Hyperoxic reperfusion after global cerebral ischemia promotes inflammation and long-term hippocampal neuronal death.

In this study we tested the hypothesis that long-term neuropathological outcome is worsened by hyperoxic compared to normoxic reperfusion in a rat global cerebral ischemia model. Adult male rats were anesthetized and subjected to bilateral carotid arterial occlusion plus bleeding hypotension for 10 min. The rats were randomized to one of four protocols: ischemia/normoxia (21% oxygen for 1 h), ischemia/hyperoxia (100% oxygen for 1 h), sham/normoxia, and sham/hyperoxia. Hippocampal CA1 neuronal survival and activation of microglia and astrocytes were measured in the hippocampi of the animals at 7 and 30 days post-ischemia. Morris water maze testing of memory was performed on days 23-30. Compared to normoxic reperfusion, hyperoxic ventilation resulted in a significant decrease in normal-appearing neurons at 7 and 30 days, and increased activation of microglia and astrocytes at 7, but not at 30, days of reperfusion. Behavioral deficits were also observed following hyperoxic, but not normoxic, reperfusion. We conclude that early post-ischemic hyperoxic reperfusion is followed by greater hippocampal neuronal death and cellular inflammatory reactions compared to normoxic reperfusion. The results of these long-term outcome studies, taken together with previously published results from short-term experiments performed with large animals, support the hypothesis that neurological outcome can be improved by avoiding hyperoxic resuscitation after global cerebral ischemia such as that which accompanies cardiac arrest.

Neuroprotection by acetyl-L-carnitine after traumatic injury to the immature rat brain.

Traumatic brain injury (TBI) is the leading cause of mortality and morbidity in children and is characterized by reduced aerobic cerebral energy metabolism early after injury, possibly due to impaired activity of the pyruvate dehydrogenase complex. Exogenous acetyl-L-carnitine (ALCAR) is metabolized in the brain to acetyl coenzyme A and subsequently enters the tricarboxylic acid cycle. ALCAR administration is neuroprotective in animal models of cerebral ischemia and spinal cord injury, but has not been tested for TBI. This study tested the hypothesis that treatment with ALCAR during the first 24 h following TBI in immature rats improves neurologic outcome and reduces cortical lesion volume. Postnatal day 21-22 male rats were isoflurane anesthetized and used in a controlled cortical impact model of TBI to the left parietal cortex. At 1, 4, 12 and 23 h after injury, rats received ALCAR (100 mg/kg, intraperitoneally) or drug vehicle (normal saline). On days 3-7 after surgery, behavior was assessed using beam walking and novel object recognition tests. On day 7, rats were transcardially perfused and brains were harvested for histological assessment of cortical lesion volume, using stereology. Injured animals displayed a significant increase in foot slips compared to sham-operated rats (6 ± 1 SEM vs. 2 ± 0.2 on day 3 after trauma; n = 7; p < 0.05). The ALCAR-treated rats were not different from shams and had fewer foot slips compared to vehicle-treated animals (2 ± 0.4; n = 7; p< 0.05). The frequency of investigating a novel object for saline-treated TBI animals was reduced compared to shams (45 ± 5% vs. 65 ± 10%; n = 7; p < 0.05), whereas the frequency of investigation for TBI rats treated with ALCAR was not significantly different from that of shams but significantly higher than that of saline-treated TBI rats (68 ± 7; p < 0.05). The left parietal cortical lesion volume, expressed as a percentage of the volume of tissue in the right hemisphere, was significantly smaller in ALCAR-treated than in vehicle-treated TBI rats (14 ± 5% vs. 28 ± 6%; p < 0.05). We conclude that treatment with ALCAR during the first 24 h after TBI improves behavioral outcomes and reduces brain lesion volume in immature rats within the first 7 days after injury.

Cyclophilin D is expressed predominantly in mitochondria of gamma-aminobutyric acidergic interneurons.

Brain mitochondria are relatively resistant to calcium-induced mitochondrial permeability transition (MPT), with heterogenic response to the insult. The cause for this heterogeneity is not clear, so we studied the distribution of a key regulator of the MPT, cyclophilin D (cypD), within the rat brain by using immunohistology and Western blotting. Motor and parietal cortex, hippocampus, striatum, substantia nigra, ventral tegmental area, septum, and mammillary nucleus displayed a strong immunoreactivity to cypD within specific subpopulation of neurons. The staining was punctate and intense, particularly in perinuclear regions of cells. Apart from neurons, a subpopulation of astrocytes and NG2-positive cells showed higher cypD immunoreactivity. Double staining of cypD with cytochrome oxidase confirmed the mitochondrial specificity of cypD immunoreactivity. The neurons with high levels of cypD also expressed glutamate decarboxylase (GAD) and the calcium binding protein parvalbumin or calbinding D-28k, identifying these cells as interneurons. Western blots confirmed our immunohistochemical findings, showing significantly higher levels of cypD in crude mitochondria of substantia nigra compared with cortex or striatum. Furthermore, nonsynaptic mitochondria representing mainly mitochondria from cell bodies of neurons and glia have about 16% higher levels of cypD compared with synaptic mitochondria that are localized in presynaptic buttons. These data suggest that the underlying factor of heterogenic response of isolated brain mitochondria to MPT-inducing insults can be the different expression levels of cypD, with mitochondria originated from interneurons as the most sensitive.

Pup exposure elicits hippocampal cell proliferation in the prairie vole.

