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Axons of retinal ganglion cells are insulted in the optic nerve early in DBA/2J glaucoma.

Howell GR, Libby RT, Jakobs TC, Smith RS, Phalan FC, Barter JW, Barbay JM, Marchant JK, Mahesh N, Porciatti V, Whitmore AV, Masland RH, John SW - J. Cell Biol. (2007)

Bottom Line: We show that proximal axon segments attached to their cell bodies survive to the proximity of the lamina.In contrast, axon segments in the lamina and behind the eye degenerate.These experiments provide strong evidence for a local insult to axons in the optic nerve.

View Article: PubMed Central - PubMed

Affiliation: The Jackson Laboratory, Bar Harbor, ME 04609, USA.

ABSTRACT
Here, we use a mouse model (DBA/2J) to readdress the location of insult(s) to retinal ganglion cells (RGCs) in glaucoma. We localize an early sign of axon damage to an astrocyte-rich region of the optic nerve just posterior to the retina, analogous to the lamina cribrosa. In this region, a network of astrocytes associates intimately with RGC axons. Using BAX-deficient DBA/2J mice, which retain all of their RGCs, we provide experimental evidence for an insult within or very close to the lamina in the optic nerve. We show that proximal axon segments attached to their cell bodies survive to the proximity of the lamina. In contrast, axon segments in the lamina and behind the eye degenerate. Finally, the Wld(s) allele, which is known to protect against insults to axons, strongly protects against DBA/2J glaucoma and preserves RGC activity as measured by pattern electroretinography. These experiments provide strong evidence for a local insult to axons in the optic nerve.

