Del Mar Photonics - Newsletter Fall 2010 - Newsletter Winter 2010
Femtosecond Laser-Induced Microvascular Clots Trigger Alzheimer's Disease
Nozomi Nishimura, Joan Zhou, Costantino Iadecola, and Chris B. Schaffer
Authors use femtosecond-laser ablation to lesion cortical microvessels in transgenic mouse models of Alzheimer’s Disease. Aβ plaques and blood flow were imaged in vivo with 2-photon microscopy. Aβ accumulated after lesions which stalled blood flow.
Del Mar Photonics offer variety of lasers that can be used for cost effcetive neurobiology research. E-mail us today to ask ask about application of lasers in Alzheimer's Desease Research
Neuroscience 2010 annual meeting in San Diego, California.
Abstracts from Neuroscience Nanosymposia where lasers are used in research.
To request information about Del Mar Photonics lasers for applications in laser
imaging, laser scanning, laser ablation, speckle imaging, laser capture
microdissection, laser axotomy, laser Doppler flowmetry, laser injury and other
contact our neuroscince application group
(abstracts in pdf)
Title: Cholesterol accumulates in the vicinity of amyloid deposits in brain tissue
Authors: *S. SOLÉ DOMÈNECH1, P. SJÖVALL5, V. VUJOCEVIć2, S. SALVE3, A. CODITA4, M. SCHALLING3, F. M. LAFERLA6, L. GIMÉNEZ-LLORT7, P. NILSSON8, L. TERENIUS2, B. JOHANSSON3; 2Dept. of Clin. Neurosci., 3Dept. of Mol. Med., 4Dept. of Neurobiology, Care Sci. and Society, 1Karolinska Institutet, Stockholm, Sweden; 5Dept. of Chem. and Materials Technol., SP Tech. Res. Inst. of Sweden, Borås, Sweden; 6Dept. of Neurobio. and Behaviour, Univ. of California Irvine, Irvine, CA; 7Dept. of Psychiatry and Forensic Med., Univ. Autònoma de Barcelona, Barcelona, Spain; 8Dept. of Chem., Linköpings Universitet, Linköping, Sweden
Abstract: Amyloid beta (Aβ) aggregation plays a central role in the
pathogenesis of Alzheimer‘s disease (AD), but increasing evidence suggests that
altered cholesterol (Ch) homeostasis may also be implicated in the disease
etiology1. The aim of our study is to clarify the role of Ch in the aggregation
process of Aβ. In the present work we combined time-of-flight
secondary ion mass spectrometry (ToF-SIMS) with confocal laser scanning
microscopy (CLSM) to study Ch distribution around Aβ deposits in
mouse (3xTgAD transgenic model) and human AD brain tissue. Aβ deposits were
identified by p-FTAA, an amyloid-specific fluorescent probe and visualized by
CLSM imaging. CLSM images were used as guiding templates for ToF-SIMS
experiments. ToF-SIMS analyses were performed on the immediately adjacent,
mirror-image section in order to evaluate the Ch/lipid profile. The sections
subjected to ToF-SIMS were thereafter incubated with p-FTAA and imaged by CLSM.
Colocalization between Aβ and Ch was studied in overlaid ToF-SIMS and CLSM
images obtained from the same section. In some cases, Ch accumulations could be
observed in the vicinity of Aβ deposits, within a distance of 0 to 50 μm from
the plaque core. As an example, images of a 3xTgAD mouse brain section (approx.
