INTRODUCTION

Dementia results in a number of neurodegenerative diseases which consequence into the poor development of multiple cognitive domains and badly affects the task achievements necessary for living an independent life. It has been estimated recently, that globally there are 46 million people suffering from dementia. Additionally, it is also expected that each 10 million individuals add to this list and worldwide expanse of dementia will outrun 1 trillion dollars this year. Alzheimer Diseases (AD) is the most ordinary type of dementia which, in the common population, contribute to 3/4 of all the cases of dementia. As the stealthy pathological pathways of neurodegeneration have always been very difficult to achieve and the clinically used therapies based on neurotransmitter are not effective in the treatment of the disease, the expectations are that in the next 30 years the number of patients of the AD will reach to 106.8 million in the whole world. It suggests that there is urgent need to explore the pathology of disease causing.

The mechanisms involved in cognitive and motor behavioral deficiency during age yet to be studied however, it is quite obvious from the available literature that inflammation and oxidative stress are among the possible reasons. The long term exposure to the oxidative stress and inflammation are considered to be the factors involved in cognitive decline and performance of motor diagnosed in neurodegenerative diseases.  Oxidative stress is the major factor causing AD. As compared to other organs, brain is the most sensitive one for oxidative stress. In AD, due to enhanced levels of metals, ?-amyloid peptides, inflammation and mitochondrial dysfunction the neuronal components such as proteins, nucleic acids and lipids can be oxidized. Oxidative stress cause AD by developing deposition of ?-amyloid and tau hyperphosphorylation followed by synapses and neuronal loss. In short, it is suggested that oxidative stress is the most contributing agent responsible for the pathological process of AD. It makes the antioxidants very useful for the treatment of AD.

In family Rosacea, a genus of Rubus fruits such as raspberry and blackberry grow in Asia, Europe and North America. They contain significant amount of phenolic compounds due to which they are supposed to possess physiological properties. In cells, the reactive oxygen species are produced as a result of biochemical reactions and fruits extracts from raspberry and blackberry have antioxidant roles against those reactive oxygen species. Previously, it has been found that phenolic compounds have possible good effects including anti-inflammatory, chemo-protective and antioxidant activities. Due to these benefits, the naturally available polyphenolics in diets have gained enough attention as a candidate of therapeutic alternative for AD. Studies of epidemiology have shown that there is a relationship between the polyphenolic rich diets consumption and control of AD as well as other neurological diseases. The polyphenolics’ potential to cross the wall of mammals’ intestines and blood brain barrier enhances their biological properties. Additionally, the ability of the polyphenolics compounds to interact with proteins and other peptides is one of the major features of these compounds. This interaction can be very valuable and beneficial biologically, in general and in Alzheimer’s disease, especially.

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EXPERIMENTAL DESIGN

DIETS PREPARATION

The fruits are first freeze-dried and then added to the control diet in the powder form. The amount of components in the control diet is adjusted according to the fruit powder. For powder preparation of raspberry, it is first homogenized with deionized water.

ANIMAL MODELS

There are many genetic models of mice available that can be chosen for studying AD or otherwise they can be made models of AD by injecting lipopolysaccharide into their peritoneal cavity. On the bases of previous studies, there is a proof that exposure of human primary brain cells to the lipopolysaccharide of Bacteroides fragilis is very powerful in induction of the NF-kB, a pro-inflammatory transcription factor (p50/p65). This is the complex known for the initiation of the pathogenic pathways expression which is involved in inflammation of neurodegeneration.

OPEN FIELD TEST

It is done for the recording and quantitatively assessment of the movement and exploratory behavior of mice inside open field arena. The open field test involves an arena surrounded by walls. A lamp illuminates the arena. As animal explores the arena, the camera fixed above the arena and the computerized tracking system records its behavior. The linearity and path length is determined along with the number of times the rearing event is done. The comparison between exploration of the arena’s center and the periphery by the animal is also made.

OBJECT RECOGNITION TEST

Object recognition test is done to evaluate the recognition memory of mice. The object recognition test examines the neural process integrity needed for the recall and storage of previously found ‘known object’ encouraging investigation of ‘unknown object’ without the involvement of negative reinforcement. For the acquisition stage of the object recognition test mouse is placed into the open field arena. There are two random objects present inside the open field arena. The objects are secured to the floor of the arena. The investigation of either object is recorded and it is defined as the number of times an animal positioned its snout from the object.  The test stage of the object recognition test consists of an individual exploration of the field by mouse. The field contains one object formerly happened upon by the mouse during the acquisition stage of the test and one ‘new’ object that the animal face for the first time. Murine investigation of the two objects is recorded in the same way as in the acquisition stage. The recognition index is determined for the mouse in both the acquisition stage as well as test stage of the task. The recognition index is defined as the amount of the time spent by the mouse investigating wither object (tA or tB) divided by the amount of time spent for investigating both the objects (tA or tB/tA+tB) × 100.

DETERMINATION OF THIOFLAVIN-S-POSITIVE AMYLOID PLAQUES

In mice, within the dentate gyrus, cortex and sub-regions of hippocampus, number of plaques of thiofleavin-S-positive are counted. From the cranial vault the brain of the animal is excised by dissecting carefully. Average number of the thioflavin-S positive plaques is determined within the brain.

MICROVASCULAR COROSION CASTING

Scanning Electron Microscopy is the best way to study the three-dimensional structure of the vascular network. Resin, a solidifying material is used for the casting of vascular corrosion. Blood is drained from the vessels for prevention of the blockage due to blood clotting. For this, blood vessels are perfused using a phosphate buffer saline or any other physiological buffer. After, the resin is filled in the blood vessels of interest. The resin is let for solidification inside the vessels network which result in the vessels filled with solid plastic material. Potassium hydroxide, a corrosive chemical, is used for dissolution of surrounding tissue in such a way that corrosion dissolve only tissue without harming resin. At the end three-dimensional network of blood vessels is obtained.

SCANNING ELECTRON MICROSCOPIC EVALUATION                      

            The solution of potassium hydroxide is used for soaking the casted brain. Until removal of all of the tissue around the vasculature has been dissolved completely, the soaking solution is renewed every day. The dd.H2O is used for washing of resin-casted sample. The washed specimen is the placed inside a dust-proof container, interiorly lined with tissue paper or filter paper. The specimens are air-dried and protected by aluminum stub. The samples are coated homogeneously with a fine layer of gold and palladium for the examination of scanning electron microscopy.

QUANTITAVIE ANALYSIS OF MICROVASCULAR ULTRASTRUCTURE

Within the brain specimens, the cortical vasculature areas are imaged using a scanning electron microscope. The images obtained are processed in ImageJ software for calibration. The diameter and lengths of the cortical capillaries are measured using the software.

DETERMINING THE UACR IN COLLECTED URINE

Urine samples are collected from mice and is stored in the sterilized collection vessels. Within each urine sample of mice, the albumin concentration is determined through albumin ELISA technique. The concentration of creatinine is also determined with the help of enzymatic assay.

Statistical analysis

Graphpad Prism software is used for the comparison of means independent values. Kolmogorov-Smirnov assessment method is used for the normality of the distribution. Data is presented in the form of graphs and the Graphpad Prism software is used for the generation of the graphs.

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