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Wlds profoundly protects from glaucomatous damage. (A) The IOP distributions of mice of each genotype overlap extensively. The boxes show the upper and lower quartiles and the bars show the extremes including outliers. The centerline of each diamond is the mean and the upper and lower points of each diamond represent the 95% confidence intervals of the mean. Mice with a single copy of the Wlds fusion gene are hemizygous (Hemi). At both 9 and 12 mo, there were no significant differences in IOP (ANOVA) between the neuroprotected, Wlds mice (see below), and mice of any other genotype. At 10.5 mo, the wild-type (WT) mice had an unusually high skewing of their IOP compared with typical values at this age (see Anderson et al., 2005; Libby et al., 2005a), and their IOP was significantly higher than mice of all other genotypes (P < 0.04 for all comparisons). This skewing did not account for the decreased axon loss in Wlds mice. This is clear because (1) there was extensive overlap between all genotypes, and (2) the IOP distribution of Wlds mice was extremely similar (P = 0.7) to Bax+/− heterozygotes mice (Het), whose axons were not protected (see below). (B) Axon protection in Wlds mice. Distributions of optic nerve damage in mice of the indicated genotypes are shown at two important time points (Materials and methods). The Wlds allele significantly rescues axons from glaucomatous degeneration at 12 mo of age (P < 0.0001, chi square comparing Wlds Hemi to their WT littermates). Importantly, Wlds increased the number of eyes with no detectable glaucoma. Bax+/− heterozygosity conferred no protective effect when compared with wild-type mice (right). Though not statistically significant, there tended to be a greater degree of protection in D2.WldsBax+/− double mutants (right), compared with Wlds single mutants (left). The number of eyes analyzed for each genotype at 10.5 mo was typically ≥40 but 20 for D2.Bax+/− heterozygotes. At 12 mo, typically ≥63 eyes were studied for each genotype but 21 for D2.Bax+/− heterozygotes. (C–F) Wlds preserves RGC axons and optic nerve morphology as seen in semi-thin sections. Most wild-type (C) and D2.Bax+/− nerves (E) had severe glaucoma with very substantial axon loss (see Fig. 3) and extensive glial scarring. In contrast, the majority of D2.Wlds (D) and D2.WldsBax+/− nerves (F) had no or early glaucoma, with <5% of axons damaged (Fig. 3) and no evidence of glial hypertrophy or scarring. (G) Axon counts for a randomly selected sample of DBA/2J and D2.Wlds nerves with no or early glaucoma (n = 6 for each genotype). The averages are not statistically different (P = 0.235), and none of the D2.Wlds nerves had decreased axon counts. Because Wlds more than doubled the number of eyes with no or early glaucoma, half of these counted eyes were rescued. (H–J) Retinal flatmounts showing that in Wlds protected eyes RGC somata survive (H, preglaucoma; I, severe glaucoma; J, no or early glaucoma). (K) This was confirmed by counting RGC layer cells in eyes with no or early glaucoma. Again, half of the D2-Wlds eyes were rescued and none had cell numbers below the range of wild-type values (n = 10 each genotype, P > 0.1). As expected, in both wild-type and D2.Wlds mice with severe optic nerve damage the majority of RGCs were lost. The retinal images shown are from a matched region of the superior, peripheral retina. (L) Despite the profound rescue, a mild degree of somal shrinkage (∼10%) was seen in D2.Wlds eyes with no detectable glaucoma. The shrinkage appears to occur generally across cells of different sizes (see Fig. S4). (M) Wlds strongly preserved PERG amplitude, a measure of RGC activity, in randomly selected mice (number of eyes = 16, 14, 18 for D2-Gpnmb+, DBA/2J, and D2.Wlds, respectively). Bars, 100 μm.
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fig9: Wlds profoundly protects from glaucomatous damage. (A) The IOP distributions of mice of each genotype overlap extensively. The boxes show the upper and lower quartiles and the bars show the extremes including outliers. The centerline of each diamond is the mean and the upper and lower points of each diamond represent the 95% confidence intervals of the mean. Mice with a single copy of the Wlds fusion gene are hemizygous (Hemi). At both 9 and 12 mo, there were no significant differences in IOP (ANOVA) between the neuroprotected, Wlds mice (see below), and mice of any other genotype. At 10.5 mo, the wild-type (WT) mice had an unusually high skewing of their IOP compared with typical values at this age (see Anderson et al., 2005; Libby et al., 2005a), and their IOP was significantly higher than mice of all other genotypes (P < 0.04 for all comparisons). This skewing did not account for the decreased axon loss in Wlds mice. This is clear because (1) there was extensive overlap between all genotypes, and (2) the IOP distribution of Wlds mice was extremely similar (P = 0.7) to Bax+/− heterozygotes mice (Het), whose axons were not protected (see below). (B) Axon protection in Wlds mice. Distributions of optic nerve damage in mice of the indicated genotypes are shown at two important time points (Materials and methods). The Wlds allele significantly rescues axons from glaucomatous degeneration at 12 mo of age (P < 0.0001, chi square comparing Wlds Hemi to their WT littermates). Importantly, Wlds increased the number of eyes with no detectable glaucoma. Bax+/− heterozygosity conferred no protective effect when compared with wild-type mice (right). Though not statistically significant, there tended to be a greater degree of protection in D2.WldsBax+/− double mutants (right), compared with Wlds single mutants (left). The number of eyes analyzed for each genotype at 10.5 mo was typically ≥40 but 20 for D2.Bax+/− heterozygotes. At 12 mo, typically ≥63 eyes were studied for each genotype but 21 for D2.Bax+/− heterozygotes. (C–F) Wlds preserves RGC axons and optic nerve morphology as seen in semi-thin sections. Most wild-type (C) and D2.Bax+/− nerves (E) had severe glaucoma with very substantial axon loss (see Fig. 3) and extensive glial scarring. In contrast, the majority of D2.Wlds (D) and D2.WldsBax+/− nerves (F) had no or early glaucoma, with <5% of axons damaged (Fig. 3) and no evidence of glial hypertrophy or scarring. (G) Axon counts for a randomly selected sample of DBA/2J and D2.Wlds nerves with no or early glaucoma (n = 6 for each genotype). The averages are not statistically different (P = 0.235), and none of the D2.Wlds nerves had decreased axon counts. Because Wlds more than doubled the number of eyes with no or early glaucoma, half of these counted eyes were rescued. (H–J) Retinal flatmounts showing that in Wlds protected eyes RGC somata survive (H, preglaucoma; I, severe glaucoma; J, no or early glaucoma). (K) This was confirmed by counting RGC layer cells in eyes with no or early glaucoma. Again, half of the D2-Wlds eyes were rescued and none had cell numbers below the range of wild-type values (n = 10 each genotype, P > 0.1). As expected, in both wild-type and D2.Wlds mice with severe optic nerve damage the majority of RGCs were lost. The retinal images shown are from a matched region of the superior, peripheral retina. (L) Despite the profound rescue, a mild degree of somal shrinkage (∼10%) was seen in D2.Wlds eyes with no detectable glaucoma. The shrinkage appears to occur generally across cells of different sizes (see Fig. S4). (M) Wlds strongly preserved PERG amplitude, a measure of RGC activity, in randomly selected mice (number of eyes = 16, 14, 18 for D2-Gpnmb+, DBA/2J, and D2.Wlds, respectively). Bars, 100 μm.

Mentions: To further assess the role of axonal injury in glaucoma, we developed a strain of DBA/2J mice with the Wlds allele (D2.Wlds, Materials and methods). The Wlds gene dominantly protects axons from degeneration induced by axonal trauma (Lunn et al., 1989; Perry et al., 1990, 1991; Ribchester et al., 1995; Mack et al., 2001). Because Bax heterozygosity (Bax+/−) protects the RGC somata in DBA/2J glaucoma, we also determined if inheriting both the Wlds and Bax− mutations would be beneficial (Fig. 9).