3.15 mm from bregma) are shown in figure 1. The brain hippocampus is visualized
in figure 1a by DAPI staining of neuronal nuclei (blue). The region enriched
with Aβ deposits, visualized by p-FTAA (yellow-green), is delineated by a white
rectangle. A magnified
Title: Mild sensory stimulation completely protects the adult rodent cortex from ischemic stroke
Authors: *C. C. LAY, M. DAVIS, C. CHEN-BEE, R. FROSTIG; Neurobio. & Behavior, Univ. of California, Irvine, Irvine, CA
Abstract: Mild sensory stimulation following permanent occlusion of the
middle cerebral artery (pMCAO) has been shown to be completely neuroprotective
24 hours after ischemic onset in an animal model of stroke (Lay et al. 2010),
but precisely when functional recovery took place during the post-occlusion 24hr
period remained unclear. Here, we quantify the return of cortical function
immediately following ischemic onset using intrinsic signal
optical imaging, electrophysiological recording, laser speckle imaging of blood
flow, and histological analysis in order to assess the relationship
between protective sensory stimulation, recovery of cortical function, and
reperfusion within the ischemic territory. By examining cortical function (i.e.
spatial extent and amplitude of whisker functional representation) within the somatosensory cortex, we determined that 90 minutes of intermittent whisker stimulation delivered within 2 hours following pMCAO resulted in a return of function to pre-occlusion levels, and was accompanied by a significant reperfusion of the ischemic region via collateral vessels. In contrast, animals which received the identical whisker stimulation three hours after pMCAO never regained cortical function and sustained a major cortical infarct. Therefore, complete protection from impending ischemic stroke is possible within two hours of stroke onset whereas irreversible damage occurs when whisker stimulation is initiated three hours post-pMCAO. In summary, we found that the return of cortical function to pre-pMCAO levels by single whisker stimulation occurred 90 minutes following pMCAO, and was accompanied by a reperfusion of the imperiled region. These findings demonstrate the existence of a stimulus-induced neurovascular plasticity that confers complete protection of the ischemic territory; and that sensory induced protection is bounded within the first two hours (acute phase) of ischemia.
Title: Complement component C3 and its receptor complement receptor type 3 mediate the phagocytosis and clearance of Abeta by microglia
Authors: *H. FU1, B. LIU1, J. L. FROST1, S. HONG1, M. JIN1, I. M. COSTANTINO2, M. C. CARROLL3, T. N. MAYADAS4, D. J. SELKOE1, C. A. LEMERE1; 1Dept. of Neurol., Ctr. For Neurologic Diseases, Brigham & Women's Hosp., Harvard Med. Sch., BOSTON, MA; 2Dartmouth Col., Hanover, NH; 3Dept. of Pediatrics (Pathology), Immune Dis. Institute, Children‘s Hospital, Harvard Med. Sch., BOSTON, MA; 4Dept. of Pathology, Brigham and Women‘s Hospital, Harvard Med. Sch., BOSTON, MA
Abstract: Alzheimer‘s disease (AD) is the most common form of dementia in the
elderly and is characterized by extracellular amyloid plaques and intracellular
neurofibrillary tangles. The up-regulated production and activation of
complement components and their receptors are found
within and around cerebral amyloid plaques in AD patients. Microglia, the principal immune effector cells in the CNS, can defend against pathogens through phagocytosis via complement component C3 and/or its receptor complement receptor type 3 (Mac-1, CD11b). We previously reported that the deficiency of C3 accelerated cerebral Aβ deposition and neuronal loss, possibly through inhibition of microglia-mediated uptake and clearance of Aβ. In the present study, we provide direct evidence that C3 and Mac-1 mediate the phagocytosis and clearance of fibrillar Aβ (fAβ) by microglia in vitro and in vivo. Using flow cytometry and confocal laser scanning microscope, we found that both primary mouse microglia and immortal murine microglial cell line were able to take up fAβ42 in a concentration- and time-dependent manner. The fAβ42 taken up by microglia was colocalized with lysosomal markers. Uptake of fAβ42 by primary microglia from mouse pups genetically deficient for C3 or Mac-1 was significantly decreased, compared with that of primary microglia from wild type C57BL/6 mouse pups. The knockdown of C3 or Mac-1 in primary microglia by transient transfection of siRNA against C3 or Mac-1 significantly inhibited fAβ42 uptake compared to cells transfected with control siRNA. In vivo, we found that Iba-1 and CD68 positive microglial cells took up cortically microinjected fAβ42, most of which was colocalized with the lysosomal marker, LAMP-1. Five days after the surgery, the fluorescent signal of fAβ left in the brain of C3 or Mac-1 knockout mice was significantly higher than that in wild-type mice. Together, these results demonstrate that C3 and its receptor Mac-1 are involved in the phagocytosis and clearance of Αβ by microglia, providing evidence consistent with a beneficial role for microglia in AD pathogenesis. Attempts to develop therapies aimed at removing Aβ by activating the beneficial role of microglia are warranted.
