three consecutive Nissl-stained sections at the coronal position were used for cell counts and the average of cells was calculated. In the hippocampus, neurons were counted in the small pyramidal cell layer of CA1 region, the large pyramidal cell layer of CA3 area, and the granule cell layer of DG part. All samples were photographed at a magnification of ×400 within the identical areas in the hippocampus by a Nikon microscope. Neurons with abnormal morphologies contain massive shrunken and hyperbasophilic features were defined as dark neurons.

[19] 155w Three effects, i.e., dose, times, and dose × times, were surveyed by two-way analysis of variance (ANOVA), and interactions between all groups were considered to determine whether there was a significant effect of different doses or the effect of time duration every day for 5 days on the spatial learning. One-way ANOVA was used for comparison of behavioral criteria during training for the 5-day trained animals. The probe data and histological results were analyzed by oneway ANOVA. Dunnett comparison posttest was performed to assess differences between groups relative to control. Cell counting was analyzed by one-way analysis of variance, and Tukey posttest was used to compare difference between tested and control groups. The percentage of GFAP-positive cells was calculated. The significance between groups was analyzed by GraphPad Prism 5 software and SPSS 16v. Statistically significant differences were accepted at P < 0.05. All error bars in figures were resulting from mean ± standard deviation (SD).

[1] 85w Nissl-stained dark neurons (DN) were determined in the CA1, CA3, CA4, and DG regions of the hippocampus. The percentage of the number of dark neuron to the total neurons was calculated. The maximum number of DN was observed in CA1, CA3, CA4, and DG of STZ 2.25 and 3 mg/kg treatment which was significantly increased in these areas (F 3, 20 = 4.496, P < 0.01; F 3,20 = 4.451, P < 0.001; F 3, 20 = 0.655, P < 0.001, respectively) (Fig. 8a, b).