Mitochondrial Transfusion Improves Mitochondrial Function Through Up-regulation of Mitochondrial Complex II Protein Subunit SDHB in the Hippocampus of Aged Mice.

The mitochondrial theory of aging is characterized by mitochondrial electron transport chain dysfunction. As a hallmark of aging, an increasing number of investigations have attempted to improve mitochondrial function in both aging and age-related disease. Emerging from these attempts, methods involving mitochondrial isolation, transfusion, and transplantation have taken center stage. In particular, mitochondrial transfusion refers to the administration of mitochondria from healthy tissue into the bloodstream or into tissues affected by injury, disease, or aging. In this study, methods of mitochondrial isolation and transfusion were developed and utilized. First, we found a significant decrease (p < 0.05) in the expression of mitochondrial complex proteins (I-V) in aged (12 months old) mouse brain tissue (C57BL/6 mice) in comparison to healthy young brain tissue (1 month old). To investigate whether healthy young mitochondria taken from the liver could improve mitochondrial function in older animals, we intravenously injected mitochondria isolated from young C57BL/6 mice into aged mice from the same strain. This study, for the first time, demonstrates that mitochondrial transfusion significantly (p < 0.05) improves mitochondrial function via the up-regulation of the mitochondrial complex II protein subunit SDHB in the hippocampus of aged mice. This result has identified a role for mitochondrial complex II in the aging process. Therefore, mitochondrial complex II could serve as a putative target for therapeutic interventions against aging. However, more importantly, methods of mitochondrial transfusion should be further tested to treat a variety of human diseases or disorders and to slow down or reverse processes of aging.

Biochemical label-free tissue imaging with subcellular-resolution synchrotron FTIR with focal plane array detector.

The critical questions into the cause of neural degeneration, in Alzheimer disease and other neurodegenerative disorders, are closely related to the question of why certain neurons survive. Answers require detailed understanding of biochemical changes in single cells. Fourier transform infrared microspectroscopy is an excellent tool for biomolecular imaging in situ, but resolution is limited. The mid-infrared beamline IRENI (InfraRed ENvironmental Imaging) at the Synchrotron Radiation Center, University of Wisconsin-Madison, enables label-free subcellular imaging and biochemical analysis of neurons with an increase of two orders of magnitude in pixel spacing over current systems. With IRENI's capabilities, it is now possible to study changes in individual neurons in situ, and to characterize their surroundings, using only the biochemical signatures of naturally-occurring components in unstained, unfixed tissue. We present examples of analyses of brain from two transgenic mouse models of Alzheimer disease (TgCRND8 and 3xTg) that exhibit different features of pathogenesis. Data processing on spectral features for nuclei reveals individual hippocampal neurons, and neurons located in the proximity of amyloid plaque in TgCRND8 mouse. Elevated lipids are detected surrounding and, for the first time, within the dense core of amyloid plaques, offering support for inflammatory and aggregation roles. Analysis of saturated and unsaturated fatty acid ester content in retina allows characterization of neuronal layers. IRENI images also reveal spatially-resolved data with unprecedented clarity and distinct spectral variation, from sub-regions including photoreceptors, neuronal cell bodies and synapses in sections of mouse retina. Biochemical composition of retinal layers can be used to study changes related to disease processes and dietary modification.

Evidence for the involvement of calbindin D28k in the presenilin 1 model of Alzheimer's disease.

Pathological hallmarks of Alzheimer's disease include memory deficits, accumulation of amyloid beta (Abeta) plaques, the appearance of neurofibrillary tangles, and dysregulation of calcium homeostasis, which has been linked to mutations in the presenilin gene that code for presenilin (PS) proteins. PSs are a family of multi-pass transmembrane proteins where normal presenilins (PS1 and PS2) are highly localized in the endoplasmic reticulum (ER). Several past studies have explored alterations in long-term potentiation (LTP), a proposed molecular correlate of memory, and in behavioral tests of spatial memory in a variety of PS1 models. These reports suggest that calcium plays a role in these alterations, but mechanistic explanations for changes in LTP and in behavioral tests of memory are still lacking. To test the hypothesis that calcium-related mechanisms, such as changes in calcium buffering, are associated with alterations in LTP and memory, we utilized in vitro experimental paradigms of LTP in hippocampal slices obtained from the PS1-M146V transgenic mouse model of Alzheimer's disease (AD). We also used the in vivo Morris water maze (MWM), a test for hippocampal dependent spatial memory. In addition, we used cellular assays to explore molecular mechanisms. We confirm that PS1 mutations (M146V) enhance LTP. We also find increases in some parameters of the MWM, and alterations in other parameters, such as path length indicating impairment in cognitive functioning in PS1-M146V mice. In addition, these findings are observed in association with increased calbindin D28K expression in the CA1 hippocampus of PS1-M146V mice.