Title: Hilar and CA3 contributions to recurrent excitatory connectivity of dentate granule cells in a model of temporal lobe epilepsy--A laser scanning glutamate uncaging study
Authors: *W. ZHANG1, J. R. HUGUENARD2, P. S. BUCKMASTER1; 1Dept Comparative
Med., Stanford Univ., STANFORD, CA; 2Neurol., Stanford Univ., Stanford,
Abstract: Enhanced recurrent excitation in dentate granule cells has been intensively investigated as a potentially pro-epileptic change in temporal lobe epilepsy (TLE). Previous studies suggest that neurons in the hilus contribute little, if any, to recurrent excitation of granule cells in epileptic animals, most likely because of the loss of mossy cells and amputation of axons in slice preparations. However, other studies suggest that mossy cells and CA3 pyramidal cells might sprout axons and synapse with granule cells after deafferentation lesions and in epilepsy models. Furthermore, hilar ectopic granule cells are generated in models of temporal lobe epilepsy, and they have been shown to project axon collaterals to the molecular layer where the dendrites of normally positioned granule cells are located. In this study, we sought to test the hypothesis that neurons in regions other than the granule cell layer contribute to enhanced recurrent excitation of granule cells in epileptic pilocarpine-treated rats. Laser scanning photostimuli randomly, systematically, and focally activated neurons in the granule cell layer, hilus, and proximal CA3 pyramidal cell layer in a grid pattern with 60 μm spacing. By uncaging glutamate on neurons in scanning areas while recording evoked EPSCs of granule cells (Vhold = -70 mV), we obtained maps of excitatory connectivity in 400 μm-thick slices (control = 6, epileptic = 11 rats) and measured the proportion of stimulation sites in each region that evoked EPSCs. For all regions combined, more excitatory synaptic responses were evoked in epileptic rats (7 ± 1% of stimulation sites) than in controls (3 ± 1%, P<0.05). Consistent with previous reports, a higher proportion of sites in the granule cell layer evoked EPSCs in epileptic rats compared to controls (7 ± 2% versus 3 ± 2%). However, in addition to the granule cell layer, stimulation sites in the hilus (4 ± 2% in control, 7 ± 2% in epileptic) and proximal CA3 pyramidal cell layer (2 ± 1% in control, 7 ± 3% in epileptic) also demonstrated increased probabilities of evoking EPSCs in granule cells in epileptic animals. These findings suggest that neurons in the hilus and proximal CA3 region contribute to enhanced recurrent excitatory connectivity of dentate granule cells in TLE.
