Objective

Determine whether early changes in hippocampal capillaries occur before Aβ plaque buildup and memory decline in Alzheimer’s disease, and assess whether protecting these capillaries with Ferulic Acid can prevent or slow early Alzheimer’s progression.

Research Gap

• Current Alzheimer’s treatments focus on removing Aβ plaques.
• However, these treatments do not stop memory decline, suggesting plaques may not be the earliest cause.
• The hippocampus, critical for memory, may first experience reduced blood flow due to:
  • Capillary narrowing, or
  • Loss of capillary density.
• This impaired microcirculation could then trigger Aβ accumulation, accelerating disease progression.

Methodology

Animals Used in the Study

• APP/PS1 Transgenic Mice:
  These mice carry two human genes (APPswe and PSEN1dE9) that lead to the formation of Aβ plaques, making them a widely used animal model for Alzheimer’s disease (AD).

• Wild-Type Littermate Controls:
  Genetically normal mice born from the same breeding pairs were used as controls to compare against the APP/PS1 group.

• C57BL/6 (C57) Mice:
  These mice were also included for additional comparison or experimental purposes.

Animal Housing and Care Conditions

• Group-housed (4–5 mice per cage)
• Food and water provided ad libitum (free access)
• 12-hour light / 12-hour dark cycle
• Temperature-controlled environment to ensure consistent living conditions

Ferulic Acid (FA) Treatment Protocol

• Subjects:
  6-month-old APP/PS1 Alzheimer’s model mice received Ferulic Acid (FA) treatment for 30 days.

• About Ferulic Acid:
  FA is a natural antioxidant with anti-inflammatory effects, making it a candidate for protecting brain microvascular health.

Administration Method

• FA was added to the drinking water (not injected).
• Mice remained in normal home cage conditions (4–5 mice per cage).
• Each mouse could drink freely (ad libitum).

Pre-Treatment Measurements

Before starting FA treatment, researchers:

1. Weighed each mouse, and
2. Measured water intake per cage over 3 days

Result: Each mouse drank ~3.5–5 mL water/day.

This allowed calculation of FA concentration so that each mouse received:

• ≈ 20 mg FA per kg of body weight per day

Maintaining Accurate Dosage

• Water bottles were replaced twice weekly with freshly prepared FA solution.
• Water intake was rechecked regularly to adjust the FA concentration if needed.

Control Groups

• Both APP/PS1 mice and wild-type (WT) mice in control groups received plain drinking water (no FA).
• This ensured any observed effects were due to FA treatment, not other factors.

Morris Water Maze Test

The Morris Water Maze is used to assess spatial learning and memory in mice.

Apparatus

• Circular tank: 120 cm in diameter, filled with water at 21–22°C.
• The water is made opaque using plastic particles to hide a submerged platform.
• Platform: 10 cm in diameter, positioned just below the water surface.

Testing Procedure

1. Spatial Learning (Training Phase) — 5 Days

Each mouse learns to find the hidden platform.

• Mouse is placed into the tank facing the wall.
• Four different starting points are used.
• 4 trials per day with 10-minute rests between trials.
• 60 seconds allowed to locate the platform.

Outcomes:

• If the mouse finds the platform, it remains there for 10 seconds.
• If it fails, it is guided to the platform and stays on it for 15 seconds.

Learning Measure:

• Average escape latency (time taken to find the platform).

2. Probe Trial (Memory Test) — 24 Hours Later

• The platform is removed.
• Mouse is released from the opposite side of the previous platform location.
• It swims freely for 60 seconds.

Memory Measure:

• Time spent in the target quadrant where the platform used to be.
• More time in the target quadrant = Better memory.

Immunohistochemistry (IHC)

Immunohistochemistry is used to visualize specific cells, proteins, or structures in the brain by using antibodies that bind to target molecules. These antibodies are then detected with fluorescent dyes, allowing researchers to see where and how much of the molecule is present under a microscope.