Models of brain injury and alterations in synaptic plasticity.

Animal models are crucial for understanding human pathophysiological processes and for understanding how connections are injured, lost, or even regenerated and/or repaired. When animal models are used in conjunction with theoretical computational models, an ideal combination is achieved that potentially yields insight and encourages the formation of new theories concerning connectionism, cognitive functioning, and synaptic mechanisms. Mechanisms regulating glutamate receptor activation and intracellular calcium levels are important for normal synaptic transmission. These mechanisms (and others) are also critical during and after brain injury when the potential exists for these mechanisms to function pathologically. Interestingly enough, the regulation of glutamate receptor activation and intracellular calcium levels is also involved in normal processes of neuronal and synaptic plasticity. In addition, studies have shown that neurotrophins and cytokines, which are released after brain injury, can be neuroprotective and may also be important in synaptic plasticity. Furthermore, synaptic plasticity is a phenomenon thought by many to be necessary for memory encoding. If this is the case, then research described in this review has significant scientific merit concerning plasticity and memory and clinical benefit for understanding pathophysiologic processes associated with brain injury and memory impairment. This paper reviews the application of experimental animal models of brain injury for simulating conditions of stroke, trauma, and epilepsy (and/or seizure generation) and the associated cellular mechanisms of brain injury. The paper also briefly addresses the advantage of using computational models in combination with experimental models for hypothesis building and for aiding in the interpretation of empirical data. Finally, it reviews studies concerning brain injury and synaptic plasticity.

Scientific visualization in drug discovery and development.

Scientific visualization has progressed significantly over the last century since the discovery of X-rays in 1895 and is used widely in many industries. In the pharmaceutical industry, there is a growing need for visualizing disease progression (or reversal) and for visualizing the effectiveness of a drug in animal models and in humans during clinical studies. Improvements in imaging technology could assist in decreasing the time from proof-of-concept to when a drug is finally launched. Many new drugs are entering the pipeline and progress in visualization technology should enhance the objective determination for predicting success or failure in a drug program. "A picture is worth a thousand words." Therefore, by collecting images, which provide information on molecular events, gene expression, biochemical subsystems, anatomical modifications and physiological perturbations, we positively and creatively alter our perception in our explorations of anatomy, disease mechanisms and drug interventions.

Long-term potentiation in the presence of NMDA receptor antagonist arylalkylamine spider toxins.

The role of the NMDA receptor (NMDAR) in long-term potentiation (LTP) is now well established. All potent NMDAR antagonists known to date inhibit the induction of LTP at the Schaffer collateral-CA1 pyramidal cell synapse in rat hippocampus, regardless of their site and mechanism of action. Arylalkylamine toxins are noncompetitive NMDAR antagonists in the mammalian central nervous system (CNS). The synthetic toxins argiotoxin-636 (Arg-636), Joro spider toxin (JSTX-3), alpha-agatoxin-489 and -505 (Agel-489 and Agel-505) and philanthotoxin-433 (delta-PhTX) were found in the present study to have no effect on the induction of LTP in the Schaffer collateral-CA1 pyramidal cell pathway in rat hippocampal slices maintained in vitro. Arylalkylamine toxins represent a class of potent NMDAR antagonists that fail to affect hippocampal LTP, and thus provide novel structural leads for the development of NMDAR antagonists that do not impair cognition.

Cyclosporin ameliorates traumatic brain-injury-induced alterations of hippocampal synaptic plasticity.

Although traumatic brain injury (TBI) often results in impaired learning and memory functions, the underlying mechanisms are unknown and there are currently no treatments that can preserve such functions. We studied plasticity at CA3-CA1 synapses in hippocampal slices from rats subjected to controlled cortical impact TBI. Long-term potentiation (LTP) of synaptic transmission was markedly impaired, whereas long-term depression (LTD) was enhanced, 48 h following TBI when compared to unoperated and sham control rats. Post-TBI administration of cyclosporin A, a compound that stabilizes mitochondrial function, resulted in a highly significant amelioration of the impairment of LTP and completely prevented the enhancement of LTD. Our data suggest that alterations in hippocampal synaptic plasticity may be responsible for learning and memory deficits resulting from TBI and that agents such as cyclosporin A that stabilize mitochondrial function may be effective treatments for TBI.

Diffusion and high resolution MRI of traumatic brain injury in rats: time course and correlation with histology.

