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

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.

Visualization and quantification of NAD(H) in brain sections by a novel histo-enzymatic nitrotetrazolium blue staining technique.

A histo-enzymatic technique for visualizing and quantifying endogenous NAD(H) in brain tissue was developed, based on coupled enzymatic cycling reactions that reduce nitrotetrazolium blue chloride to produce formazan. Conditions were used where the endogenous level of nicotinamide adenine dinucleotides (NAD(H)) was the rate limiting factor for formazan production. Spontaneous degradation of NAD(+) that occurs during incubation of thawed tissue was minimized by the addition of nicotinamide mononucleotide, an inhibitor of NAD(+) glycohydrolases. Cryostat sections of brains obtained from rats immediately after decapitation and 30 min later were used to determine the effects of ischemia alone on brain NAD(H) levels and neuroanatomic distribution. The ischemic insult resulted in a greater than 50% decline in the rate of formazan generation in the CA1 pyramidal neuronal layer of the hippocampus and in the parietal cortex and striatum, but not in the CA3 and dentate gyrus (DG) subregions of the hippocampus. The ischemia-induced changes in NAD(H) levels were confirmed by utilizing spectrofluorimetric measurements of NAD(H) present in perchloric acid extracts of brain samples. This new histo-enzymatic technique is suitable for visualizing and quantifying relative NAD(H) levels in the brain. This assay could prove useful in identifying region-selective NAD(H) catabolism that may contribute to neurodegeneration.

Oximetry-guided reoxygenation improves neurological outcome after experimental cardiac arrest.

BACKGROUND AND PURPOSE: Current guidelines suggest that cardiac arrest (CA) survivors should be ventilated with 100% O(2) after resuscitation. Breathing 100% O(2) may worsen neurological outcome after experimental CA. This study tested the hypothesis that graded reoxygenation, with oximetry guidance, can safely reduce FiO(2) after resuscitation, avoiding hypoxia while promoting neurological recovery. METHODS: Mature dogs underwent 10 minutes of CA and restoration of spontaneous circulation with 100% O(2.) Animals were randomized to 1-hour additional ventilation on 100% FiO(2) or to rapid lowering of arterial O(2) saturation to <96% but >94% with pulse oximeter guidance. Animals were awakened at hour 23, and the neurological deficit score (0=normal; 100=brain-dead) was measured. Reanesthetized animals were perfusion-fixed and the brains removed for histopathology. RESULTS: The neurological deficit score was significantly better in oximetry (O) dogs. O dogs appeared aware of their surroundings, whereas most hyperoxic (H) animals were stuporous (neurological deficit score=43.0+/-5.9 [O] versus 61.0+/-4.2 [H]; n=8, P<0.05). Stereological analysis revealed fewer injured CA1 neurons in O animals (cresyl violet: 35.5+/-4.3% [O] versus 60.5+/-3.3% [H]; P<0.05). There were also fewer fluoro-Jade B-stained degenerating CA1 neurons in O animals (3320+/-267 [O] versus 6633+/-356 [H] per 0.1 mm(3); P<0.001). CONCLUSIONS: A clinically applicable protocol designed to reduce postresuscitative hyperoxia after CA results in significant neuroprotection. Clinical trials of controlled normoxia after CA/restoration of spontaneous circulation should strongly be considered.