1. Preparing the Brain Tissue

1. Mice were anesthetized to ensure no pain.
2. The circulatory system was flushed with a salt solution (PBS) to remove blood.
3. PFA (paraformaldehyde) was perfused to fix and preserve the brain tissue.
4. Brains were removed and placed into a sucrose solution to prevent structural damage.
5. The brain was sectioned into thin slices using a cryostat or microtome.

2. Staining the Brain Slices

• Slices were washed and placed in a blocking solution to reduce non-specific binding.
• Slices were incubated overnight with primary antibodies targeting:
  • Aβ (amyloid plaques)
  • Iba1 (microglia)
  • GFAP (astrocytes)
  • Collagen IV (blood vessels)
• Then, fluorescent secondary antibodies were applied so the labeled structures glow under the microscope.

3. Imaging the Samples

• Slices were stained with DAPI, which labels cell nuclei (blue).
• Slices were mounted on glass slides.
• A confocal microscope was used to obtain high-resolution fluorescent images.

4. Quantification and Analysis

• Fluorescence intensity was measured to determine protein levels.
• Capillary diameter was measured using ImageJ software by drawing lines across blood vessel cross-sections.
• Multiple slices and many vessels per mouse were analyzed to ensure accuracy and reliability.

Thioflavin S Staining (for Amyloid Plaques)

Thioflavin S (ThS) is a fluorescent dye that specifically binds to amyloid plaques.
When viewed under a microscope, ThS-stained plaques glow bright green, making them easy to identify and quantify.

Procedure

1. Washing the Brain Slices

• Brain tissue sections were washed three times to remove any remaining solutions.

2. Staining with Thioflavin S

• Slices were placed in Thioflavin S solution (0.05 mg/mL).
• Staining was performed in the dark for 10 minutes to prevent dye degradation.

3. Rinsing

• Slices were washed with 50% ethanol to remove excess dye.
• Then washed again with PBS to clean and stabilize the tissue.

4. Mounting and Imaging

• Slices were mounted onto microscope slides.
• Imaging was performed using a confocal microscope, providing clear, high-resolution fluorescent images of Aβ plaque deposits.

Surgery and ET-1 Injection (Hippocampal Hypoperfusion Model)

To model reduced blood flow (hypoperfusion) in the hippocampus, the researchers injected Endothelin-1 (ET-1) directly into the CA1 region.
ET-1 is a peptide that constricts blood vessels, thereby decreasing local blood flow. This allows researchers to study how reduced circulation influences Alzheimer’s disease progression.

Step-by-Step Procedure

1. Anesthesia and Preparation

• Mice were deeply anesthetized with pentobarbital sodium (80 mg/kg, intraperitoneal).
• Body temperature was maintained at 37 ± 0.5°C using a heating pad.
• The scalp was shaved and disinfected with:
  • Povidone-iodine
  • 70% ethanol

2. Positioning and Injection

• The mouse’s head was secured in a stereotaxic apparatus for precise targeting.
• A glass micropipette was guided into the CA1 region of the hippocampus using the following coordinates:
  • AP: −2.0 mm
  • ML: ±1.5 mm
  • DV: −1.7 mm
• Injection details:
  • Solution: ET-1 or vehicle (control)
  • Volume: 1 μL
  • Concentration: 1 μg/μL
  • Injection rate: 0.1 μL/min (slow to prevent tissue damage)

3. Recovery

• After injection, mice were placed in a quiet recovery area.
• They were allowed to heal and stabilize before further testing.

Lectin Perfusion (Blood Vessel Labeling)

Lectin perfusion is used to label brain blood vessels.
Tomato lectin binds specifically to endothelial cells, which line blood vessels.
When linked to a fluorescent dye, the vessels glow under a microscope, making it possible to study their structure.

Procedure

1. Anesthesia

• Mice were deeply anesthetized with pentobarbital sodium (80 mg/kg, intraperitoneal).

