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Progressive ganglion cell loss and optic nerve degeneration in DBA/2J mice is variable and asymmetric.

Schlamp CL, Li Y, Dietz JA, Janssen KT, Nickells RW - BMC Neurosci (2006)

Bottom Line: Degeneration appears to follow a retrograde course with axons dying from their proximal ends toward the globe.Some nerves also exhibit focal preservation of tracts of axons generally in the nasal peripheral region.Similar to what we observe in the optic nerves, ganglion cell loss is often asymmetric between the eyes of the same animal.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, WI, USA. clschlamp@wisc.edu

ABSTRACT

Background: Glaucoma is a chronic neurodegenerative disease of the retina, characterized by the degeneration of axons in the optic nerve and retinal ganglion cell apoptosis. DBA/2J inbred mice develop chronic hereditary glaucoma and are an important model system to study the molecular mechanisms underlying this disease and novel therapeutic interventions designed to attenuate the loss of retinal ganglion cells. Although the genetics of this disease in these mice are well characterized, the etiology of its progression, particularly with respect to retinal degeneration, is not. We have used two separate labeling techniques, post-mortem DiI labeling of axons and ganglion cell-specific expression of the betaGeo reporter gene, to evaluate the time course of optic nerve degeneration and ganglion cell loss, respectively, in aging mice.

Results: Optic nerve degeneration, characterized by axon loss and gliosis is first apparent in mice between 8 and 9 months of age. Degeneration appears to follow a retrograde course with axons dying from their proximal ends toward the globe. Although nerve damage is typically bilateral, the progression of disease is asymmetric between the eyes of individual mice. Some nerves also exhibit focal preservation of tracts of axons generally in the nasal peripheral region. Ganglion cell loss, as a function of the loss of betaGeo expression, is evident in some mice between 8 and 10 months of age and is prevalent in the majority of mice older than 10.5 months. Most eyes display a uniform loss of ganglion cells throughout the retina, but many younger mice exhibit focal loss of cells in sectors extending from the optic nerve head to the retinal periphery. Similar to what we observe in the optic nerves, ganglion cell loss is often asymmetric between the eyes of the same animal.

Conclusion: A comparison of the data collected from the two cohorts of mice used for this study suggests that the initial site of damage in this disease is to the axons in the optic nerve, followed by the subsequent death of the ganglion cell soma.

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Summary of the scoring criteria for DiI-labeled nerves and X-gal stained retinas. (A) Exemplar of scored optic nerves. Only the left nerve is shown for 5 individual mice. The scores range from 1 for label from the globe to the chiasm, to 5 for no signs of label. Younger mice typically exhibited nerves that were scored 1–2, while older mice typically had nerves showing some level of degeneration (3–5). Bar = 0.5 mm. (B) A photomicrograph of a retina (OD) stained for βGEO activity taken from a 10.5 month old mouse. This particular example appears to have 2 wedges of cell loss, one superior and one temporal, at different stages of degeneration. (C) A cartoon of a flatmounted retina where each quadrant is separated into 4 regions and given a score based on the intensity of stain present. The quadrants represented are (clockwise): SN, superonasal; IN, inferonasal; IT, inferotemporal; ST, superotemporal. The scores in each region of the quadrants represent the scores given by 1 masked observer for the retina in (B). The average of all the scores in each quandrant then becomes the total score for that quadrant, and the average of these 4 scores becomes the final total score for that retina.
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Figure 11: Summary of the scoring criteria for DiI-labeled nerves and X-gal stained retinas. (A) Exemplar of scored optic nerves. Only the left nerve is shown for 5 individual mice. The scores range from 1 for label from the globe to the chiasm, to 5 for no signs of label. Younger mice typically exhibited nerves that were scored 1–2, while older mice typically had nerves showing some level of degeneration (3–5). Bar = 0.5 mm. (B) A photomicrograph of a retina (OD) stained for βGEO activity taken from a 10.5 month old mouse. This particular example appears to have 2 wedges of cell loss, one superior and one temporal, at different stages of degeneration. (C) A cartoon of a flatmounted retina where each quadrant is separated into 4 regions and given a score based on the intensity of stain present. The quadrants represented are (clockwise): SN, superonasal; IN, inferonasal; IT, inferotemporal; ST, superotemporal. The scores in each region of the quadrants represent the scores given by 1 masked observer for the retina in (B). The average of all the scores in each quandrant then becomes the total score for that quadrant, and the average of these 4 scores becomes the final total score for that retina.