Title: Microarray analysis of subtypes of pyramidal and nonpyramidal neurons from auditory cerebral cortex in schizophrenia
Authors: J. F. SMILEY1,2, H. M. CHAO1,3, A. J. DWORK4,5, M. J. ALLDRED1,3, *I. ELAROVA1, D. C. JAVITT1,3, S. D. GINSBERG1,3; 1Nathan Kline Inst., ORANGEBURG, NY; 2City Col., New York, NY; 3NYU Med. Sch., New York, NY; 4New York Psychiatric Inst., New York, NY; 5Columbia Univ., New York, NY
Abstract: Schizophrenia is associated with altered neurotransmission by both glutamatergic and GABAergic neurons in the cerebral cortex, and there is evidence that specific cortical pathways and neuronal subtypes are selectively vulnerable in this disease. To further characterize the specificity of schizophrenia pathology, we used microarray analysis to compare mRNA expression levels in isolated neuron subtypes of auditory association cortex, in schizophrenia (n=15) and age-matched nonpsychiatric (n=15) brains. Laser capture microdissection (LCM) was used to isolate calbindin (CB) and parvalbumin (PV) subtypes of GABAergic interneurons, as well as glutamatergic pyramidal neurons of lower layer III. These cell types represent distinct components of cortical circuitry; parvalbumin neurons are mainly involved in feed-forward inhibition, calbindin cells are more involved in feedback or modulatory inhibition, and layer III pyramidal cells are mainly feed-forward principal neurons. RNA was harvested from homogeneous cell populations acquired via LCM, amplified via terminal continuation (TC) RNA amplification, and hybridized to custom-designed microarrays containing 576 transcripts relevant to neuroscience and neuropsychiatric disorders. Preliminary analysis showed that pyramidal neurons have about twice as many significantly altered genes in schizophrenia compared to either CB or PV neurons. CB neurons were distinguished from the other cell types by their general tendency of reduced expression for most differentially regulated genes. An analysis of the types of genes changed in schizophrenia was obtained using Chi-squared analysis to look for clusters of changes within 21 predefined gene ontology groups on the microarray. Among the most significant group changes were the monoamine-related transcripts, including dopamine and norepinephrine receptors, which were especially up-regulated in CB and PV neurons. Overall, the findings indicate that the profile of gene expression changes in the cerebral cortex in schizophrenia is strikingly different in distinct neuronal subpopulations, and some changes will be overlooked by measuring expression changes in the whole cerebral cortex.
Title: Hydrogen peroxide promotes peripheral sensory axon regeneration after epidermal injury
Authors: *S. RIEGER1,2, A. SAGASTI2; 1Los Angeles, CA; 2Molecular, Cell and Developmental Biol., Univ. of California Los Angeles, Los Angeles, CA
Abstract: Peripheral sensory neurons regenerate spontaneously in response to injury, but regeneration can be limited, thereby often leading to incomplete functional recovery. In zebrafish, peripheral sensory neurons innervate the epidermis between 18 and 36 hpf. If a sensory axon is severed using laser axotomy during that developmental window it can reinnervate its former territory, but, if axotomy occurs after 48 hpf, regenerating axons avoid the denervated regions. The ability of zebrafish to regenerate tissues in response to injury prompted us to explore whether axon and tissue regeneration are related. At 78 hpf, when axotomized axons are normally no longer capable of reinnervating uninjured tissue, we amputated the caudal fin and used time-lapse confocal imaging to visualize the behavior of injured GFP-expressing Rohon-Beard (RB) sensory axons over 12h. We found that RB axons always regenerated into the wound site, suggesting that global fin injury can overcome growth inhibitors present at this stage. We further found that injury often promoted growth of all axonal branches, but only when the axon was injured close to the amputation site. To search for the source of regeneration signals, we laser damaged a few keratinocytes, followed by axotomy near the ablation site. Axotomized axons were indeed capable of regeneration. The ability of damaged keratinocytes to induce axon regeneration was not only restricted to the fin but also occurred in the head for trigeminal axons. To identify potential molecules mediating injury-dependent axon regeneration, we tested whether hydrogen peroxide (H2O2) was involved, as high concentrations of this reactive oxygen species are produced along the wound margins of the injured epithelium. Indeed, we observed that the addition of H2O2 to the media promoted peripheral sensory axon regeneration following axotomy in uninjured fins. Conversely, inhibition of H2O2 production via morpholino knockdown of duox1 completely prevented axon growth following fin amputation. We also found that in duox1 morphants, inhibition of IκB kinase (IKK) significantly rescued peripheral sensory axon regeneration, suggesting that H2O2 may be a negative regulator of IKK in this process. Together these findings suggest a novel function for H2O2 in promoting axon regeneration following epithelial injury.
