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Comparison of longitudinal in vivo measurements of retinal nerve fiber layer thickness and retinal ganglion cell density after optic nerve transection in rat.

Choe TE, Abbott CJ, Piper C, Wang L, Fortune B - PLoS ONE (2014)

Bottom Line: RGCs were labeled by retrograde transport of fluorescently conjugated cholera toxin B (CTB) from the superior colliculus 48 hours prior to ONT or sham surgery.In ONT-CTB labeled fellow eyes, RNFLT increased by 18%, 52% and 36% at weeks 2, 3 and 4 (p<0.0001), but did not change in fellow ONT-eyes sans CTB.The strong fellow eye effect observed in eyes contralateral to ONT, only in the presence of CTB label, consisted of a dramatic increase in RNFLT associated with retinal microgliosis.

View Article: PubMed Central - PubMed

Affiliation: Discoveries in Sight Research Laboratories, Devers Eye Institute and Legacy Research Institute, Legacy Health, Portland, Oregon, United States of America.

ABSTRACT

Purpose: To determine the relationship between longitudinal in vivo measurements of retinal nerve fiber layer thickness (RNFLT) and retinal ganglion cell (RGC) density after unilateral optic nerve transection (ONT).

Methods: Nineteen adult Brown-Norway rats were studied; N = 10 ONT plus RGC label, N = 3 ONT plus vehicle only (sans label), N = 6 sham ONT plus RGC label. RNFLT was measured by spectral domain optical coherence tomography (SD-OCT) at baseline then weekly for 1 month. RGCs were labeled by retrograde transport of fluorescently conjugated cholera toxin B (CTB) from the superior colliculus 48 hours prior to ONT or sham surgery. RGC density measurements were obtained by confocal scanning laser ophthalmoscopy (CSLO) at baseline and weekly for 1 month. RGC density and reactivity of microglia (anti-Iba1) and astrocytes (anti-GFAP) were determined from post mortem fluorescence microscopy of whole-mount retinae.

Results: RNFLT decreased after ONT by 17% (p<0.05), 30% (p<0.0001) and 36% (p<0.0001) at weeks 2, 3 and 4. RGC density decreased after ONT by 18%, 69%, 85% and 92% at weeks 1, 2, 3 and 4 (p<0.0001 each). RGC density measured in vivo at week 4 and post mortem by microscopy were strongly correlated (R = 0.91, p<0.0001). In vivo measures of RNFLT and RGC density were strongly correlated (R = 0.81, p<0.0001). In ONT-CTB labeled fellow eyes, RNFLT increased by 18%, 52% and 36% at weeks 2, 3 and 4 (p<0.0001), but did not change in fellow ONT-eyes sans CTB. Microgliosis was evident in the RNFL of the ONT-CTB fellow eyes, exceeding that observed in other fellow eyes.

Conclusions: In vivo measurements of RNFLT and RGC density are strongly correlated and can be used to monitor longitudinal changes after optic nerve injury. The strong fellow eye effect observed in eyes contralateral to ONT, only in the presence of CTB label, consisted of a dramatic increase in RNFLT associated with retinal microgliosis.

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Representative images from each group of experimental eyes, fellow control eyes and naïve control eyes acquired by confocal microscopy.Increased microglial (Iba-1 positive) cell density was evident in Group-1 ONT eyes, their fellow eyes and in the Group-2 ONT eyes (sans CTB labeling of RGCs). The majority of microglia within the anterior most plexus exhibited morphological characteristics consistent with the ‘activated’ state, including enlarged, rounded or ameboid soma and retracted processes. See text for additional details (arrows). Scale bar = 0.19 mm.
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pone-0113011-g008: Representative images from each group of experimental eyes, fellow control eyes and naïve control eyes acquired by confocal microscopy.Increased microglial (Iba-1 positive) cell density was evident in Group-1 ONT eyes, their fellow eyes and in the Group-2 ONT eyes (sans CTB labeling of RGCs). The majority of microglia within the anterior most plexus exhibited morphological characteristics consistent with the ‘activated’ state, including enlarged, rounded or ameboid soma and retracted processes. See text for additional details (arrows). Scale bar = 0.19 mm.