Mentions: Axons in the optic nerves of D2 mice were labeled post-mortem with DiI crystals (Molecular Probes, Eugene, OR) using a modification of the procedure described by Plump et al [28]. Briefly, adult mice between 6 and 21 months of age were euthanized. The heads were removed and fixed in 4% paraformaldehyde in Phosphate Buffered Saline (PBS) for 1 hr at room temperature. After fixation, the heads were skinned and a 270° incision was made around the circumference of the globe at the limbus of each eye to allow the corneas to flip open. The lenses were removed, and crystals of DiI were embedded into the optic nerve head of each eye using watchmaker's forceps. To keep the crystals in place, the lenses were replaced and the corneas folded back into position. Heads were incubated for 2 weeks in PBS containing 0.1% sodium azide at 37°C to allow the DiI to diffuse along the axon plasma membranes. After incubation, the skullcap and underlying brain tissue was removed to expose the optic nerves from the globe to the chiasm. Fluorescent label in the nerves was visualized using a Leica MZ FL III fluorescent dissecting microscope with a digital camera attachment (Leica, Bannockburn, IL). To estimate the staining intensity of each nerve, individual nerves were scored by 2 masked observers using a 5 point scale ranging from label extending from the globe to the optic chiasm (score of 1) to no detectable label in the entire nerve (score of 5). An exemplar of the 5 different scores is shown in Figure 11. A weighted Kappa statistic showed a high level of agreement between the 2 observers (κ = 0.818, 95% CI = 0.697 to 0.939).


Progressive ganglion cell loss and optic nerve degeneration in DBA/2J mice is variable and asymmetric.

Schlamp CL, Li Y, Dietz JA, Janssen KT, Nickells RW - BMC Neurosci (2006)

Summary of the scoring criteria for DiI-labeled nerves and X-gal stained retinas. (A) Exemplar of scored optic nerves. Only the left nerve is shown for 5 individual mice. The scores range from 1 for label from the globe to the chiasm, to 5 for no signs of label. Younger mice typically exhibited nerves that were scored 1–2, while older mice typically had nerves showing some level of degeneration (3–5). Bar = 0.5 mm. (B) A photomicrograph of a retina (OD) stained for βGEO activity taken from a 10.5 month old mouse. This particular example appears to have 2 wedges of cell loss, one superior and one temporal, at different stages of degeneration. (C) A cartoon of a flatmounted retina where each quadrant is separated into 4 regions and given a score based on the intensity of stain present. The quadrants represented are (clockwise): SN, superonasal; IN, inferonasal; IT, inferotemporal; ST, superotemporal. The scores in each region of the quadrants represent the scores given by 1 masked observer for the retina in (B). The average of all the scores in each quandrant then becomes the total score for that quadrant, and the average of these 4 scores becomes the final total score for that retina.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1621073&req=5