Although widely employed in studies of cerebral ischemia, the use of diffusion-weighted imaging (DWI) for traumatic brain injury (TBI) has been both limited and primarily confined to the first few hours after injury. Therefore, the present study examined the temporal evolution of magnetic resonance imaging (MRI) signal changes from hours to weeks after moderate fluid-percussion TBI in rats. We used isotropic diffusion along three directions and high resolution (HR) spin-echo pulse sequences to visualize DWI and HR MRI changes, respectively. Late changes were compared to histopathological and neurological outcome. A significant decrease (P<0.05) in the apparent diffusion coefficients (ADC) below preinjury levels was found in the left cortex and left hippocampus (ipsilateral to injury) at 1-2 h post-TBI. At 2 weeks post-TBI, ADCs were significantly elevated (P<0.05) above preinjury levels in both cortex and hippocampus. Regions of hypo- and hyperintensity detected in HR MRI scans also showed evidence of tissue damage by histological evaluation. Neurological assessment indicated that such changes were observed at a level of injury which produced moderate impairment 2 weeks after the insult. These results indicate that alterations in DWI and HR MRI signals occur both early (hours) and late (weeks) after lateral fluid-percussion injury. Furthermore, the study showed that DWI was sensitive to MR signal change at 1-2 h post TBI (in select ROIs), whereas HR scans showed MR signal change primarily at later time points (3-4 h and later). Moreover, regions which demonstrate late changes are associated with histological damage and neurological impairment. The study demonstrates the utility of MRI to detect early changes, in some cases, that are predictive of long-lasting damage verified histologically.

Evidence for the involvement of TNF and NF-kappaB in hippocampal synaptic plasticity.

The cytokine tumor necrosis factor-alpha (TNF), well-known for its roles in cellular responses to tissue injury, has recently been shown to be produced in response to physiological activity in neuronal circuits. TNF stimulates receptors in neurons linked to the activation of the transcription factor NF-kappaB, and recent findings suggest that this signaling pathway can modulate neuronal excitability and vulnerability of neurons to excitotoxicity. Because data indicate that TNF is produced, and NF-kappaB activated, under conditions associated with learning and memory, we performed experiments in the hippocampal slice preparation aimed at elucidating roles for TNF and NF-kappaB in modulating synaptic plasticity. Whereas stimulation of Schaffer collateral axons at a frequency of 1 Hz induced long-term depression (LTD) of synaptic transmission in region CA1 of wild-type mice, LTD did not occur in slices from TNF receptor knockout mice. Stimulation at 100 Hz induced long-term potentiation (LTP) in slices from both wild-type mice and mice lacking TNF receptors. Basal transmission was unaltered in mice lacking TNF receptors. Pretreatment of slices from wild-type mice with kappaB decoy DNA prevented induction of LTD and significantly reduced the magnitude of LTP. Collectively, these data suggest important roles for TNF and signaling pathways that modulate NF-kappaB activity in regulation of hippocampal synaptic plasticity.

Unilateral hypoxic-ischemic injury in the neonatal rat brain evaluated by in vivo MRI. Correlation with histopathology and neuroprotection by MK-801.

RATIONALE AND OBJECTIVES: MRI was used for the in vivo evaluation of unilateral hypoxic-ischemic brain injury and the evaluation of MK-801 in the neonatal rat. METHODS: T2-weighted scans were obtained during the acute phase of HI injury and 3 months later. Histology was performed to correlate MRI signal changes with pathology. Finally, the effectiveness of MK-801 to limit brain injury was regionally assessed in vivo using T2-weighted MRI. RESULTS: Injury visualized by MRI at 72 hours after hypoxia correlated strongly with histopathologic analysis. Transient injury was identified. MK-801 significantly reduced the lesion extent at the level of the hippocampus. Patterns of unilateral versus bilateral neonatal brain injury were found to differ. CONCLUSIONS: The study demonstrates unique patterns of brain injury not seen in adult animal hypoxia-ischemia studies, and the sensitivity of the corpus callosum to hypoxia-ischemia. MK-801, although neuroprotective, did not offer any selective neuroprotective benefit.

Magnetic resonance imaging of hypoxic-ischemic brain injury in the neonatal rat.