2. Injection of Fluorescent Lectin

• A fluorescent tomato lectin solution (0.5 mg/mL, 100 μL) was injected directly into the heart (intracardiac injection).
• The lectin circulated through the bloodstream and bound to endothelial cells throughout the brain’s vasculature.

3. Perfusion and Brain Collection

• The mice were perfused to:
  • Flush out blood, and
  • Fix the tissues for preservation.
• The brains were carefully removed and sectioned into thin slices.

4. Imaging and Analysis

• The fluorescently labeled vessels were visualized using a fluorescence or confocal microscope.
• Researchers measured:
  • Vessel density
  • Capillary structure
  • Vessel diameter

Ferulic Acid–Biotin Injection and Immunostaining

This experiment investigated whether Ferulic Acid (FA) can bind to the ETRA receptor in the hippocampus.
To visualize FA under the microscope, the researchers used FA chemically linked to biotin (FA-biotin).
Biotin can be detected with fluorescent probes, allowing co-localization of FA and ETRA.

1. Injection into the Hippocampus

• FA-biotin: 1 μL at 10 μg/μL
• Control: Biotin-only (1 μL at 5 μg/μL)
• Injection rate: 0.1 μL/min (slow to prevent tissue damage)
• After injection, mice were allowed 40 minutes for FA-biotin to diffuse through the tissue.

2. Brain Collection and Sectioning

• Mice were euthanized.
• Brains were collected, fixed, and cut into thin slices for immunostaining (same procedure as earlier).

3. Immunostaining Procedure

• Brain slices were incubated overnight at 4°C with a primary antibody against ETRA (1:800).
• After washing, slices were incubated with:
  • Secondary antibody (1:1000) → to visualize ETRA
  • Streptavidin–Alexa Fluor 488 (1:1000) → to visualize FA-biotin

Why both signals are needed:

• Secondary antibody highlights ETRA receptors.
• Streptavidin-Alexa 488 binds to biotin, revealing the location of FA.

This allowed the researchers to determine whether FA co-localizes with ETRA in the hippocampus.

4. Final Imaging Steps

• Slices were stained with DAPI to label cell nuclei (blue).
• Slices were mounted on slides and coverslipped.
• Confocal microscopes (e.g., Nikon A1 or Olympus FV3000) were used to capture high-resolution images.

ELISA and β-Secretase Activity Assay

Sample Preparation

• Hippocampal tissue was homogenized in ice-cold RIPA lysis buffer (Beyotime Biotech).
• The buffer contained:
  • PMSF
  • Protease inhibitor
  • Phosphatase inhibitor

These additives prevent protein degradation during processing.

Aβ Quantification (ELISA)

• Levels of Aβ₁–₄₀ and Aβ₁–₄₂ were measured from the hippocampal tissue lysates.
• Measurements were performed using ELISA kits, following the manufacturer’s protocols:
  • Aβ₁–₄₂: R&D Systems (Cat. No. DAB142)
  • Aβ₁–₄₀: R&D Systems (Cat. No. DAB140B)

β-Secretase (BACE1) Activity Assay

• BACE1 enzymatic activity was assessed using fresh hippocampal extracts.
• Conducted with the β-Secretase Activity Fluorometric Assay Kit:
  • BioVision (Cat. No. K360-100)
• The assay was performed according to the manufacturer’s instructions.

Western Blot Analysis

Western blotting is used to measure how much of a specific protein is present in tissue samples.
In this study, proteins from the hippocampus were analyzed to compare levels between:

• Normal (WT) mice
• Alzheimer’s model (APP/PS1) mice
• FA-treated APP/PS1 mice

Step-by-Step Procedure

1. Protein Extraction

• Hippocampal tissue was collected and homogenized in RIPA lysis buffer, which breaks open cells.
• Protease and phosphatase inhibitors were added to prevent protein degradation.
• Samples were centrifuged to remove debris.
• The clear supernatant contained the proteins used for analysis.