Axons of retinal ganglion cells are insulted in the optic nerve early in DBA/2J glaucoma.

Howell GR, Libby RT, Jakobs TC, Smith RS, Phalan FC, Barter JW, Barbay JM, Marchant JK, Mahesh N, Porciatti V, Whitmore AV, Masland RH, John SW - J. Cell Biol. (2007)

Wlds profoundly protects from glaucomatous damage. (A) The IOP distributions of mice of each genotype overlap extensively. The boxes show the upper and lower quartiles and the bars show the extremes including outliers. The centerline of each diamond is the mean and the upper and lower points of each diamond represent the 95% confidence intervals of the mean. Mice with a single copy of the Wlds fusion gene are hemizygous (Hemi). At both 9 and 12 mo, there were no significant differences in IOP (ANOVA) between the neuroprotected, Wlds mice (see below), and mice of any other genotype. At 10.5 mo, the wild-type (WT) mice had an unusually high skewing of their IOP compared with typical values at this age (see Anderson et al., 2005; Libby et al., 2005a), and their IOP was significantly higher than mice of all other genotypes (P < 0.04 for all comparisons). This skewing did not account for the decreased axon loss in Wlds mice. This is clear because (1) there was extensive overlap between all genotypes, and (2) the IOP distribution of Wlds mice was extremely similar (P = 0.7) to Bax+/− heterozygotes mice (Het), whose axons were not protected (see below). (B) Axon protection in Wlds mice. Distributions of optic nerve damage in mice of the indicated genotypes are shown at two important time points (Materials and methods). The Wlds allele significantly rescues axons from glaucomatous degeneration at 12 mo of age (P < 0.0001, chi square comparing Wlds Hemi to their WT littermates). Importantly, Wlds increased the number of eyes with no detectable glaucoma. Bax+/− heterozygosity conferred no protective effect when compared with wild-type mice (right). Though not statistically significant, there tended to be a greater degree of protection in D2.WldsBax+/− double mutants (right), compared with Wlds single mutants (left). The number of eyes analyzed for each genotype at 10.5 mo was typically ≥40 but 20 for D2.Bax+/− heterozygotes. At 12 mo, typically ≥63 eyes were studied for each genotype but 21 for D2.Bax+/− heterozygotes. (C–F) Wlds preserves RGC axons and optic nerve morphology as seen in semi-thin sections. Most wild-type (C) and D2.Bax+/− nerves (E) had severe glaucoma with very substantial axon loss (see Fig. 3) and extensive glial scarring. In contrast, the majority of D2.Wlds (D) and D2.WldsBax+/− nerves (F) had no or early glaucoma, with <5% of axons damaged (Fig. 3) and no evidence of glial hypertrophy or scarring. (G) Axon counts for a randomly selected sample of DBA/2J and D2.Wlds nerves with no or early glaucoma (n = 6 for each genotype). The averages are not statistically different (P = 0.235), and none of the D2.Wlds nerves had decreased axon counts. Because Wlds more than doubled the number of eyes with no or early glaucoma, half of these counted eyes were rescued. (H–J) Retinal flatmounts showing that in Wlds protected eyes RGC somata survive (H, preglaucoma; I, severe glaucoma; J, no or early glaucoma). (K) This was confirmed by counting RGC layer cells in eyes with no or early glaucoma. Again, half of the D2-Wlds eyes were rescued and none had cell numbers below the range of wild-type values (n = 10 each genotype, P > 0.1). As expected, in both wild-type and D2.Wlds mice with severe optic nerve damage the majority of RGCs were lost. The retinal images shown are from a matched region of the superior, peripheral retina. (L) Despite the profound rescue, a mild degree of somal shrinkage (∼10%) was seen in D2.Wlds eyes with no detectable glaucoma. The shrinkage appears to occur generally across cells of different sizes (see Fig. S4). (M) Wlds strongly preserved PERG amplitude, a measure of RGC activity, in randomly selected mice (number of eyes = 16, 14, 18 for D2-Gpnmb+, DBA/2J, and D2.Wlds, respectively). Bars, 100 μm.
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fig9: Wlds profoundly protects from glaucomatous damage. (A) The IOP distributions of mice of each genotype overlap extensively. The boxes show the upper and lower quartiles and the bars show the extremes including outliers. The centerline of each diamond is the mean and the upper and lower points of each diamond represent the 95% confidence intervals of the mean. Mice with a single copy of the Wlds fusion gene are hemizygous (Hemi). At both 9 and 12 mo, there were no significant differences in IOP (ANOVA) between the neuroprotected, Wlds mice (see below), and mice of any other genotype. At 10.5 mo, the wild-type (WT) mice had an unusually high skewing of their IOP compared with typical values at this age (see Anderson et al., 2005; Libby et al., 2005a), and their IOP was significantly higher