RATIONALE AND OBJECTIVES: Magnetic resonance (MR) imaging was used for the in vivo evaluation of bihemispheric hypoxic-ischemic (HI) injury in the neonatal rat. METHODS: Seven-day-old rats underwent sham surgery (n = 7) or bilateral carotid artery ligation and hypoxia (30-45 min) (n = 8). T2-weighted imaging was used to study the temporal evolution of injury. Histopathology was used to correlate injury with MR signal changes. RESULTS: T2-weighted images exhibited considerable anatomic detail (0.2 mm resolution in-plane). The cortex, dorsolateral striatum and thalamus were affected, while the hippocampus was spared. Magnetic resonance signal change was seen as early as 1.5 hrs post-HI (lesion extent, 27%-39%), and reached a maximum at 48 hrs (37%-49%). Magnetic resonance imaging estimation of injury at 72 hours after HI was compared with histopathology and correlated well (r = 0.98). CONCLUSIONS: The study demonstrates the feasibility of magnetic resonance imaging for in vivo evaluation of neonatal brain injury and that vulnerability in the neonatal hippocampus is strikingly different than in adult HI models.

The differential optomotor response of the four-eyed fish Anableps anableps.

The perception of motion is important for the survival and reproduction of many animals, including fish. In the laboratory, support for this idea comes from the observation that many fish show a tendency to follow a series of stripes revolving around a circular aquarium. This response, known as the optomotor response (OMR), is recognized as an innate behavior in many species. The 'four-eyed' fishes of the genus Anableps are an unusual fish from Central and South America and actually have only two eyes. Each eye is divided into upper and lower halves internally and externally. This peculiar dual visual system allows Anableps to feed on creatures that swim or land near or on the water surface or to flee from flying predators attacking from above. It was hypothesized that Anableps should also possess the OMR. We used the OMR as a test to investigate potential differential visual processing in Anableps on normal and 'blinded' fish (the eyes are actually covered--not physically blinded). It was found that the OMR does exist in Anableps and that the strength of this response is dependent on the visual field being tested--a stronger OMR was seen as a result of visual stimulation from the aerial environment.

Localization of [125I]omega-conotoxin GVIA binding in human hippocampus and cerebellum.

The peptide toxin omega-conotoxin GVIA (omega-CgTx) has been shown to be a high affinity ligand for N-type calcium channels in the brain. We have employed [125I]omega-CgTx to localize N-type channels in human hippocampus and cerebellum using autoradiography. Ten micron thick slide-mounted tissue sections of human cerebellum and hippocampus were labeled with [125I]omega-CgTx under various conditions. Specific binding to human cerebellum was virtually irreversible and saturable. It was displaceable by the N-channel antagonist, omega-conotoxin MVIIA, but not by L- or P-channel ligands. Binding sites were heterogeneously distributed with denser binding in the molecular layer than the granule cell layer of cerebellum and with specific laminar patterns evident in the hippocampus. [125I]omega-CgTx should be a useful tool for the study of N-type calcium channels in human brain tissue.

Arylamine spider toxins antagonize NMDA receptor-mediated synaptic transmission in rat hippocampal slices.

The effects of arylamine spider toxins on synaptic transmission in rat hippocampal slices were investigated. Two different responses were monitored: the AMPA receptor-mediated population spike recorded in control buffer (selectively antagonized by DNQX) and the NMDA receptor-mediated EPSP recorded in nominally magnesium-free buffer containing 20 microM DNQX (selectively antagonized by AP5, AP7, and dizocilpine (MK-801)). The synthetic arylamine spider toxins JSTX-3, argiotoxin-636, and argiotoxin-659 were 26 to 73 times more potent at antagonizing the NMDA receptor-mediated EPSP (IC50 values ranging from 12 to 24 microM) than the AMPA receptor-mediated population spike (IC50 values ranging from 612 to 878 microM). These results indicate that arylamine spider toxins are selective antagonists of NMDA receptors in the mammalian CNS.

Arylamine toxins from funnel-web spider (Agelenopsis aperta) venom antagonize N-methyl-D-aspartate receptor function in mammalian brain.

The venom of the North American funnel-web spider Agelenopsis aperta contains a variety of arylamine toxins (the alpha-agatoxins) that paralyze insects by blocking glutamatergic neuromuscular transmission. We have tested six synthetic alpha-agatoxins for their ability to antagonize glutamate receptor function in mammalian brain. These compounds produce, at submicromolar concentrations, noncompetitive inhibition of N-methyl-D-aspartate (NMDA) receptor-mediated elevations in the concentration of cytosolic free calcium in cultured rat cerebellar granule neurons. In contrast, the alpha-agatoxins are relatively weak antagonists of elevations in the cytosolic free calcium concentration induced by non-NMDA receptor agonists. The alpha-agatoxins also produce reversible suppression of the NMDA receptor-mediated excitatory postsynaptic potential in rat hippocampal slices at concentrations that have little effect on the non-NMDA receptor-mediated population spike. We conclude that the alpha-agatoxins are selective and reversible noncompetitive antagonists at NMDA receptors in mammalian brain.