2. Sample Preparation

• Protein samples were mixed with loading buffer and heated to:
  • Unfold the proteins
  • Give them a negative charge

This ensures proteins separate by size during electrophoresis.

3. Gel Electrophoresis (SDS-PAGE)

• Samples were loaded into a polyacrylamide gel.
• An electric current pushed proteins through the gel:
  • Smaller proteins move faster
  • Larger proteins move slower
• This separates proteins by size.

4. Protein Transfer

• Separated proteins were transferred to a membrane (similar to “printing” the protein pattern onto paper).

5. Blocking

• The membrane was incubated in a blocking buffer to prevent non-specific binding.

6. Antibody Labeling

1. Primary antibody was added:
   • Binds specifically to the protein of interest (e.g., APP).
2. Secondary antibody was added:
   • Binds to the primary antibody
   • Contains HRP, an enzyme that produces a chemiluminescent signal.

7. Detection and Quantification

• The membrane was developed, and bands representing the protein appeared.
• Images were captured using a chemiluminescence imaging system.
• ImageJ software was used to measure band intensity:
  • Brighter band = higher protein level

Purpose

This method allows researchers to determine whether Ferulic Acid treatment changes protein expression, such as reducing APP levels or other Alzheimer’s-related proteins, which may reflect therapeutic effects.

Transmission Electron Microscopy (TEM)

What is TEM?
Transmission Electron Microscopy uses electrons instead of light to see extremely small structures—much smaller than what a normal microscope can show.
In this study, TEM was used to observe tiny blood vessels (capillaries) in the hippocampus.

They compared capillary structure in:

• Wild-type (WT) mice — normal controls

• APP/PS1 mice (AD model) — Alzheimer’s disease model

This helps determine whether AD affects small blood vessels in the brain.

Procedure Overview

1. Tissue Collection

   • Small hippocampal samples were taken from WT and AD mice.

2. Fixation
   To preserve cell structure:

   • Glutaraldehyde
   • Paraformaldehyde

3. Staining (Contrast Enhancement)
   Heavy-metal stains help structures appear darker under TEM:

   • Potassium ferrocyanide
   • Osmium tetroxide
   • Uranyl acetate
   • Lead nitrate

4. Dehydration

   • Tissue water was removed using ethanol and acetone.

5. Embedding

   • Tissue was embedded in resin, forming a hard block.

6. Ultrathin Sectioning

   • Slices were cut ~70 nm thick using an ultramicrotome.

7. Imaging

   • Sections were examined using the TEM, which sends electrons through the sample to produce high-resolution images.

Purpose / Why This Matters

TEM allows researchers to:

• Examine capillary diameter, wall thickness, and structural integrity.
• Identify microvascular changes associated with Alzheimer’s disease.

This is important because:

Changes in tiny blood vessels may occur early in Alzheimer’s — possibly before memory loss and plaque buildup.

Laser Speckle Contrast Imaging (LSCI)

Purpose:
LSCI was used to measure cerebral blood flow (CBF) in live mice.
A laser is shined onto the brain surface, and a special camera detects how quickly blood cells are moving:

• Fast blood flow → brighter signal
• Slow blood flow → darker signal

This allows real-time visualization and comparison of blood flow between groups.

Procedure

1. Anesthesia

   • Mice were anesthetized to prevent movement.

2. Positioning

   • The mouse was placed securely in a stereotaxic holder.

3. Skull Exposure

   • The scalp was carefully removed to expose the intact skull surface.

4. Imaging

   • A laser speckle imaging camera was aimed at the skull.
   • Blood flow was recorded in the same brain region for all animals to ensure consistency.

What Was Measured

Researchers compared:

• Wild-type (WT) mice vs APP/PS1 (AD) mice
• Blood flow before and after Ferulic Acid (FA) treatment
• Effects of Endothelin-1 (ET1) — a vasoconstrictor (narrows blood vessels)

Jugular Vein (Peripheral Vessel) Test

This tested whether FA could reverse ET1-induced vasoconstriction in a major vessel.

