This trip would not have been possible without the help of my thesis advisor, Dr. 150 4.5 Visual function is differentially affected in KO and control mice exposed to graded IOP elevation.
Astrocyte Structure and Function
- Astrocyte Characterization
- Astrocyte Function
- Astrocyte Stress Response
- Astrocyte Gap Junctions and Chemical Synapses
However, astrocytes are vital in maintaining homeostasis and general function of the nervous system. Changes in the metabolic profile of the brain are often associated with neuropathology and are explored in more detail in subsequent chapters.
Neuron/Astrocyte Metabolic Interactions
- Glucose Distribution and Uptake
- Glycogen
- Canonical Glucose Metabolism: Glycolysis and Mitochondrial Oxidative
- Lactate Shuttle
- Alterations During Disease
Due to the limited nature of energy stores in the brain, neurons are highly dependent on an uninterrupted supply of energy substrates from the circulation. This reaction is the source of the glycosyl residues that donate to the glycogen molecule.
Neuronal Alterations in Early Neurodegeneration
Transport Deficits
In contrast, dynein family members move toward the minus end, thus mediating retrograde transport. Any damage to mitochondria or related changes in their distribution can have a profound effect on axonal transport and thus maintenance of distal parts of a.
Mitochondrial Changes
This is supported by the fact that non-coding regions of mtDNA in the brain accumulate two to four times more mutations than coding regions176. This resulted in increased DNA damage in the same age-regulated genes, and was partially reversed by the antioxidant vitamin E.
Morphological Alterations
This correlates with human findings demonstrating that oxidative damage plays an important role in the cognitive decline that accompanies aging180. Within the axonal compartment, early changes include changes in the ratio of oligodendrocytes to neurons.
Optic Neuropathies
The Visual System
Bipolar cells provide a graded signal to retinal ganglion cells (RGCs), which then translate that signal into action potentials transmitted through the optic nerve to multiple brain regions. The first and most predominant are the Müller glia, which extend vertically through the retina from the distal border of the outer nuclear layer to the inner edge of the retina.
Links between Optic Neuropathies and Other Neurodegenerative Diseases
Glaucoma Pathogenesis and Study
- Clinical Presentation of Glaucoma
- RGCs and their Susceptibility to Glaucomatous Neurodegeneration
- Modeling Glaucoma in Rodents
- Inter-orbital Interactions
In the healthy eye (left), aqueous humor flows between the lens and iris through the pupil and exits the eye through draining structures in the iridocorneal angle (see inset, left). Astrocytes in the contralateral retina also express significantly more GFAP and occupy a larger portion of the retina260,261.
Connexin 43 and Neurodegeneration
Connexins
Gap junctions are degraded after both junctional membranes are internalized in one of the adjacent cells269. There is still debate as to whether the connexosome is the only mechanism through which the hole.
Metabolite Transfer Through Gap Junctions
Oxidative stress is the result of an imbalance between reactive oxygen species (ROS) generated as a result of energy expenditure and antioxidant activity, which is supposed to reduce their impact. Whether gap junction remodeling is protective or detrimental likely depends on the severity of the stressor and the length of time the cell is exposed to it.
Connexin Expression in the Nervous System
A main function of this network is the transport of lactate between astrocytes and the neuronal axons that oligodendrocytes surround6. A prevailing hypothesis states that microglial connexins largely serve as a link with extracellular space through which microglia can monitor the prevailing conditions in the region in which they reside.
Hypothesis and Specific Aims
Central Hypothesis
Aims
Through the experiments in Aim 2, we determined the necessary components of the mechanism for the redistribution of metabolites during early chronic stress. In objective 3, we assessed the impact of redistribution of metabolites on axonal structure and function in both visual streams.
Introduction
Early RGC axonopathy in animal models of glaucoma is characterized by enlargement of the optic nerve concurrent with expansion of individual axons, accumulation of. Astrocyte remodeling in early stages of neurodegeneration is closely linked to both changes in metabolic capacity and energy reserves.
Materials and Methods
- Animals
- Tissue Preparation and Imaging
- Nerve and Axon Quantification
- Quantification of Glial Area and Process Organization
- Tracing Anterograde Axonal Transport
- Statistical Analysis
Finally, the outer border of the nerve was demarcated from the original mounting and eroded to exclude pia and dura mater. This took into account any irregularities in the shape of the outer border of the nerve, creating a consistent area for each division.
Results
Optic Nerve and Axon Expansion Precede Axon Loss
Density was then compared to the average axon cross-sectional area for increasingly inclusive areas of density (Figure 2.2). This is about twice the smallest average axon area in our sample.
Retraction of Astrocyte Processes with Early Axon Expansion
Next, we calculated the CoM of the glial distribution for nerves with average axon areas below 0.50 m2 (Figure 2.5, left panel). This is consistent with movement of glial processes out of the center and toward the edge of the nerve, as shown for the nerve in Figure 2.4B.
