My goal is to discuss DAI and its upstream and downstream consequences through the use of traditional as well as modern imaging approaches, with emphasis on the unique capabilities of modern imaging for unmasking this complex pathobiology.
YFP under the control of Thy1 promoter (neuron specific)
Transgene insertion location and/or copy number results in unique expression patterns
YFP-H expresses YFP throughout cerebrum, but..
In neocortex expression is limited to Layer V pyramidal neurons
Feng et al., 2000
Mouse Central Fluid Percussion Injury Model
A circular craniotomy made along sagittal suture midway between bregma and lambda
Leur-Loc syringe hub affixed to craniotomy site using cyanoacrylate
Moderate severity (1.65-1.75)
No intraparenchymal hemorrhage
Utilization of YFP-H Mice Allows the Continued Visualization of Axonal Damage and its Sequelae Over Time 24H 24H
cFPI in Thy1-YFP-H Mice Results in TAI in Layer V Greer et al. J. Neurosci. 31:5089, 2011
Sham 1h 3h 12h 24h 48h Wang et al. J. Neurotrauma, 8: 2011 P321
12 hour 200 μm 20μm 20μm
Scale Bar: 500µm
Technique Extends the Temporal Window for Studying Axotomized Processes and Sustaining Somata 30m
Phospho-c-Jun Expression is Induced Preferentially within Layer V Pyramidal Neurons Sustaining TAI Greer et al. J. Neurosci 30: 5089,2011
Phospho-c-Jun Expression is Induced Preferentially within Layer V Pyramidal Neurons Sustaining TAI
TAI Induces Persistent Neuronal Atrophy
Importantly, in some cases, these Fluorophore-containing elements can be targeted for TEM analysis.
Specifically, antibodies to the Alexa 488 Fluorophore can be used with subsequent use of avidin/biotin/peroxidase strategies to generate an electron dense reaction product
Alternatively, antibodies with the appropriate chromagen targeted to the protein of interest can be used to yield an electron dense reaction product
Ultrastructural Evidence of Reactive Change
The use of antibodies to fluorophores with their subsequent conversion to an electron dense reaction product Farkas et el. J. Neurosci 26:3130, 2006 VCU
Alternatively, photoconversion using confocal laser scanning microscopy can be employed for subsequent TEM analysis.
Specifically, using region of interest scanning capabilities of the confocal microscope, together with an acousto-optical filter a controlled and regional specific reaction product can be generated in the presence of diaminobenzidine.
Tozer et al. J. Neurosci. Methods 164:240, 2006
Bioimaging in determining drug targets
Ca 2+ Na + Na + Ca 2+ Procaspase 9 Procaspase 3 Calpain Calcineurin Cyto c Apaf-1
Illuminating brain tissue protein interactions using confocal and two-photon excitation fluorescent resonance energy transfer microscopy-FRET Mills et al. J. Biomed Optics 8:347-356
Ultrafast Pulsed Lasers for MPLSM High peak power Low average power Short pulse width
average power laser = 10 mW
pulse rate = 100 MHz
pulsewidth = 100 fsec.
