Cellular alterations in human traumatic brain injury: changes in mitochondrial morphology reflect regional levels of injury severity

Abstract

Mitochondrial dysfunction may be central to the pathophysiology of traumatic brain injury (TBI) and often can be recognized cytologically by changes in mitochondrial ultrastructure. This study is the first to broadly characterize and quantify mitochondrial morphologic alterations in surgically resected human TBI tissues from three contiguous cortical injury zones. These zones were designated as injury center (Near), periphery (Far), and Penumbra. Tissues from 22 patients with TBI with varying degrees of damage and time intervals from TBI to surgical tissue collection within the first week post-injury were rapidly fixed in the surgical suite and processed for electron microscopy. A large number of mitochondrial structural patterns were identified and divided into four survival categories: normal, normal reactive, reactive degenerating, and end-stage degenerating profiles. A tissue sample acquired at 38 hours post-injury was selected for detailed mitochondrial quantification, because it best exhibited the wide variation in cellular and mitochondrial changes consistently noted in all the other cases. The distribution of mitochondrial morphologic phenotypes varied significantly between the three injury zones and when compared with control cortical tissue obtained from an epilepsy lobectomy. This study is unique in its comparative quantification of the mitochondrial ultrastructural alterations at progressive distances from the center of injury in surviving TBI patients and in relation to control human cortex. These quantitative observations may be useful in guiding the translation of mitochondrial-based neuroprotective interventions to clinical implementation.

FIG. 1.

Human brain mitochondrial ultrastructural patterns. (A–C) Normal survival category (A, Case #12, Penumbra zone; B, Case #1, Far zone; C, Case #1, Penumbra zone); (D–G) Normal reactive category (D, Case #12, Far zone; E, Case #13, Far zone; F, Case#11, Far zone; G, Case #1, Penumbra zone); (H–M) Reactive/degenerating category (H, Case #13, Far zone; I, Case #12, Far zone; J, Case #16, Far zone; K, Case #14, Penumbra zone; L, Case #17, Far zone; M, Case #2, Near zone; arrow shows mitochondria-derived vesicles). Scale bar equals 500 nm.

FIG. 2.

End-stage degenerating mitochondrial profiles. (A) Case #12, Far zone. High amplitude swollen mitochondrion with electron-lucent matrix. (B) Case# 13, Far zone. Swollen collapsing mitochondrion. (C) Case #10, Near zone. Swollen mitochondrion with matrical dense inclusions. (D) Case #22, Near zone. Mitochondrion with dark diffusely granular matrix. (E) Case #7, Near zone. Spherical mitochondrion with segmental area loss of cristate structure and matrical densities is enveloped by double membranous authophagic vacuole (mitophagy). Scale bar equals 500 nm.

FIG. 3.

Accumulation of mitochondria in bulbous enlargements of neuronal processes present in injured cortex, Far zone. (A) Case #12, Far zone. Disorientation of microtubules and mitochondria are predominantly accumulated in the periphery of the process enlargement. (B) Case #21, Far zone. Tightly packed mitochondria in a process enlargement.

FIG. 4.

Ultrastructural pathology of injured cortex at early injury phase (less than 1 day post-injury; Case #1). (A) Near zone. Neuropil with frequent swollen processes showing high amplitude swollen mitochondria (arrows). Inner membrane-associated dense granular inclusions are present in a mitochondrion (black arrow). (B) Far zone. Slight to moderate cytoplasmic swellings are noticeable. Mitochondria are primarily normal (black arrow) and normal reactive (white-head arrow). (C) Penumbra zone. Slight to moderate cytoplasmic swelling. Mitochondria are predominantly normal orthodox (black arrow) or normal reactive (white head arrow). (D) Far zone. Neuropil with swollen processes showing mitochondrial changes. Note the teardrop-shaped mitochondrial profiles with thin thread-like extension (arrows) suggestive of a thread-grain transition. Scale bars equal 500 nm.

FIG. 5.

Ultrastructural pathology of injured cortex at early injury phase (less than 1 day post-injury; Case #2). (A) Far zone. (B) Near zone. A and B: Total disruption of tissue architecture and end-stage degenerating mitochondria (arrows) present. Scale bars equal 500 nm.

FIG. 6.

Ultrastructural pathology of injured cortex at late injury phase (approximately 3 days post-injury and later; Case #14). (A,B) Penumbra zone. (C) Far zone. (D) Near zone. (A) Neuronal cell body showing a group of mitochondria that exhibit abnormally spherical profiles with clear matrix space and disruption of cristate architecture (black arrows). Another group of mitochondria exhibit normal reactive structure (white-head arrows). N-nucleus. (B) Area of neuropil with slight to moderate swelling of neuronal processes. Mitochondria exhibit normal reactive (white-head arrows) and reactive/degenerating (black arrow) profiles. (C) Neuropil with frequent swollen processes showing normal reactive (white-head arrows) and reactive/degenerating (black arrows) mitochondrial changes. (D) Total disruption of tissue architecture and end-stage degenerating mitochondria (arrows) present. Scale bars equal 500 nm.

Fig. 7.

