Long ‐ term behavioral , histological , biochemical and hematological evaluations of amyloid beta ‐ induced Alzheimer ’ s disease in rat

1 Cancer Biology Research Center, Cancer research Institute of Iran, Tehran University of Medical Sciences, Tehran, Iran, 2 Vali-e-Asr Reproductive Health research center, Tehran University of Medical Sciences, Tehran, Iran, 3 Dept. of Pathology, School of Medicine, Imam Khomeini Hospital Complex, Tehran University of Medical Sciences, Tehran, Iran, 4 Islamic Azad University, Tehran Medical Branch, Tehran, Iran, 5 Neuroimaging and Analysis Group, Research Center for Molecular and Cellular imaging, Tehran University of Medical Sciences, Tehran, Iran, 6 International Graduate School of Neuroscience, Ruhr University, Bochum, Germany, 7 Biosensor Research Center, Endocrinology and Metabolism Molecular-Cellular Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran, * E-mail: Amanpour_s@tums.ac.ir


INTRODUCTION
Irreversible loss of memory and cognitive functions are associated with Alzheimer's disease (Duleu et al. 2010).The symptoms begin with short-term memory loss and evolve toward time and space confusion, depression, speech disorders (aphasia), withdrawal, mood change, mental problems, and respiratory disorders (Alzheimer's Association 2010, Rainon et al. 2016).Ab-normal formation and accumulation of Aβ peptides and tau proteins in the brain are two important neuropathological features of Alzheimer's disease (AD), with microscopic features such as neuritic plaques and intracellular neurofibrillary tangles, respectively (Davies et al. 2009).These deposits formation is associated with damage to the brain blood vessels and synapses, lack of neurotransmitters, death of neurons, and loss of memory and learning problems (Frozza et al. 2009).In addition, AD reduces the brain blood flow speed (Hamel et al. 2004, Iadecola et al. 2004), which is due to Aβ deposit on the vessels tunica, which reduces their elasticity (Petrella et al. 2003).In animal studies, intra-hippocampal injection of Aβ has resulted in memory loss, learning problems, and cholinergic system dysfunction (Lu et al. 2009).Since 1993, numerous studies have been performed on animals in which AD was induced with beta-amyloids of different lengths and other compounds such as streptozotocin, ibotenic acid and insulin amyloid fibrils (Cleary et al. 1995, Kheirbakhsh et al. 2015, Limón et al. 2012, Shi et al. 2011, Yaghmaei et al. 2013).
Aβ is generated by the action of beta-and gammasecretase enzymes on the amyloid precursor protein (APP).It has an oxidative activity which increases abnormal active oxygen and nitrogen, while decreasing endogen antioxidants, and causing neurons degeneration (Yan et al. 2014).
Reactive oxygen species and oxidative stress are known mediators in neurodegenerative disorders affecting the brain function, and cause lipids and proteins oxidation and cellular damage in the central nervous system.On the other hand, insulin signal pathway is important for regulating feeding behaviors and cognitive functions (Jhoo et al. 2004).Decrease of insulin levels in the brain and central nervous fluids and impairments in the insulin receptors function have been reported in AD patients (Hoyer et al. 2000).Increase in the blood sugar level, which cause oxidative stress and lipid peroxidation in some brain regions including hippocampus, is a result of disturbance in neuronal insulin signals.These facts demonstrate that there is a close relationship between AD and diabetes (Lupien et al. 2003, Sima et al. 2005).
Amyloid plaques contain other proteins in addition to Aβ, such as Apolipoprotein E (APOE) which is encoded by another AD developing gene (Duleu et al. 2010).Apolipoprotein E4 is the main genetic risk factor of AD and cause high accumulation of amyloids in the brain before AD symptoms appearance (Polvikoski et al. 1995).
In this study, we compared the amyloid plaques density in the beginning of AD, and one year after intrahippocampal injection of Aβ in animal model.Our main aim was to observe an AD model for a relatively long period in order to assess its characteristics over the long run.To this end, hematologic and blood biochemistry parameters, brain and vital organs histopathology, and memory loss were assessed during one year.

Animals and experimental groups
The study was carried out in accordance with the European Communities Council Directive of 22 September, 2010 (2010/63/EU) for care of laboratory animals and after approval of the Local Ethic Committee of Tehran University of Medical Sciences.
Fourteen male Wistar rats (6 months old at the time of surgery, 250-300 g, from Pasteur Institute of Iran) were used in this study.Animals were housed in a pathogenand temperature-controlled (22±2) room with a constant 12-h light/dark cycle where they had access to food and water ad libitum.
Animals were divided into control and Aβ-treated group with 7 animals in each group.Forty two days after surgery, 3 animals from each group were chosen randomly to examine the disorder occurrence.The remaining were tested for histology, hematology, and biochemistry characteristics after one year.