Title: Elucidating the role of microglia in secondary degeneration following spinal cord injury in real-time using two-photon microscopy
Authors: *D. P. STIRLING, K. A. CUMMINS, P. K. STYS; Clin. Neurosciences, Hotchkiss Brain Institute/ Univ. of Calgary, Calgary, AB, Canada
Abstract: Spinal cord injury (SCI) induces a robust inflammatory response mediated in part by the rapid activation of microglia, however, whether they play a destructive or protective role after SCI remains unclear. To better understand the role of microglia following SCI we utilized two-photon microscopy to ablate cervical dorsal column axons (primary injury) from live murine spinal cord preparations and documented the subsequent changes in axon, myelin and microglia as the lesion evolved (secondary injury) over time. Towards this goal we generated double transgenic mice that express EGFP (enhanced green fluorescent protein) in microglia (CX3CR1-GFP+/-), YFP (yellow fluorescent protein) in axons (Thy1-YFP+), and applied lipophilic fluorescent dyes to visualize myelin. In control conditions, time-lapse recordings of live spinal cord white matter revealed parallel-aligned YFP+ axon cylinders ensheathed in myelin with largely constant diameters along the length of the axon. Few spheroids or other morphological signs of axonal degeneration were present. In support, microglia were highly ramified and frequently extended/retracted processes. In contrast to baseline conditions, transected axons formed end bulbs that began to swell both rostral and caudal to injury within 5 minutes of ablation. Microglia responded immediately to laser ablation by extending processes several microns to wall of the ablation site. As microglia density increased over time, transected axons formed end bulbs that ―died back‖ from the ablation site both rostral and caudal to injury. Swelling of axonal end bulbs partially detached from their myelin sheath were a common finding remote to the ablation site along with axonal spheroids tightly encased in myelin. In distinction, large swollen empty tubes of myelin remained at the border of the ablation site. Fiber loss adjacent to the ablation site, and in close apposition with microglia, was a prominent feature; Lesion width at 4 hours post-ablation was increased by ~70% versus ~ 26% at 5 minutes after injury. These data suggest that significant numbers of axons that were spared by the original laser ablation succumbed to secondary degeneration in a delayed fashion. We conclude that laser ablation is a useful model to examine spinal microglial activation and changes in myelin and axons in real-time using two-photon microscopy. Studies are currently underway to examine the effects of microglial modulators on axon and myelin sparing following axonal injury.
Title: Shedding light on brain mitochondrial function in vivo under systemic or local oxygen deficiency
Authors: *A. MAYEVSKY1,2,3, E. BARBIRO-MICHAELY4,2, M. MANDELBAUM-LIVNAT4,2, A. LIVNAT4,2; 1Ramat-Gan, Israel; 2The Leslie and Susan Gonda Multidisciplinary Brain research center, 3The Mina & Everard Goodman Fac. of Life-Sciences, 4The Mina & Everard Goodman faculty of life sciences, Bar-Ilan Univ., Ramat-Gan, Israel
Abstract: Normal mitochondrial function is a critical factor in maintaining cellular homeostasis in the brain as well as in various organs of the body. Due to the involvement of mitochondrial dysfunction in many pathophysiological conditions, real-time in vivo monitoring of the mitochondrial metabolic state is crucially important. This type of monitoring in animal models as well as in patients provides real-time data that can help interpret experimental results or optimize patient treatment. The monitoring of NADH redox state in the brain provides the most important information on the metabolic state of the mitochondria in terms of energy production and intracellular oxygen levels. This study presents the responses of the rat cerebral tissue metabolic and hemodynamic state, to systemic as well as focal ischemia. The models that were tested included systemic hemorrhage and middle cerebral artery occlusion (MCAO). Hemodynamic changes were evaluated by laser Doppler flowmetry and NADH by the fluorometric technique. Hemorrhage was induced in rats (n=9) by bleeding until mean