Figure 11: Summary of the scoring criteria for DiI-labeled nerves and X-gal stained retinas. (A) Exemplar of scored optic nerves. Only the left nerve is shown for 5 individual mice. The scores range from 1 for label from the globe to the chiasm, to 5 for no signs of label. Younger mice typically exhibited nerves that were scored 1–2, while older mice typically had nerves showing some level of degeneration (3–5). Bar = 0.5 mm. (B) A photomicrograph of a retina (OD) stained for βGEO activity taken from a 10.5 month old mouse. This particular example appears to have 2 wedges of cell loss, one superior and one temporal, at different stages of degeneration. (C) A cartoon of a flatmounted retina where each quadrant is separated into 4 regions and given a score based on the intensity of stain present. The quadrants represented are (clockwise): SN, superonasal; IN, inferonasal; IT, inferotemporal; ST, superotemporal. The scores in each region of the quadrants represent the scores given by 1 masked observer for the retina in (B). The average of all the scores in each quandrant then becomes the total score for that quadrant, and the average of these 4 scores becomes the final total score for that retina.
Mentions: Axons in the optic nerves of D2 mice were labeled post-mortem with DiI crystals (Molecular Probes, Eugene, OR) using a modification of the procedure described by Plump et al [28]. Briefly, adult mice between 6 and 21 months of age were euthanized. The heads were removed and fixed in 4% paraformaldehyde in Phosphate Buffered Saline (PBS) for 1 hr at room temperature. After fixation, the heads were skinned and a 270° incision was made around the circumference of the globe at the limbus of each eye to allow the corneas to flip open. The lenses were removed, and crystals of DiI were embedded into the optic nerve head of each eye using watchmaker's forceps. To keep the crystals in place, the lenses were replaced and the corneas folded back into position. Heads were incubated for 2 weeks in PBS containing 0.1% sodium azide at 37°C to allow the DiI to diffuse along the axon plasma membranes. After incubation, the skullcap and underlying brain tissue was removed to expose the optic nerves from the globe to the chiasm. Fluorescent label in the nerves was visualized using a Leica MZ FL III fluorescent dissecting microscope with a digital camera attachment (Leica, Bannockburn, IL). To estimate the staining intensity of each nerve, individual nerves were scored by 2 masked observers using a 5 point scale ranging from label extending from the globe to the optic chiasm (score of 1) to no detectable label in the entire nerve (score of 5). An exemplar of the 5 different scores is shown in Figure 11. A weighted Kappa statistic showed a high level of agreement between the 2 observers (κ = 0.818, 95% CI = 0.697 to 0.939).

Bottom Line: Degeneration appears to follow a retrograde course with axons dying from their proximal ends toward the globe.Some nerves also exhibit focal preservation of tracts of axons generally in the nasal peripheral region.Similar to what we observe in the optic nerves, ganglion cell loss is often asymmetric between the eyes of the same animal.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, WI, USA. clschlamp@wisc.edu

ABSTRACT

Background: Glaucoma is a chronic neurodegenerative disease of the retina, characterized by the degeneration of axons in the optic nerve and retinal ganglion cell apoptosis. DBA/2J inbred mice develop chronic hereditary glaucoma and are an important model system to study the molecular mechanisms underlying this disease and novel therapeutic interventions designed to attenuate the loss of retinal ganglion cells. Although the genetics of this disease in these mice are well characterized, the etiology of its progression, particularly with respect to retinal degeneration, is not. We have used two separate labeling techniques, post-mortem DiI labeling of axons and ganglion cell-specific expression of the betaGeo reporter gene, to evaluate the time course of optic nerve degeneration and ganglion cell loss, respectively, in aging mice.

Results: Optic nerve degeneration, characterized by axon loss and gliosis is first apparent in mice between 8 and 9 months of age. Degeneration appears to follow a retrograde course with axons dying from their proximal ends toward the globe. Although nerve damage is typically bilateral, the progression of disease is asymmetric between the eyes of individual mice. Some nerves also exhibit focal preservation of tracts of axons generally in the nasal peripheral region. Ganglion cell loss, as a function of the loss of betaGeo expression, is evident in some mice between 8 and 10 months of age and is prevalent in the majority of mice older than 10.5 months. Most eyes display a uniform loss of ganglion cells throughout the retina, but many younger mice exhibit focal loss of cells in sectors extending from the optic nerve head to the retinal periphery. Similar to what we observe in the optic nerves, ganglion cell loss is often asymmetric between the eyes of the same animal.

Conclusion: A comparison of the data collected from the two cohorts of mice used for this study suggests that the initial site of damage in this disease is to the axons in the optic nerve, followed by the subsequent death of the ganglion cell soma.

Show MeSH
Related in: MedlinePlus