Passive avoidance apparatus (shuttle box)
The shuttle box consisted of a light and a dark compartment of the same size (20×20×30 cm each), separated by a retractable door (7×9 cm).Behavioral measurements were carried out at 42, 145, 320, 365 days and one year after surgery.In each measurement, animals were trained one day and tested the next day (Everss et al. 1998).In training step, each animal was put in the lightened section of the box for 5 seconds.The door was opened after what the animal could enter the dark section.After entrance the door was closed immediately, and the animal was allowed to explore the dark part for 10 s.The procedure was repeated after 30 s, but the animal received electrical stimulation from the dark compartment floor for 5 s.The procedure was repeated with 2 min intervals until the animal showed a delay before entering the dark part.That delay was considered as a sign of successful learning.On the test day, electrical stimulation was not applied.Animals were positioned in the lightened section, the door was opened after 5 s, and the delay duration before entering the dark section was recorded as a criterion for memorizing.The maximum allowed delay was 300 s (Urban et al. 1995).

Histology and hematological study
365 days after Aβ injection, remaining animals were anesthetized by intraperitoneal injection of ketamine and xylazine (60 and 20 mg/kg body weight, respec-tively).Cardiac puncture was then performed to make a terminal blood collection for hematologic and biochemistry measurements.Vital organs such as brains, hearts, kidneys, livers, colons, and spleens were excised and kept in a solution of 10% formalin.Six µm thick sections were prepared from the samples.Brain tissues were stained using Thioflavin S (Sigma and St Louis, MO, USA) for fluorescence microscopy while other tissues were stained with haematoxylin and eosin for routine light microscopy evaluations.

Data analysis
Results were expressed as mean ±S.E.M.In order to make multiple comparisons between groups, ANOVA method followed by Tukey's post hoc test were used, with p<0.05 taken as statistically significant.In the case of insulin levels, Independent Samples t-Test was used.

Evaluation of the shuttle box test data
Comparison of the animals delay times to enter the dark chamber showed that the longer the disease persisted, the more impairment of learning and memory was observed.Delay time's data corresponding to days 365 and 320 following induction of AD were different at the most significance level (p<0.001) in comparison with day 42.Data of day 365 after AD induction also demonstrated significant change in comparison to the control group (p<0.05) (Fig. 1).In other words, retention time of short-term memory is decreased during one year, which is indicative of the upward progression of the disease.

Hematology and biochemistry results
Evaluation of hematology factors and com plete blood count with differential (CBC-Diff) after one year did not show any significant difference in the AD rats (Fig. 2).However, evaluation of the biochemical factors of serum indicated marked changes.According to the statistical analysis, triglyceride (TG), hepatic enzyme, aspartate aminotransferase (AST or OT1), and glucose (Glue) level demonstrated highly significant change compared to the control group (p<0.001).Alkaline phosphatase (ALP1) and alanine aminotransferase (ALT) also indicated marked differences compared to the control group (Fig. 3).Further, insulin hormone increased compared to the control group (Fig. 4).

Histology results
Comparison of the brain tissues of animals one year post AD induction with the day 42 tissues demonstrated increase in size of the amyloid plaques (Fig. 5).It should be mentioned that higher brightness of the plaques states the increased concentration of beta amyloid, and is indicative of neural pathways disturbance and exacerbation of the disease (Fig. 5).Assessment of other tissues illustrated notable disorders.In liver and kidney of animals, amorphous deposits resulting from amyloid bodies were observed (Fig. 6).In these tissues, inflammation and changes in blood vessels are observed: monocular inflammatory cells are found between hepatocytes (Fig. 6A-1), and abnormal congested blood vessels are detected (Fig. 6A-2).