1. The jugular vein was surgically exposed.
2. Baseline blood flow was recorded using saline.
3. ET1 was applied → blood flow decreased (vessel constriction).
4. The vein was then treated with either:
   • Saline (control), or
   • Ferulic Acid (FA)

If FA increased blood flow, this indicates FA can relax blood vessels and improve circulation.

Why This Matters

This experiment helps determine:

• Whether reduced cerebral blood flow is an early feature of Alzheimer’s disease.
• Whether ET1 contributes to microvascular narrowing.
• Whether Ferulic Acid can restore blood flow and potentially improve brain function.

Key Insight:
Improving blood flow may be one way Ferulic Acid helps protect the brain in Alzheimer’s disease.

Ischemic Insult and TTC Staining

This part of the experiment is about causing a small stroke in a specific part of the mouse’s brain (the hippocampus) on purpose. A stroke happens when blood flow is blocked, and brain cells don’t get enough oxygen.

They do this so they can test whether Ferulic Acid (FA) can protect the brain from this kind of damage.

Step-by-Step (Simple Version)

1. Rose Bengal Injection
   The mouse is given a dye called rose bengal.
   This dye makes the brain cells react to blue light.

2. Anesthesia
   The mouse is put to sleep so it feels no pain.

3. Creating a Mini-Stroke (Ischemic Insult)
   They shine blue light onto a specific spot in the hippocampus.
   The rose bengal + blue light clogs small blood vessels → blood flow stops → a tiny stroke happens.

4. Give Ferulic Acid (FA)
   After the stroke, they give FA to see:

   • Does FA reduce the damage?
   • Does FA help brain cells survive?

5. TTC Staining (To See the Damage)
   24 hours later, the brain is sliced and dipped into a chemical called TTC.

How TTC Staining Works

• Healthy brain tissue turns red
• Dead or damaged tissue (from the stroke) stays white

So scientists can:

• Measure how big the damaged area is
• Compare with and without FA treatment

Why They Did This

They want to test if Ferulic Acid helps protect the brain by:
✅ Reducing damage caused by low blood flow
✅ Possibly keeping blood vessels more open
✅ Preventing cell death

This supports the idea that FA may help treat early Alzheimer’s, which also involves blood flow problems.

Time-of-Flight Magnetic Resonance Angiography (TOF-MRA)

Purpose:
TOF-MRA was used to visualize and quantify blood vessels in the brain without using contrast dyes.
The method takes advantage of flowing blood:

• Flowing blood appears bright
• Stationary tissue appears darker

This allows researchers to map the cerebral vasculature in vivo.

Procedure

1. Anesthesia

   • Mice were anesthetized using isoflurane to prevent movement during scanning.

2. Positioning

   • Each mouse was placed in the MRI scanner.
   • The head was positioned inside a dedicated small animal head coil to enhance signal quality.
   • Body temperature and vital signs were continuously monitored throughout imaging.

3. TOF-MRA Scanning

   • No contrast agent was required.
   • Imaging parameters (e.g., TR, TE, flip angle, field of view, slice thickness) were optimized to highlight arterial blood flow.
   • Images were acquired to capture 3D vascular networks in the brain.

Image Processing and Vessel Quantification

After image acquisition:

1. Iterative Threshold Segmentation Algorithm

   • Used to distinguish blood vessels (bright) from background tissue (darker).

   • Simplified algorithm steps:

     1. Choose an initial intensity threshold.
     2. Divide pixels into two groups: vessel vs. background.
     3. Compute the average intensity of each group.
     4. Update the threshold based on these averages.
     5. Repeat until the threshold stabilizes.

   • The final threshold isolates the blood vessels.

2. Vascular Volume Measurement

   • The segmented vascular regions were quantified to calculate total cerebral blood vessel volume.

