J Pathol Transl Med.  2017 May;51(3):292-305. 10.4132/jptm.2017.02.17.

An Experimental Infarct Targeting the Internal Capsule: Histopathological and Ultrastructural Changes

Affiliations
  • 1Department of Pathology, Chonnam National University Medical School and Research Institute of Medical Sciences, Gwangju, Korea. mclee@jnu.ac.kr
  • 2Department of Forensic Medicine, Chonnam National University Medical School and Research Institute of Medical Sciences, Gwangju, Korea.
  • 3Department of Medical System Engineering and School of Mechatronics, Gwangju Institute of Science and Technology, Gwangju, Korea.

Abstract

BACKGROUND
Stroke involving the cerebral white matter (WM) has increased in prevalence, but most experimental studies have focused on ischemic injury of the gray matter. This study was performed to investigate the WM in a unique rat model of photothrombotic infarct targeting the posterior limb of internal capsule (PLIC), focusing on the identification of the most vulnerable structure in WM by ischemic injury, subsequent glial reaction to the injury, and the fundamental histopathologic feature causing different neurologic outcomes.
METHODS
Light microscopy with immunohistochemical stains and electron microscopic examinations of the lesion were performed between 3 hours and 21 days post-ischemic injury.
RESULTS
Initial pathological change develops in myelinated axon, concomitantly with reactive change of astrocytes. The first pathology to present is nodular loosening to separate the myelin sheath with axonal wrinkling. Subsequent pathologies include rupture of the myelin sheath with extrusion of axonal organelles, progressive necrosis, oligodendrocyte degeneration and death, and reactive gliosis. Increase of glial fibrillary acidic protein (GFAP) immunoreactivity is an early event in the ischemic lesion. WM pathologies result in motor dysfunction. Motor function recovery after the infarct was correlated to the extent of PLIC injury proper rather than the infarct volume.
CONCLUSIONS
Pathologic changes indicate that the cerebral WM, independent of cortical neurons, is highly vulnerable to the effects of focal ischemia, among which myelin sheath is first damaged. Early increase of GFAP immunoreactivity indicates that astrocyte response initially begins with myelinated axonal injury, and supports the biologic role related to WM injury or plasticity. The reaction of astrocytes in the experimental model might be important for the study of pathogenesis and treatment of the WM stroke.

Keyword

Stroke; White matter; Models, animal; Pathology; Ultrastructure

MeSH Terms

Astrocytes
Axons
Coloring Agents
Extremities
Glial Fibrillary Acidic Protein
Gliosis
Gray Matter
Internal Capsule*
Ischemia
Microscopy
Models, Animal
Models, Theoretical
Myelin Sheath
Necrosis
Neurons
Oligodendroglia
Organelles
Pathology
Plastics
Prevalence
Recovery of Function
Rupture
Stroke
White Matter
Coloring Agents
Glial Fibrillary Acidic Protein
Plastics

Figure

  • Fig. 1. A 28-gauge needle with an optical fiber (A, inset) inserted stereotactically into the posterior limb of the internal capsule (B), 2.0 mm posterior, 3.1 mm lateral to midline, and 7.2 mm deep from bregma, to produce the infarction.

  • Fig. 2. A series of frozen sections with Nissl staining to measure infarct volume, marked with red dotted line, are present (A). Graphic presentation of the volumes reveals no significant difference between the moderate recovery group (MRG) and poor recovery group (PRG) (B).

  • Fig. 3. Light microscopy findings of stroke involving the poster limb of the internal capsule (PLIC). Coronal sectioning reveals a poorly defined focal lesion characterized by loss of hematoxylin and eosin (H&E) staining (A) and increased glial fibrillary acidic protein (GFAP) staining (B) at 3 hours post-ischemia. A well-demarcated, pale, whitish lesion is identified by low magnification microscopy via H&E and luxol fast blue–periodic acid-Schiff (LBPAS) staining (C, D), and GFAP and neurofilament protein immunostaining (E, F) at 12 hours to 1 day post-ischemia. The focal lesion shows central necrosis surrounded by macrophages (G) phagocytized with myelinated axon debris (H) by LBPAS staining, and GFAP-positive hypertrophic astrocytes along the lesion periphery (I) at 4 days post-ischemia. The necrotic center is gradually reduced in volume, and infiltrating macrophages decrease in number at 7 days post-ischemia. However, macrophages persist, and phagocytize periodic acid-Schiff–positive myelin debris as shown through LBPAS staining (J). At 14 to 21 days post-ischemia, the infarct lesion presents as a pseudocystic cavity consisting of reactive astrocytes, macrophages, and newly formed capillaries. The motor recovery at 14 to 21 days post-ischemia is related to the completeness of axonal injury in the PLIC. In the moderate recovery group, LBPAS staining in the PLIC revealed a pseudocystic lesion surrounded by GFAP-positive reactive astrocytes (K), and partially preserved myelinated axons passing in the PLIC periphery (L). In the poor recovery group, myelinated axons in the PLIC were completely disrupted as shown by LBPAS staining (M) and were replaced by a cystic cavity surrounded by GFAP-positive reactive astrocytes (N).

  • Fig. 4. Ultrastructural findings during the early stage of stroke involving the poster limb of internal capsule (PLIC). In the control group, a myelinated axon unit in PLIC had a compact lamellar sheath closely encompassing an axon (A, ×6,000). Each axon was filled with intermediate filament structures, called neurofilaments, and mitochondria. The lesion group presents early pathologic changes; nodular swelling and loosening of the myelin sheath, empty cystic change of submyelin space, polygonal wrinkling of axons, and accentuated cleft along the nuclear membrane of an oligodendrocyte (B, ×3,000) at 3 hours post-ischemia. Progressive loosening of the myelin sheath covers crinkled axons developing characteristic laminating whorled appearance (C, ×10,000) at 6 hours post-ischemia. Axonal swelling with a reactive change of the glial cells presented at 12 hours to 1 day post-ischemia (D-G). Swollen axons full of mitochondria, vesicles, and granules surrounded by acompact myelin sheath appeared. Fibrin thrombosis in capillary lumen (asterisk), a ruptured myelin sheath with extrusion of axonal organelles (arrows), and cystic vacuoles in the vicinity of swollen axons presented (D, ×12,000). Club-shaped processes of reactive astrocytes (arrows) intimately contacted swollen axons and organelles drifted from injured myelinated axons (E, ×6,000). Degenerative changes in an oligodendrocyte appear as an isolated cell with nuclear cytoplasmic dissociation by an irregular cleft, vesicles, and swollen mitochondria in the cytoplasm, and nuclear chromatin condensation along the nuclear membrane (F, ×6,000). Note the different stages of myelinated axon injury; from swollen axons filled with electrondense organelles surrounded by a loosely arranged, laminating myelin sheath to demyelinated ghost axons formed by cystic vacuoles containing sparsely granular or myeloid debris (G, ×6,000). A series of myelinated axonal injury, from loosening myelin sheath to macrophages infiltration phagocytized myelin and other cellular debris, was present at 4 days post-ischemia (H, ×6,000).


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