DISCUSSION
The present study results demonstrated that Aβ injection in the CA1 regions of both brain's hemispheres in Wistar rats causes memory loss and amyloid plaques formation which are exacerbated over time.Aβ-treated group (experimental group) showed significant difference in learning and memory capabilities, as demonstrated by decreased entrance delay in shuttle-box test with respect to control group, both in the early days after injection and later.In addition, histological analysis confirmed the presence of amyloid plaques which is an important characteristic of Alzheimer's disease (Selkoe et al. 2016).
nervous system cells and involved in cytoskeleton, extracellular matrix, and cellular connectivity (Gandy et al. 2005).However, Aβ protein deposits cause inflammation, oxidative reactions, and neurodegeneration in the brain cortex and hippocampus (Stanga et al. 2016).Aβ activates microglia and astrocytes, causing reactive oxygen generation and nitric oxide production resulting into neurons death (Vassar et al. 1999).Aβ 1-42 injection into brain gives rise to noticeable memory impairment (Delgado et al. 2005), and is accompanied by hippocampal neurodegenerative alterations (Ceccom et al. 2012).In fact, memory impairment may start from hippocampus which is involved in learning (Choi et al. 2011, Ting et al. 2007).Overall, oxidative reactions, inflammation, and neurodegeneration in the hippocampus and the cortex are considered to cause AD symptoms.
Accumulation of amyloids increases the distance between neurons and interferes with connections (Frozza et al. 2009), which could be related with our observa-  Besides direct damages in the brain, evaluation of blood and other tissues 365 days post AD induction demonstrated the occurrence of peripheral complications.Glucose, TG, insulin and OT1 levels of the AD group clearly shows a significant change in comparison with the control group.The liver enzymes ALP1 and PT1 have also significantly changed which is suggestive of other tissues degeneration alongside with brain damage.
Various conditions such as diabetes, mid-life obesity and vascular events are considered to increase the risk of AD occurrence (Fitzpatrick et al. 2009, Janson et al. 2004).Hence, factors such as elevated blood glucose levels or dyslipidemia may be considered as causative parameters in the disease.On the other hand, brain function damage may also lead to complications in other organs.The idea that AD may actually be a systemic disease has been discussed at length in a recent review, where the authors point out the "chicken or egg" problem when dealing with AD and the "whole body changes" that occur (Morris et al. 2014).Our results seem to be more in accordance with a causal effect of AD itself, which may somehow spread from the brain toward other organs.More exactly, what we see is a subsequent peripheral effect of amyloid plaques initially injected in the brain.
Further studies are needed to characterize the agents or pathways that are involved in this process.Based on previous studies, the role of oxidative stress is extremely important in AD (Tönnies and Trushina 2017).
In healthy condition, there is an equilibrium between active oxygen-nitrogen species and anti-oxygenation defense.When this equilibrated state is disturbed, oxidative stress occurs which impairs biological macromolecules such as extra-and intra-cellular proteins, and cell membrane phospholipids.Oxidative stress is involved in many pathologies such as cardiovascular diseases, dia- betes, AD, and Parkinson (Afshari et al. 2007, Duleu et al. 2010).Lipid peroxidation, which is caused by free radicals and changes the chemical characteristics, structure, and function of cell membranes plays also a role in neurodegenerative diseases (Kota et al. 2008).
Amyloid angiopathy may also occur in AD, resulting into brain blood circulation decrease (Hamel et al. 2004, Iadecola et al. 2004).Morphologic lesions related to beta amyloid accumulation in the brain vessels lead to decreased vasodilatation (Petrella et al. 2003).Damage to the brain vessels is followed by impairment of intracellular oxidative metabolism and consequent reduction in glucose, neurotransmitters, calcium and phosphate (Sultana et al. 2006).Ischemia and vascular obstruction result into a ten-fold increase of free radical production in the extracellular space of the hippocampal system and the basal ganglia (Magenta et al. 2013).The produced free radicals damage neuronal membrane and cause brain edema due to unsaturated fatty acids peroxidation in the membrane phospholipids; ultimately, neuronal death happens (Fujita et al. 2013).
In addition, a significant drop in brain glucose usage has also been reported in AD.This abnormality occurs due to dysfunction of insulin neuronal signals.Decreased insulin concentration and functional perturbation of insulin receptors were observed in postmortem examination of the brain and cerebrospinal fluid of AD patients (O'Connell et al. 2013).Even though glucose transportation into the brain is not insulin dependent, insulin passage through blood-brain barrier is linked to insulin receptors.Insulin receptors and insulin-like factors are present in neurons and astrocytes, and hippocampus and hypothalamus which play main roles in learning and memory present the highest insulin receptor density in the brain (Hoyer et al. 2000).In insulin-resistant individuals, the cells do not respond in the normal way to insulin and glucose cannot easily enter the cells.As a consequence, more and more insulin has to be secreted to assist glucose transportation into cells, resulting into insulinemia.Finally, when the body is no longer able to produce enough insulin to maintain normal glucose levels, diabetic state occurs (Baker et al. 2011).
In the present study, increased amounts of both glucose and insulin in the blood are indicative of insulin resistance in the brain cells.Glucose is the main energy source of the brain and its normal metabolism is necessary for accurate brain function and preserving cell energy as ATP molecules.A decrease in glucose levels leads to problems in cognitive functions and affects the process of amyloid precursor proteins production which results into higher accumulation of beta amyloid plaques (Rafacho et al. 2014).
Alongside with cholesterol, TGs constitute the plasma lipids.Elevated levels of blood TG, even in the absence of other risk factors, are directly linked with higher probability of cardiovascular pathologies, nephropathies and pancreatitis (Wenk et al. 1989).Increased blood TG is known to be one of main causes of metabolic syndrome.Other key pathologic factors include increased insulin level in the blood (Clearfield et al. 2014, Nordestgaard andVarbo 2014), hypertension, glucose tolerance disorder, abdominal obesity (Yamaoka et al. 2014) and increased blood lipids.Besides two main health problems, namely diabetes and cardiovascular diseases, metabolic syndrome has numerous other consequences such as liver steatosis, cirrhosis, chronic kidney failure, albuminuria, hyperuricemia and gout, polycystic ovary syndrome, sleep apnea, cognitive disorder and dementia (Dietrich et al. 2014, Parapid et al. 2014).
Considerable increases were observed in the liver enzymes of the animal model of AD.The most sensitive and commonly used diagnostic liver enzymes are aminotransferases AST, OT1, and ALT (PT1) which are released into blood circulation upon liver damage.Consequently, elevation of these enzymes is indicative of widespread death of hepatocytes (hepatic necrosis)which is also observed in many inflammatory diseases (Choi et al. 2007, Raurich et al. 2015).High levels of aminotransferases have been reported in 90% of ischemic or toxic hepatic damages (Giboney 2005).A study indicated that in 98% of alcoholic hepatitis patients, blood AST level increased 6-7 times compared to normal levels (Dufour et al. 2000).Moreover, patients with 10-fold increase in their enzyme levels have been diagnosed with acute extrahepatic bile duct obstruction (Karim et al. 2015).Increased ALT and AST levels have been observed in 79% of hepatic cirrhosis (Ferenci et al. 2003).
ALP1 is another enzyme which has increased in the AD model compared with healthy rats.It is a nonspecific enzyme with a widespread production in all tissues.High level of ALP1 is observed in kidney tubules and urologic disorders.Its levels rises in bone growth, malignancies, fracture repair, hepatic diseases, heart failure, over-activity of parathyroid gland and many other diseases (Lucyk et al. 2015).Studies have shown that high rates of ALP1 play an important role in urologic disorders and prostate malignancies (Kamali et al. 2010).In patients with prostate malignancy, high serum level of ALP1 is attributed to intensified bone metabolism which is indicative of metastasis to the bones (Lehmann et al. 2004).High ALP1 rate has been reported as a sensitive indicator of tubular damages in patients with inflammatory bowel disease (IBD) who suffer also from kidney failure (Krones et al. 2015).Moreover, studies have demonstrated that hepatic damage resulting from medicines consumption can emerge with a cholestatic pattern or pathologic increase in ALP1 (Ghaznavi et al. 2005).Further, pathologic ALP1 levels can also be related to metastatic lymphoma and liver cancers (Oorts et al. 2016, Pratt andKaplan 2000).
In conclusion, one year after Aβ injection in the CA1 regions of both hemispheres in Wistar rats, memory loss and Alzheimer's plaques formation were seen.Other progressive pathologic events and disorders occurred in parallel with increased learning and memory impairments in liver and kidney of test animals.Based on the current results, AD state may be the cause of these peripheral disorders, which would be of importance in AD patients' condition.