Purpose in the Study

TOF-MRA allowed researchers to:

• Visualize cerebral vascular architecture in live mice.
• Compare vascular volume between:
  • Normal (WT) mice
  • Alzheimer’s model (AD) mice
  • AD mice treated with Ferulic Acid (FA)

Goal: Determine whether FA improves or protects cerebral blood supply in Alzheimer’s disease.

Molecular Docking of Ferulic Acid (FA) with Endothelin Receptor A (ETRA)

Purpose:
This analysis tested whether Ferulic Acid (FA) can directly bind to Endothelin Receptor A (ETRA)—a receptor that causes vasoconstriction and contributes to reduced cerebral blood flow in Alzheimer’s disease and ischemic injury.
If FA binds to ETRA, it may help prevent or reduce excessive vessel constriction.

Procedure

1. Protein Structure Modeling

• The 3D structure of ETRA was not available in the Protein Data Bank (PDB).
• Researchers obtained the ETRA amino acid sequence from NCBI (Reference: NP_001948.1).
• Homology modeling was performed using SWISS-MODEL.
• The closest structural template identified was 5GLI, which was used to construct a predicted ETRA receptor model.

2. Ferulic Acid Structure

• The molecular structure of FA was downloaded from PubChem (CID: 445858).
• The SMILES format was used to generate a 3D ligand structure.

3. Docking Simulation

• Docking was performed using AutoDock Vina.
• FA was computationally placed into the predicted binding pocket of ETRA.
• The software calculated:
  • Binding affinity (ΔG)
  • Best fitting pose
  • Likely molecular interactions (e.g., hydrogen bonds, hydrophobic contacts)

4. Visualization and Interaction Analysis

• Docking results were imported into Discovery Studio 3.1.
• Researchers examined:
  • Which amino acid residues interacted with FA
  • The orientation and stability of binding
  • Potential inhibition mechanism

Interpretation / Why This Matters

This docking study helps determine whether FA could regulate blood flow by interacting with ETRA.

If FA binds to ETRA, it may:

• Reduce vasoconstriction
• Improve cerebral blood flow
• Protect against vascular damage seen in Alzheimer’s and ischemic injury

Thus, the docking experiment supports the mechanistic explanation behind the physiological improvements observed in FA-treated mice.

RNA Sequencing (RNA-seq)

Purpose:
To determine how ferulic acid (FA) treatment alters gene expression in the brains of Alzheimer’s disease (AD) mice.
Two brain regions were analyzed:

• Hippocampus
• Cortex

These regions are highly affected in AD pathology.

Workflow Overview

1. Tissue Collection & RNA Extraction

• Hippocampus and cortex tissues were dissected.
• Samples were preserved in RNAlater and stored at −80°C.
• Total RNA was extracted using TRIzol reagent.

2. Library Preparation & Sequencing

• RNA libraries were constructed using the NEB Ultra RNA Library Prep Kit.
• Sequencing was performed on Illumina HiSeq 4000:
  • 150 bp paired-end reads

3. Data Preprocessing and Alignment

4. Differential Expression Analysis

Differential gene expression was performed in R using limma (empirical Bayes).

Cutoff criteria:

• Fold change ≥ 1.5
• p-value ≤ 0.05

Key comparisons:

1. AD vs WT → Genes altered in disease.
2. AD-FA vs AD → Genes rescued by FA.
3. AD-FA vs WT → Degree of recovery toward normal state.

Visualization:

• Heatmaps generated using pheatmap
• Clustering performed with ward.D2 linkage.

5. Functional Interpretation

Focus was placed on biological pathways relevant to AD:

Key Purpose / Interpretation

This RNA-seq analysis assessed whether FA treatment can reverse disease-associated gene expression abnormalities, particularly those involving:

• Neuroinflammation
• Cerebrovascular dysfunction
• Synaptic impairment

If FA shifts AD gene expression patterns toward WT levels, it supports FA as a neuroprotective and vasoprotective therapeutic candidate.