Ischemic Stroke & Ultrastructural Analysis

Photothrombotic Stroke Model

Focal unilateral stroke was induced in the somatosensory cortex (SSC) corresponding to the forelimb representation in adult mice (3 and 18 months, n = 5). Rose Bengal (110 mg/kg, IP) was administered as a photosensitizing agent, followed by targeted illumination with a green laser (532 nm, 17 mW, 15 min) through a thinned skull window. No craniotomy was needed — the skull was thinned to ~50% thickness with a dental drill under stereotaxic guidance.

Following 72 hours of recovery, mice were deeply anesthetized and prepared for transcardial perfusion fixation to preserve ultrastructure for both light and electron microscopy.

Perfusion, Fixation & Embedding

Transcardial perfusion with heparinized PBS (5 min) followed by 2% glutaraldehyde + 2% paraformaldehyde in 0.15M cacodylate buffer. Post-fixation in 1% OsO₄-potassium ferrocyanide (2 h), en bloc uranyl acetate staining (2 h), ascending ethanol dehydration (50–100%), and infiltration with Spurr's resin. Tissue embedded in BEEM capsules at 60 °C for 48 h.

Coronal sections (200 µm) cut on a Leica VT1000S vibratome. Under stereomicroscope, 1 mm² tissue blocks microdissected from the peri-infarct zone and contralateral control hemisphere.

Toluidine Blue Light Microscopy

Semithin sections (~0.5 µm) were cut with glass knives on a Reichert-Jung UltraCut E ultramicrotome, stained with toluidine blue, and imaged on an Olympus BX51 light microscope with a DP73 camera at 4800 × 3600 px resolution. These sections served as orientation for subsequent ultrathin sectioning and quality control.

Cortical Layer Architecture

Toluidine blue staining reveals the full laminar architecture of the adult mouse somatosensory cortex (layers 1–6). Capillaries (Cap), pyramidal neurons (Pyr), and glial cells (GC) are readily identified in the control hemisphere.

Ischemic Tissue Zones

The photothrombotic lesion creates three distinct zones visible at low magnification: the intact zone (normal cytoarchitecture), the peri-infarct zone (PI — compromised but recoverable tissue), and the necrotic core (complete tissue destruction with pyknotic nuclei).

Peri-Infarct Zone Detail

Higher magnification of the ischemic border region reveals the gradual transition from intact tissue to necrotic core. Within the peri-infarct zone, there is a progressive loss of recognizable capillaries, decreased concentration of pyramidal neurons, and compromised blood-brain barrier morphology evidenced by perivascular vacuoles.

Transmission Electron Microscopy

Ultrathin sections (70–80 nm) were cut with a Diatome diamond knife on the UltraCut E, collected on 200-hex copper mesh grids, and imaged at 80 kV on a JEOL JEM-1400 TEM equipped with a Gatan Orius SC1000 CCD camera (4008 × 2672 px). Key subcellular structures were pseudocolored in Adobe Photoshop to enhance visualization and enable morphometric quantification in ImageJ.

Pseudocolored Ultrastructure Overview

Low-magnification pseudocolored EM panorama showing the neuropil of the contralateral (control) SSC. Pyramidal neurons (blue) display characteristic round morphology with prominent nucleoli, surrounded by astrocytic processes (yellow) and mitochondria (red). Synaptic contacts with pre- and post-synaptic elements visible throughout the neuropil.

Synaptic Classification

Synapses were classified and pseudocolored for morphometric analysis. Presynaptic terminals (green) contain synaptic vesicles and interface with postsynaptic structures (blue). The dendrite shown here features multiple synaptic contacts including both excitatory (round vesicles, large PSD) and inhibitory (flattened vesicles) types.

Control Pyramidal Neurons

In the contralateral (uninjured) hemisphere, pyramidal neurons display round, electron-lucent morphology with intact nuclear envelopes, prominent nucleoli, and well-organized cytoplasmic organelles. Astrocytes (wavy arrows) occupy relatively limited space in the surrounding neuropil.

Peri-Infarct Neuronal Degeneration

Progressive neuronal degeneration is observed with increasing proximity to the infarct core. Distal PI neurons show early cytoplasmic darkening with partial organellar preservation and microglia encroachment. Proximal PI neurons display advanced degeneration — swollen mitochondria, disrupted endoplasmic reticulum, and vacuolization of the surrounding neuropil.

Necrotic Core & Infarct Zone

Within the necrotic core, neurons show complete loss of organellar integrity — barren dendritic processes, no identifiable synapses, and pyknotic glial cell nuclei. This zone is characterized by widespread degenerating neuropil tissue and the complete absence of functional synaptic structures.

Astrocytic Inflammation & Mitochondrial Changes

Astrocytes responded dramatically to ischemic injury, showing increased process area and reactive morphology in the peri-infarct zone. Astrocytic processes were traced and pseudocolored yellow for quantification, revealing glycogen granule accumulation, swollen mitochondria, and extensive perivascular endfeet coverage around capillaries.

Reactive Astrocytosis

Pre- & Post-Synaptic Structures

Morphometric Quantification

Over 600 synapses were identified and counted across 50 test fields (6.58 × 4.39 µm each) at 30,000× magnification. Subcellular structures were traced in Adobe Photoshop and quantified via thresholding in ImageJ. Contralateral hemisphere served as paired control for each animal (n = 5).