energy per pulse = average power/pulse rate = 0.1 nJ
peak power = energy per pulse/pulsewidth = 1000W
duty cycle = pulsewidth x pulse rate = 0.001%
period (time between pulses) = 1/pulse rate = 100 msec
Multi-photon (2-photon) laser scanning microscope
fixed stage upright (in situ / physiology)
3 confocal detectors
(2 descanned + META)
2 direct (non-descanned) detectors
transmitted light detector
Argon (458, 476, 488, 514 nm)
yellow diode (561 nm)
red HeNe (633 nm)
Ti:sapphire (710 - 990 nm) (MP)
high NA (oil & water) IR objective lenses
+ dipping IR objective lenses
standard translating stage
Luigs & Neumann 380FM workstation
AMPI Master 8 VP timing unit
Multiclamp 700 amplifier
pre-amplifier - A/D board
peristaltic perfusion pump
Zeiss LSM 510 META NLO
Spinning Disc confocal microscope
High-speed multi-dimensional confocal imaging
Ca 2+ imaging
Axotomized and intact neurons are easily identified in the living slice and labeled with biocytin during whole cell patch clamp recordings Axotomy at both short (A) and long (B, C) distances from the soma could be easily identified by the bulb on the axon in the live slice prior to patch clamping recordings. Two focus levels of same image are seen in B. Greer & Jacobs, Poster 320
Both intact and axotomized neurons are healthy Parameters shown are typically measured when assessing the physiological health of recorded neurons. For these ‘health’ parameters, injured neurons were not different from controls. Although there was a trend towards an increase in input resistance for some injured neurons, suggesting possible cell shrinkage, this was not significant. Data shown are from the following number of neurons: 15 control, 9 axotomized and 6 intact one day after injury, and 6 axotomized and 6 intact two days after injury.
Axotomized neurons show a decrease in intrinsic excitability at 1 day that recovers by 2 days after injury. Intact neurons show an increase in intrinsic excitability at 2 days post injury Membrane properties reflecting intrinsic excitability show a decrease for axotomized neurons one day after injury and an increase for other groups relative to controls. During depolarizing current steps, intact neurons from injured brains tended to fire at increased frequencies relative to control cells (A, B). A shows examples from individual neurons. The slope of the plot of frequency versus injected current was significantly different between experimental groups (B, 1-way ANOVA, p<0.05, LSD post-hoc). The rheobase (lowest current to produce action potentials) was significantly greater in axotomized neurons one day after injury (C, 1-way ANOVA, p<0.05, LSD post-hoc). The primary instantaneous frequency = 1/(interspike interval for first 2 action potentials responding to injected current). Only non-doublet RS neurons were used to calculate this measure. There was a trend toward a lower frequency for axotomized neurons one day after injury (D-F), while there was a significant increase in this measure for intact neurons two days after injury (D-F, 1-way ANOVA, p<0.05, LSD post-hoc). D shows examples of the response to 400 (upper) and 200 (lower) pA current injections. E shows examples of the plot of primary frequency versus current injection for individual neurons. Data shown are from the following number of neurons: 15 control, 9 axotomized and 6 intact one day after injury, and 6 axotomized and 6 intact two days after injury.
Andras Buki, M.D. Ph.D.
Daniel Erb, Ph.D.
Orsi Farkas, M.D. Ph.D.
Guoyi Gao, M.D
John Greer, M.D. Ph.D. student
Brian Kelley, M.D. Ph.D.
Hiro Koizumi, M.D. Ph.D.
Jonathan Lifshitz, Ph.D.
Tina Marmarou, Ph.D.
Melisa McGinn, Ph.D.
Yasukata Oda, M.D. Ph.D.
David Okonkwo, M.D. Ph.D.
Ted Pettus, Ph.D.
Linda Phillips, Ph.D
Tom Reeves, Ph.D.
Rich Singleton, M.D. Ph.D.
James Stone, M.D. Ph.D.
Eiichi Suehiro, M.D. Ph.D.
Yugi Ueda, M.D. Ph.D.
Joan Wang, M.D.
Enoch Wei, Ph.D.