Fig. 1 .
Fig. 1.Comparing delay times to enter the dark compartment after 5 min in the test day in the initial stages of AD and during a year post AD induction.*** (P<0.001)* (P<0.05)(The Control group was evaluated after 42 days).

Fig. 2 .
Fig. 2. Hematology parameters of the animals, one year after AD induction.For statistical analysis, Anova method followed by Tukey's post hoc test was used.WBC: White blood cell, LYMPH: Lymphocyte, Neut: Percentage of neutrophils, RBC: red blood cell, HGB: Hemoglobin, HCT: Compact RBC volume, MCV: The average blood cell volume, MCH: Mean weight of hemoglobin in a red blood cell, MCHC: Mean concentration of hemoglobin in red blood cells, RDW-CV: The range of red blood cell size changes, P-LCR: The ratio of large platelets, PLT: Platelet, PDW: Scale of platelet volume, MPV: Average platelet volume.

Fig. 4 .
Fig. 4. Evaluating blood insulin of the AD animals, one year post AD induction.For statistical analysis, Independent Samples t-Test was used.

Fig. 5 .
Fig. 5. Comparing brain section of control group, AD animals following 42 days and 365 days post AD induction.Amyloid plaques are indicated by arrows.A and B: control groups.C, D and E: the animal brain tissue 42 days post AD induction and F, G and H, brain tissues 365 days post AD induction.Hippocampus area is shown with×40 magnification e.

Fig. 6 .
Fig. 6.Comparison of animals liver (1) and kidney (2) tissues with control group one year after Aβ injection.(A) optical microscopy; tip of the arrows indicates necrotic lesions in the tissue.(A-1) see monocular inflammatory cells, (A-2) see congested blood vessels.(B) Fluorescence microscopy, arrows tips indicate amyloid deposits in the tissue.