arterial pressure (MAP) decreased to 40mmHg. Then after there was no further interference for 30 minutes after which the withdrawn blood was re-infused and monitoring proceeded for 2 hours. Focal cerebral ischemia (MCAO) was induced according to a modified standard protocol (n=9) with several modifications. The MCA was occluded for 10 minutes, following by 60 minutes of reperfusion. Cerebral blood flow (CBF) and NADH were recorded from the lateral site (core) and the medial site (penumbra) on the right hemisphere. In the hemorrhagic model, although the small intestine showed significant deteriorations both in tissue blood flow and mitochondrial function during the beginning of the hemorrhagic phase, the brain was protected and NADH remained stable. Whereas, following focal cerebral ischemia (MCAO) CBF levels decreased and NADH increased in both core and penumbra although the deterioration was larger in the core compared to the penumbra. In both models mitochondrial dysfunction was associated with decreased cerebral perfusion and full recovery was associated with full reperfusion. In addition, the results from the hemorrhagic model points towards the advantage of monitoring NADH and tissue blood flow in less-vital organs, such as the small intestine, versus vital organs such as the brain, in order to detect systemic deterioration in an early phase before vital organs are affected. In conclusion, these results demonstrate the potential of mitochondrial NADH monitoring for the evaluation of tissue viability under systemic as well as focal deterioration.
Title: ABCD1 deficiency impairs mononuclear phagocytic cells: implications for neurodegeneration
Authors: *F. EICHLER, K. GAROFALO, B. SCHMIDT, N. ELPEK, T. MEMPEL, J. EL KHOURY; Massachusetts Genl Hosp., BOSTON, MA
Abstract: ABCD1 is an ATP-binding cassette transporter protein involved in fatty acid metabolism. Mutations in ABCD1 cause X-linked adrenoleukodystrophy (ALD), a genetic disorder of the peroxisome associated with devastating inflammatory demyelination in childhood. The role of ABCD1 in other neurological disorders is unknown. We previously reported that microglial apoptosis in perilesional white matter represents an early stage in lesion evolution in cerebral ALD. We investigated the peripheral mononuclear and brain microglial cell response in a mouse model with targeted inactivation of the ABCD1 gene. Using a sterile peritonitis model we assessed monocyte recruitment in ABCD1 -/- mice. We also investigated the role of ABCD1 in acute experimental autoimmune encephalitis (EAE). In order to study ABCD1-/- brain microglia in vivo, we crossed the ABCD1 -/- mouse with a mouse in which the chemokine receptor CX3CR1 has been replaced by the GFP reporter gene. Using in vivo two-photon microscopy we then studied the response of brain microglia to laser injury. In all three scenarios mononuclear phagocytic cells demonstrated aberrant function. Following thioglycolate stimulation only 50% of cells were found within the peritoneum of the ABCD1-/- mouse (30% macrophages compared to 60% in wild type). The ABCD1-/- mouse also showed an attenuated response to EAE induction. This manifested in both a delayed onset by 1-2 days and decreased severity of disability (mean maximum score of 1.9+/-0.33 versus 4.6+/-0.4). Lastly, following laser injury ABCD1 -/- microglial processes showed defective convergence of processes upon the site of injury as measured by difference in fluorescence intensity. These observations confirm that there is an aberrant innate immune response in ALD. These data also suggest that ABCD1 could be involved in regulating the mononuclear phagocytic response in other neurodegenerative disorders. We are in the process of investigating the molecular pathways disrupted by deficiency of the ABCD1 protein.
A Primer on the brai n and nervous system
Del Mar Photonics - Newsletter Fall 2010 - Newsletter Winter 2010
To request information about Del Mar Photonics lasers for applications in laser imaging, laser scanning, laser ablation, speckle imaging, laser capture microdissection, laser axotomy, laser Doppler flowmetry, laser injury and other techniques please contact our neuroscince application group
Del Mar Photonics, Inc.
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