Image formation in TEM
column under vacuum
electron beam focused by magnetic lenses
electrons interact with stained sample
elastic (2) & inelastic (3) scattering of electrons
scattered electrons blocked by aperture
non-scattered electrons (1) strike phosphorescent screen or film or CCD
APP positive swelling and disconnection
Kelley et al., Poster Number 64
Proximal YFP Swellings, Not Distal Contain -APP
CALPAIN MEDIATED SPECTRIN PROTEOLYSIS (CMSP) AND NFC CO-LOCALIZE AT FOCAL SITES OF TAI Calpain has been shown to target spectrin a major structural link of the actin cytoskeleton to the axolemma. AB38 (CMSP) and NFC show a co-localization. Buki et al., 1999
Early Reactive Sprouting in Phospho-c-Jun+ Neurons
Evidence of Reactive Sprouting Early Post Injury * * * ** ** * ** * * C A 2D
Technique Extends the Temporal Window for Studying Axotomized Processes and Sustaining Somata 14D 14D 7D 7D * *
Early Morphological Evidence for Regeneration at Later Time Points Post-Injury
DIFFERENTIAL RESPONSE OF AXONS TO TBI Stone, J.R., Singleton, R.H., and Povlishock, J.T. Experimental Neurology 172, 320 – 331 (2001) C-APP RMO14
YFP-positive neurons show a variety of action potential firing patterns. Two days after injury, intact neurons are less likely to show bursts of action potentials Most YFP recorded neurons were non-adapting, non-doublet regular-spiking (RS) pyramidal neurons. A small percentage of YFP-labeled neurons were of the previously described intrinsically-bursting (IB) type (A, B, Connors, 1990). Some RS neurons had an initial doublet of action potentials, followed by non-adapting, single action potentials (C). These were similar to those previously reported for YFP-H neurons (Yu, 2008). Non-doublet RS neurons were also similar to those previously described for layer V somatosensory neurons in YFP-H mice (D, Sugino). A series of hyperpolarizing currents produced rectification and a prominent sag of the voltage response that is likely due to hyperpolarization-activated current (E arrow). A transient calcium (T) current also appeared to be present in some cells as indicated by the action potentials produced after return to rest from hyperpolarization (E). All examples shown (A-E) are from controls. Intact neurons recorded two days after injury, only produced single action potentials, thus this population had a significantly different percentage of the three cell types compared to the control population (F, z-test, p<0.05). The other injured groups showed these three neuronal types in percentages similar to controls. Data shown are from the following number of neurons: 20 control, 10 axotomized and 8 intact one day after injury, and 7 axotomized and 6 intact two days after injury. The IB cell type was excluded from subsequent analyses.
Changes in intrinsic properties of regular-spiking neurons suggest modifications of potassium channels Action potential amplitude was measured with respect to the -60 mV resting membrane potential (RMP), where the cells were held with small constant depolarizing or hyperpolarizing currents when necessary. The action potential amplitude was increased in axotomized neurons one and two days after injury and in intact neurons one day after injury (A, B). Lower dashed line in A indicates -60 mV resting membrane potential, while upper dashed line identifies the peak for the control cell shown. There was no change in either action potential rise time (C) or action potential threshold (D), measures sensitive to changes in numbers or density of sodium channels. The after-hyperpolarization (note only non-doublet RS cells were used for this measure) was decreased in the same groups showing an increase in action potential amplitude (E, F), suggesting a possible reduction in potassium channels. Data shown are from the following number of neurons: 15 control, 9 axotomized and 6 intact one day after injury, and 6 axotomized and 6 intact two days after injury.
better speed (acquisition speed limited by detector rate)
( i.e. CCD off-loading)
compatible with variety of illumination sources
poor light throughput
fixed pinhole size
better lateral resolution
better light throughput
large detection area
low dark current
high dynamic range
rapid refresh rate
slower acquisition time
Optimization of confocal images
Choice of mounting & immersion media
Choice of coverslip thickness
Selection of high N.A. objective lens
Correction for cross-talk
Selection of scan resolution
Selection of acquisition bit depth
Selection of scan speed
Averaging of acquired image
Elimination of background autofluorescence
Cyclosporin A attenuates both mitochondrial injury…
Mitochondria: Death Switch of the Cell? MPTP Smac/Diablo AIF P. Sullivan Death Triggers Ca 2+ Glutamate Bax Oxidants Caspases Cyto C Cyto C Caspase 9 Apaf-1 dATP Downstream Caspases APOPTOSIS NECROSIS ATP ROS