than mice of all other genotypes (P < 0.04 for all comparisons). This skewing did not account for the decreased axon loss in Wlds mice. This is clear because (1) there was extensive overlap between all genotypes, and (2) the IOP distribution of Wlds mice was extremely similar (P = 0.7) to Bax+/− heterozygotes mice (Het), whose axons were not protected (see below). (B) Axon protection in Wlds mice. Distributions of optic nerve damage in mice of the indicated genotypes are shown at two important time points (Materials and methods). The Wlds allele significantly rescues axons from glaucomatous degeneration at 12 mo of age (P < 0.0001, chi square comparing Wlds Hemi to their WT littermates). Importantly, Wlds increased the number of eyes with no detectable glaucoma. Bax+/− heterozygosity conferred no protective effect when compared with wild-type mice (right). Though not statistically significant, there tended to be a greater degree of protection in D2.WldsBax+/− double mutants (right), compared with Wlds single mutants (left). The number of eyes analyzed for each genotype at 10.5 mo was typically ≥40 but 20 for D2.Bax+/− heterozygotes. At 12 mo, typically ≥63 eyes were studied for each genotype but 21 for D2.Bax+/− heterozygotes. (C–F) Wlds preserves RGC axons and optic nerve morphology as seen in semi-thin sections. Most wild-type (C) and D2.Bax+/− nerves (E) had severe glaucoma with very substantial axon loss (see Fig. 3) and extensive glial scarring. In contrast, the majority of D2.Wlds (D) and D2.WldsBax+/− nerves (F) had no or early glaucoma, with <5% of axons damaged (Fig. 3) and no evidence of glial hypertrophy or scarring. (G) Axon counts for a randomly selected sample of DBA/2J and D2.Wlds nerves with no or early glaucoma (n = 6 for each genotype). The averages are not statistically different (P = 0.235), and none of the D2.Wlds nerves had decreased axon counts. Because Wlds more than doubled the number of eyes with no or early glaucoma, half of these counted eyes were rescued. (H–J) Retinal flatmounts showing that in Wlds protected eyes RGC somata survive (H, preglaucoma; I, severe glaucoma; J, no or early glaucoma). (K) This was confirmed by counting RGC layer cells in eyes with no or early glaucoma. Again, half of the D2-Wlds eyes were rescued and none had cell numbers below the range of wild-type values (n = 10 each genotype, P > 0.1). As expected, in both wild-type and D2.Wlds mice with severe optic nerve damage the majority of RGCs were lost. The retinal images shown are from a matched region of the superior, peripheral retina. (L) Despite the profound rescue, a mild degree of somal shrinkage (∼10%) was seen in D2.Wlds eyes with no detectable glaucoma. The shrinkage appears to occur generally across cells of different sizes (see Fig. S4). (M) Wlds strongly preserved PERG amplitude, a measure of RGC activity, in randomly selected mice (number of eyes = 16, 14, 18 for D2-Gpnmb+, DBA/2J, and D2.Wlds, respectively). Bars, 100 μm.
Mentions: To further assess the role of axonal injury in glaucoma, we developed a strain of DBA/2J mice with the Wlds allele (D2.Wlds, Materials and methods). The Wlds gene dominantly protects axons from degeneration induced by axonal trauma (Lunn et al., 1989; Perry et al., 1990, 1991; Ribchester et al., 1995; Mack et al., 2001). Because Bax heterozygosity (Bax+/−) protects the RGC somata in DBA/2J glaucoma, we also determined if inheriting both the Wlds and Bax− mutations would be beneficial (Fig. 9).

Bottom Line: We show that proximal axon segments attached to their cell bodies survive to the proximity of the lamina.In contrast, axon segments in the lamina and behind the eye degenerate.These experiments provide strong evidence for a local insult to axons in the optic nerve.

View Article: PubMed Central - PubMed

Affiliation: The Jackson Laboratory, Bar Harbor, ME 04609, USA.

ABSTRACT
Here, we use a mouse model (DBA/2J) to readdress the location of insult(s) to retinal ganglion cells (RGCs) in glaucoma. We localize an early sign of axon damage to an astrocyte-rich region of the optic nerve just posterior to the retina, analogous to the lamina cribrosa. In this region, a network of astrocytes associates intimately with RGC axons. Using BAX-deficient DBA/2J mice, which retain all of their RGCs, we provide experimental evidence for an insult within or very close to the lamina in the optic nerve. We show that proximal axon segments attached to their cell bodies survive to the proximity of the lamina. In contrast, axon segments in the lamina and behind the eye degenerate. Finally, the Wld(s) allele, which is known to protect against insults to axons, strongly protects against DBA/2J glaucoma and preserves RGC activity as measured by pattern electroretinography. These experiments provide strong evidence for a local insult to axons in the optic nerve.

Show MeSH
Related in: MedlinePlus