Optic Nerve Remodeling Involves Diminished Astrocyte Organization
First, each nerve was rotated to maximize the orientation of the glial processes along the horizontal axis (Figure 2.7A, B). The skeletons were decomposed by removing branch points, resulting in a population of vectors representing the orientation of each segment of astrocytic processes (Figure 2.7E).
Parallelism Reflects Early Axonal Changes
Accordingly, because nerve expansion predicts both axonal loss and axon expansion190, increased axon size (Figure 2.10D), decreased axon density (Figure 2.10E), and loss of total axon number are associated with reduced parallelism (Figure 2.10F). All data collected from a subset of nerves with the highest axon sampling density below the axon size threshold at which axon loss occurs/mm2).
Astrocyte Cytoskeletal Reorganization Depends on Location in the Nerve
DBA/2J nerves contain significantly altered GFAP CoM from D2 control nerves, and these differences occur most frequently within distal nerve segments (*; p < 0.029). Furthermore, within distal DBA/2J nerves, GFAP tends toward less parallelism than the equivalent segment of D2 control nerves (p = 0.09).
Parallelism Reflects both Astrocyte Connectivity and RGC Axonal Function
Finally, the CoM for distal segments of the DBA/2J nerve indicated edge-distributed astrocytes with increased parallelism (p = 0.005), while CoM in D2 nerves showed the opposite trend (Figure 2.12G), although not significant (p = 0 .07). Top left: Coronal section through superior colliculus of DBA/2J mouse (between white dotted lines) after intravitreal injection of CTB (green).
Early Axon Expansion involves Diminished Mitochondrial Density
For example, a small four-month-old nerve with a small axon size had mitochondria distributed throughout both axons and glial processes within the inter-axonal space (Figure 2.15A). When quantified from multiple electron micrographs of nerves with high axon density, we found that axon expansion was strongly associated with reduced mitochondrial density (Figure 2.15D).
Discussion
As parallelism increased in the distal segment, astrocytes were redistributed toward the neural crest, as indicated by a lower CoM (Figure 2.12G). In contrast, levels of Cx43 in the proximal segment predicted parallelism with no clear relationship to transport (Figure 2.14A).
Introduction
Furthermore, the optic nerve head is the location likely to be most affected by sensitivity to intraocular pressure (IOP), one of the most important risk factors. Functional and structural deficits arise early in progression in the optic nerve, even if the cell body and axon segment persist in the retina.
Materials and Methods
- Generating GFAP-Cre-ER t2 x Cx43 flox/flox Mice
- Genotyping GFAP-Cre-ER t2 x Cx43 flox/flox Mice
- Inducing and Confirming Cx43 Excision in GFAP-Cre-ER t2 x Cx43 flox/flox Mice
- Immunohistochemistry in Retinal Wholemount Tissue
- Immunohistochemistry of Retinal and Optic Nerve Cryosections
- Induction of Ocular Hypertension by Microbead Occlusion
- Glycogen Assay
- Positron Emission Tomography
- Optic Nerve Transection
- Compound Action Potential
- Statistical Analysis
Crosses resulted in a 1 in 2 chance of the desired genotype, which at this stage is a mouse that is hemizygous for a GFAP-Cre-ERt2 and heterozygous for Cx43flox (Figure 3.1). In our initial cross, we used a mouse hemizygous for GFAP-Cre-ERt2 (parent 1) and crossed with a mouse homozygous for Cx43flox.
Results
- Tamoxifen Induces Cx43 Knockout in GFAP-Cre-ER t2 x Cx43 flox/flox Mice
- Glycogen Stores Diminish Bilaterally After Unilateral IOP Elevation
- Astrocyte-Specific Cx43 KO Reverses Glycogen Distribution Pattern after Chronic
- Unilateral IOP Elevation Results in Energy Transfer between Normal and
- Energy Transfer between Normal and Glaucomatous Optic Projections Occurs
- Energy Transfer During Unilateral Glaucomatous Stress Involves Astrocyte Connexins
- Energy Transfer between Optic Projections Causes Functional Deficits in Optic Nerve
- Astrocyte-Specific Cx43 Knockout Rescues Functional Deficits in Optic Nerve Signaling
IOP was significantly elevated ipsilaterally in microbeads (Δ) compared to contralateral eyes (o) in all conditions (p<0.001). Two weeks of unilateral IOP elevation resulted in decreased ability to survive glucose depletion bilaterally (Figure 3.15C).
Discussion
Although glycogen levels were decreased in both the microsphere and the contralateral nerve, glycogen was significantly lower in the microsphere compared to the contralateral nerve. In fact, rescue in the contralateral nerve far exceeded the signal deficit in the ipsilateral nerve.
Introduction
Rapid transport of growth factors250307 as well as metabolites276 between cells and brain regions are critical for maintaining neuronal function during stress. The immune response also appears to be activated in many interconnected regions of the CNS even during a focal event262.
