Reversible Disruption of Neuronal Mitochondria by Ischemic and Traumatic Injury Revealed by Quantitative Two-Photon Imaging in the Neocortex of Anesthetized Mice

Abstract

Mitochondria play a variety of functional roles in cortical neurons, from metabolic support and neuroprotection to the release of cytokines that trigger apoptosis. In dendrites, mitochondrial structure is closely linked to their function, and fragmentation (fission) of the normally elongated mitochondria indicates loss of their function under pathological conditions, such as stroke and brain trauma. Using in vivo two-photon microscopy in mouse brain, we quantified mitochondrial fragmentation in a full spectrum of cortical injuries, ranging from severe to mild. Severe global ischemic injury was induced by bilateral common carotid artery occlusion, whereas severe focal stroke injury was induced by Rose Bengal photosensitization. The moderate and mild traumatic injury was inflicted by focal laser lesion and by mild photo-damage, respectively. Dendritic and mitochondrial structural changes were tracked longitudinally using transgenic mice expressing fluorescent proteins localized either in cytosol or in mitochondrial matrix. In response to severe injury, mitochondrial fragmentation developed in parallel with dendritic damage signified by dendritic beading. Reconstruction from serial section electron microscopy confirmed mitochondrial fragmentation. Unlike dendritic beading, fragmentation spread beyond the injury core in focal stroke and focal laser lesion models. In moderate and mild injury, mitochondrial fragmentation was reversible with full recovery of structural integrity after 1-2 weeks. The transient fragmentation observed in the mild photo-damage model was associated with changes in dendritic spine density without any signs of dendritic damage. Our findings indicate that alterations in neuronal mitochondria structure are very sensitive to the tissue damage and can be reversible in ischemic and traumatic injuries.

Rapid disruption of the cortical microcirculation after mild traumatic brain injury

“…Continuously intact microcirculation is required to meet the high energetic demands of neuronal tissue, and cellular health declines rapidly after CBF is disrupted. In stroke, multiple forms of pathophysiology emerge within 5 minutes of diminished perfusion, including ionic imbalance, structural degradation of the cytoskeleton and mitochondria, and failure of synaptic transmission (72–74). Similar cellular effects are often found in TBI (75, 76), likely due in part from energetic stress from loss of CBF.…”

“…Energetic stress may trigger diverse biochemical cascades that continue to cause cellular damage even after flow is restored (7, 76, 82). This includes loss of ionic homeostasis leading to mitochondrial dysfunction (74) and cortical spreading depolarization (83–85). Spreading depolarization increases intracellular calcium, further stressing mitochondria and limiting their ability to generate ATP, buffer intracellular calcium, and negate reactive oxygen species (74), forming a feed-forward loop that worsens oxidative stress.…”

Immune responses in stroke: how the immune system contributes to damage and healing after stroke and how this knowledge could be translated to better cures?

“…Particularly, cytotoxic CD8 + lymphocytes have been indicated to be recruited into the stroke brain as early as 3 hr following stroke while CD4 + T cells and natural killer cells are recruited within 24 hr and peak at 72 hr post‐reperfusion . In order to further characterize the timeline of T‐cell infiltration to the stroke brain, novel techniques such as multiphoton laser scanning microscopy have been applied to monitor immune responses in real time ex vivo . The first study by Stoll's group described the spatial and temporal infiltration of T cells following MCAO .…”

“…5,86 In order to further characterize the timeline of T-cell infiltration to the stroke brain, novel techniques such as multiphoton laser scanning microscopy have been applied to monitor immune responses in real time ex vivo. [87][88][89] The first study by Stoll's group described the spatial and temporal infiltration of T cells following MCAO. 90 Since these studies, there is strong support for the role of immune cells in promoting inflammation that cause secondary tissue injury in the brain following stroke.…”

Microglia monitor and protect neuronal function via specialized somatic purinergic junctions

“…S4a). We observed the fragmentation of mitochondria and an almost complete declustering of Kv2.1 proteins (38,39) in morphologically intact penumbral neurons ( Fig. 4a, b; Fig.…”