Mitochondrial profiles present in neuropil in normal cortex obtained by temporal lobectomy. (A) Neuronal process contains long orthodox mitochondria (arrows) in close association with microtubules. A spherical mitochondrion (double arrows) with slightly condensed matrix is present in an adjacent cell process. (B) Mitochondria are predominantly tubular in shape (white arrow), their matrix density is moderately condensed, and cristate architecture is well preserved. A rare large spherical mitochondrial profile (black arrow) is contained within another cell process. (C) Predominantly tubular mitochondria (white arrow) with moderately condensed matrices are present. Occasionally, cristae are parallel to the mitochondrial long axis (black arrow). A mitochondrion shows moderate rounding and fusiform shape change of a late thread-grain transition (double black arrows). All scale bars equal 500 nm.

FIG. 8.

Mitochondrial profiles present in neuronal cell bodies in normal cortex obtained by temporal lobectomy. (A) Pyramidal neuron with good preservation of nucleus (N), cytoplasm, and even distribution of organelles. (B) Fragment of neuronal cell body with well-preserved normal mitochondria (arrow). (C) Fragment of neuronal cell body showing spherical mitochondria with clear matrix space and disruption of cristate architecture (arrows). Scale bars equal A: 2 μm; B and C: 500 nm.

FIG. 9.

Toluidine blue stained semi-thin sections of human brain. (A) control cortical tissue; (B–D) Case #12 (B, injured Penumbra zone; C, injured Far zone; D, injured Near zone). A–D: original magnification×200; insertions within A–D: original magnification×550.

FIG. 10.

Ultrastructural pathology of injured cortex, Near zone (Case #12) at intermediate injury phase (between 1 and 3 days post-injury). (A) Enlarged area of ruptured cell shows advanced cytoplasmic dilution and spherical mitochondria containing multiple matrix densities and dark diffusely granulated matrix (arrows). N, nucleus. (B) High magnification of perinuclear area. Mitochondria show segmental area loss of cristate structure and soft matrical densities (arrows). N, nucleus. (C) Enlarged area of neuropil shows interstitial edema, swollen processes, cytoplasmic fragmentation, and spherical mitochondria containing multiple matrix densities in a dark diffusely granulated matrix (arrows). (D) Area of advanced cellular disruption. Mitochondria show both retention of matrix with large hard densities or soft semicrystalline densities (arrow with white head) and matrical swelling and clearing (black arrow). Scale bars for A, C, and D equal 500 nm; for B, 100 nm.

FIG. 11.

Distribution of cerebral cortex mitochondria among morphologic categories. Categories were defined as normal, normal reactive, reactive/degenerating, and end-stage degenerating. Distributions among these categories are given for control tissue and TBI tissue separated into Near, Far, and Penumbra injury zones. Measurements were made within 2D fields (44 μm²) of ultrathin sections (100 nm). The number of fields used for control, Near, Far, and Penumbra were 36, 30, 32, and 50, respectively. Total mitochondrial number counted for Control, Near, Far, and Penumbra were 712, 346, 378, and 621, respectively. Values represent the means±standard error (SEM). *p≤0.0001 when compared with control.

FIG. 12.

Ultrastructural pathology of injured cortex, Far zone (Case #12) at intermediate injury phase (between 1 and 3 days post-injury). (A) Perinuclear area showing moderate edema of cytoplasm, and mitochondrial profiles showing degenerative changes of cristate structure and clear expanded matrix (arrows). A profile shows undergoing thread-like extension of inner and outer membranes suggesting an arrested thread-grain transition (black arrow). N, nucleus. (B) Area of neuropil showing widespread edematous expansion of cell processes and synaptic terminals as well as variation in mitochondrial size, shape, and matrix density. Arrows show normal reactive mitochondria with condensed matrix. (C) Area of neuropil showing moderate swelling of cell processes. Note cell process with edematous swelling and containing condensed twisted mitochondria (arrows). Scale bars equal 500 nm.

FIG. 13.

Ultrastructural pathology of injured cortex, Penumbra zone (Case #12) at intermediate injury phase (between 1 and 3 days post-injury). (A) Neuronal cell with good preservation of nucleus (N), cytoplasm, and even distribution of organelles. Endoplasmic reticulum (ER) cisternae are in a normal configuration. Mitochondria, however, exhibit abnormally spherical profiles with clear matrix space and disruption of cristate architecture (black arrows). (B) Neuropil with numerous swollen cell processes and a wide range of mitochondrial profiles including spherical mitochondria with normal density matrix and apparent cristate proliferation (double black arrows) or with clear matrix space and disruption of cristate architecture (arrow with white head). A filamentous orthodox mitochondrion (black arrow) is also seen. (C) Swollen cell processes with advanced cytoskeletal fragmentation and contiguous spherical mitochondrial profiles suggestive of budding or fission (arrow). (D) Neuropil with frequent swollen processes showing mitochondrial changes. Note the teardrop-shaped mitochondrial profile suggestive of a late thread-grain transition (arrow). All scale bars are 500 nm.

FIG. 14.

Optical Absorbance of normal mitochondria exhibiting an orthodox conformation present in human traumatic brain injury (TBI) (Fig. 13B (black arrow)) and control cortex (Fig. 7A, arrows). Absorbance units (AU) obtained from the cytoplasm present immediately adjacent to mitochondria were defined as background absorbance and subtracted from the AU recorded for entire mitochondria. n=390 for control tissue; n=78 for TBI tissue, using both Far and Penumbra zones. ***p≤0.001.