The onset of parental behavior has profound and enduring effects on behavior and neurobiology across a variety of species. In some cases, mere exposure to a foster neonate (and a subsequent parental response) can have similar effects. In the present experiment, we exposed adult male and female prairie voles (Microtus ochrogaster) to two foster pups for 20 min and quantified cell proliferation in the dentate gyrus of the hippocampus (DG), medial amygdala (MeA) and cortical amygdala (CorA). Prairie voles are highly social rodents that typically display biparental care and spontaneous parental care when exposed to foster pups. Comparisons were made between the animals that responded parentally or non-parentally towards the pups, as well as control conditions. Cell proliferation was assessed using injections of 5-bromo-2'-deoxyuridine (BrdU) and immunocytochemical localization of this marker. The phenotype of the cells was determined using double label immunofluoresence for BrdU and TuJ1 (a neuronal marker). An increase in cell proliferation in the DG was seen in animals exposed to pups. However, animals that responded non-parentally had a greater number of BrdU labeled cells in the DG compared to those that responded parentally. The majority of BrdU labeled cells co-expressed TuJ1 across all groups. These results demonstrate that exposure to a foster pup and the behavioral reaction to it (parental or non-parental) are associated with site-specific changes in cell proliferation.

Social environment regulates corticotropin releasing factor, corticosterone and vasopressin in juvenile prairie voles.

Stressful social conditions, such as isolation, that occur during sensitive developmental periods may alter present and future social behavior. Changes in the neuroendocrine mechanisms closely associated with affiliative behaviors and stress reactivity are likely to underlie these changes in behavior. In the present study, we assessed the effects of post-weaning social housing conditions on the neuropeptides arginine vasopressin (AVP) and oxytocin (OT), and components of the hypothalamic-pituitary-adrenal axis (corticotropin releasing factor: [CRF], and corticosterone: [CORT]) in the prairie vole (Microtus ochrogaster), a socially monogamous bi-parental rodent. Following weaning at 21 days of age, prairie voles were maintained in one of three housing conditions: social isolation (isolate), paired with a same sex sibling (sibling) or paired with a stranger (stranger) of the same sex and age. Housing conditions were maintained for either 4 or 21 days. Central CRF, AVP and OT immunoreactivity (ir) were quantified and circulating plasma CORT, AVP and OT were assayed. Isolated voles had higher CRF-ir in the paraventricular nucleus of the hypothalamus (PVN) compared with sibling and stranger housed voles. Plasma CORT was significantly higher in isolates. AVP-ir was significantly lower in the PVN of isolate females compared to either sibling females or stranger females. However, AVP-ir was significantly higher in the supraoptic nucleus (SON) of isolates compared to siblings. There were no differences in central OT-ir or plasma OT. These results identify neuroendocrine mechanisms which respond to isolation and potentially modulate behavior.

Neuron-specific conditional expression of a mitochondrially targeted fluorescent protein in mice.

Mitochondrial dysfunction contributes to the pathophysiology of both acute and chronic neurodegenerative disorders. Quantification of mitochondrial bioenergetic properties generally requires the use of isolated brain mitochondria. However, the involvement of neuronal mitochondrial dysfunction in these disorders is limited by the lack of markers, and therefore isolation procedures, that distinguish neuronal compared with astrocyte mitochondria. To address this and other issues concerning neuronal mitochondria in the CNS, transgenic mice were generated that express a fluorescent protein targeted specifically to neurons. A neuron-specific promoter, CaMKIIalpha (calcium/calmodulin-dependent kinase IIalpha) driven tTA (tetracycline transactivator) mice were crossed with TRE (tetracycline responsive element) driven mitochondrial targeted enhanced yellow fluorescent protein (eYFP) mice. Expression of eYFP in the bigenic mouse brain was observed only in neuronal mitochondria of striatum, forebrain, and hippocampus and was enhanced by the removal of the tetracycline analog doxycycline (Dox) in the diet. The respiratory control ratio of synaptic and nonsynaptic mitochondria isolated from eYFP-expressing mice was the same as control mice, suggesting that neuronal mitochondria expressing eYFP maintain normal bioenergetic functions. More importantly, the development of Dox-inducible, neuron targeted mito/eYFP transgenic mice offer a unique in vivo model for delineating the participation of neuronal mitochondria in neuronal survival and death.

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.

Assessing the consequences of the pesticide methoxychlor: neuroendocrine and behavioral measures as indicators of biological impact of an estrogenic environmental chemical.

Japanese quail provide an advantageous avian model for assessing long-term biological consequences of endocrine disrupting chemicals (EDCs). These studies examined route of exposure and vulnerability to biological impact of EDCs over the life cycle in a precocial avian model, the Japanese quail. Embryonic exposure occurs with maternal deposition and methoxychlor (MXC) accumulated with maternal exposure. Egg injections of MXC or estradiol at selected stages of development impacted hypothalamic neuroendocrine systems in hatchlings and affected sexual maturation, with evidence for long-term effects on neurotransmitters and male behavior. Two-generation dietary studies were conducted to examine transgenerational effects of EDCs. Adult quail (P1) were exposed to dietary MXC (0, 0.5 and 5 ppm), with continued exposure in their offspring (F1), and control diet for all F2 chicks. Toxicological end points, including fertility, hatching success, and 14-day viability were unaffected. F1 and F2 male offspring from MXC-treated pairs MXC had impaired mating behavior and altered plasma hormones. These studies confirm neuroendocrine and behavioral measures as reliable indices of exposure to an estrogenic EDC. Moreover, maternal deposition remains a primary route of EDC exposure, with potential deleterious consequences for field birds, especially precocial species that appear to be particularly sensitive to embryonic EDC exposure.