Materials and Methods
- Induction of Ocular Hypertension by Microbead Occlusion in Staggered and Bilateral
- Tracing Anterograde Axonal Transport
- Glycogen Assay
- Psychophysical Visual Testing: Optomotor Response
- Statistical Analysis
Serial sections of the superior colliculus were imaged using a Nikon Eclipse TI microscope (Nikon . Instruments) and CTB fluorescent signal intensity was measured using custom ImagePro routines (Media Cybernetics) as previously described153,256. Data for are presented as mean ± standard error of the mean (SEM) for each treatment.
Results
- IOP Elevation in the Staggered Paradigm
- Glycogen Does Not Redistribute after Staggered IOP Elevation
- Anterograde Axonal Transport Function is Differentially Impacted in KO and Control
- Visual Function is Differentially Impacted in KO and Control Mice Exposed to
In the 2-1 staggered paradigm, only the nerve exposed to 1 week of IOP elevation contained less glycogen than bilateral naïve nerves. Furthermore, nerves in the offset paradigm exposed to 2 weeks of IOP elevation contained significantly more glycogen than their ipsilateral counterparts.
Discussion
One-week unilateral elevation of IOP did not significantly reduce anterograde transport function in control mice in either visual stream (Figure 4.4). KO mice showed more severe pathology; two weeks of elevated IOP resulted in a 45.6% loss of anterograde transport function compared to contralateral naïve.
Significance
Furthermore, the optic nerve head, the region through which unmyelinated RGC axons travel, is the site most directly affected by intraocular pressure (IOP). Therefore, not only is the optic nerve a prime model for studying interactions between astrocytes and axons in response to stress, but it is also a prime target for protective therapies in glaucoma.
Aim 1
Outcomes
As parallelism increased in the distal segment, astrocytes redistributed toward the nerve edge, as indicated by a bearing. In the distal segment, nerves with increased Cx43 also showed higher levels of intact transport, but showed no association with parallelism (Figure 2.14B).
Future Directions
Could these calcium waves generate enough of a stimulus to dictate both the necessity and directional remodeling needed to maintain the integrity of the optic nerve. Further, a greater understanding of the astrocyte response in particular may provide avenues for protective therapies for those in the early stages of pathology.
Aim 2
Outcomes
Transport of 18F-FDG by performing PET experiments in mice, each of which underwent a unilateral microbead injection and optic nerve transection in the contralateral projection. We hypothesized that metabolic isolation of each nerve from the stores of the other might rescue the vulnerability to metabolic stress in the contralateral nerve.
Future Directions
We observed radioactivity in the brain after 18F-FDG injection into one eye of WT mice. An interesting future experiment to determine the contribution of brain metabolic networks to metabolic redistribution would be to transect the optic chiasm sagittally, maintaining two intact and distinct optic projections that no longer cross.
Aim 3
Outcomes
Two weeks of IOP elevation in KO mice caused a 45.6% loss in anterograde transport function compared to the contralateral naïve nerve. Moreover, transport capacity between the KO projections exposed to 2 weeks of height in the staggered paradigm and the unilateral KO 2-week projections was not statistically significant.
Future Directions
Glial cell types
General astrocyte subtypes
The many functions of astrocytes
Grades of astrocyte reactivity following injury
Glucose processing pathways
Glycogen synthesis and breakdown
Key astrocyte/axon metabolic components
Basic retinal circuitry
Murine optic projections
Production and outflow of aqueous humor in the eye
Optic disc cupping in human glaucoma patients
Key events in RGC glaucomatous progression
A potentially neuroprotective astrocyte function during early neurodegeneration
Summary of Aims
Axons enlarge prior to degeneration
Axon expansion eventually leads to loss
Increased axon size is accompanied by astrocyte withdrawal
Method to quantify changes in glial distribution
Distribution of glial ramification as axons expand
Pathology alters glial orientation in the optic nerve
Deconstruction of astrocyte processes into representative vectors
Method to quantify overall parallelism of glial processes
Measuring the spatial distribution of astrocyte processes
Parallelism diminishes with markers of axon degeneration
Astrocytes become more parallel as axons expand early prior to loss
Astrocyte GFAP redistributes throughout the optic nerve
Diminished anterograde transport in a sample of DBA/2J nerves
RGC axon functional deficits are associated with astrocyte connectivity in DBA/2J optic
Mitochondrial density decreases as mean axon area increases
Astrocytes and the energetic environment of the myelinated optic nerve through
Generating GFAP-Cre-ER t2 x Cx43 flox/flox mice, cross 1
Generating GFAP-Cre-ER t2 x Cx43 flox/flox mice, crosses 2a and 2b
Genotyping GFAP-Cre-ER t2 x Cx43 flox/flox mice, final products
Genotyping GFAP-Cre-ER t2 x Cx43 flox/flox mice for excision, final products
Primers
GFAP-Cre ER T2 Master Mix
Cx43 flox Master Mix
PCR Reaction Program
Cx43 flox Excision Master Mix