Mentions: In contrast to the results shown in Fig. 7 for retinal microglia, there were no consistent changes observed in retinas stained with an anti-GFAP marker. Figure 8 shows representative images of retinal flat-mounts acquired by confocal microscopy in each of the experimental groups. Increased microglial (Iba-1 positive) cell density was evident in Group-1 ONT eyes, Group-1 ONT fellow eyes, and Group-2 ONT eyes that did not have RGC labeling by CTB. Microglia in both groups of ONT-fellow eyes also exhibited signs of being in an activated state (such as altered morphology: larger size, rounder or ameboid in shape, with fewer, thicker processes; arrows), particularly within the RNFL/RGC layer. However, consistent with the results of quantitative density analysis shown in Fig. 7, the most impressive subjective indications of microglial activation were observed in the fellow eyes of Group-1 animals (i.e. those in which there was a combination of ONT to the contralateral eye and bilateral CTB labeling of RGCs). Microglial density and morphology in Group-3 sham-ONT operated eyes and their fellow eyes were both comparable to naïve eyes. There were no changes in astrocyte (GFAP-positive) appearances between naïve and Group-2 eyes (without CTB labeling of RGCs, either in the ONT eye or their fellow eyes). Colocalization of CTB fluorescence and Iba-1 staining was not observed in any of the ONT, ONT-fellow, sham or sham-fellow eyes, which indicates that active microglial phagocytosis of CTB was nearing completion by the 4-week time point.


Comparison of longitudinal in vivo measurements of retinal nerve fiber layer thickness and retinal ganglion cell density after optic nerve transection in rat.

Choe TE, Abbott CJ, Piper C, Wang L, Fortune B - PLoS ONE (2014)

Representative images from each group of experimental eyes, fellow control eyes and naïve control eyes acquired by confocal microscopy.Increased microglial (Iba-1 positive) cell density was evident in Group-1 ONT eyes, their fellow eyes and in the Group-2 ONT eyes (sans CTB labeling of RGCs). The majority of microglia within the anterior most plexus exhibited morphological characteristics consistent with the ‘activated’ state, including enlarged, rounded or ameboid soma and retracted processes. See text for additional details (arrows). Scale bar = 0.19 mm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231142&req=5

pone-0113011-g008: Representative images from each group of experimental eyes, fellow control eyes and naïve control eyes acquired by confocal microscopy.Increased microglial (Iba-1 positive) cell density was evident in Group-1 ONT eyes, their fellow eyes and in the Group-2 ONT eyes (sans CTB labeling of RGCs). The majority of microglia within the anterior most plexus exhibited morphological characteristics consistent with the ‘activated’ state, including enlarged, rounded or ameboid soma and retracted processes. See text for additional details (arrows). Scale bar = 0.19 mm.
Mentions: In contrast to the results shown in Fig. 7 for retinal microglia, there were no consistent changes observed in retinas stained with an anti-GFAP marker. Figure 8 shows representative images of retinal flat-mounts acquired by confocal microscopy in each of the experimental groups. Increased microglial (Iba-1 positive) cell density was evident in Group-1 ONT eyes, Group-1 ONT fellow eyes, and Group-2 ONT eyes that did not have RGC labeling by CTB. Microglia in both groups of ONT-fellow eyes also exhibited signs of being in an activated state (such as altered morphology: larger size, rounder or ameboid in shape, with fewer, thicker processes; arrows), particularly within the RNFL/RGC layer. However, consistent with the results of quantitative density analysis shown in Fig. 7, the most impressive subjective indications of microglial activation were observed in the fellow eyes of Group-1 animals (i.e. those in which there was a combination of ONT to the contralateral eye and bilateral CTB labeling of RGCs). Microglial density and morphology in Group-3 sham-ONT operated eyes and their fellow eyes were both comparable to naïve eyes. There were no changes in astrocyte (GFAP-positive) appearances between naïve and Group-2 eyes (without CTB labeling of RGCs, either in the ONT eye or their fellow eyes). Colocalization of CTB fluorescence and Iba-1 staining was not observed in any of the ONT, ONT-fellow, sham or sham-fellow eyes, which indicates that active microglial phagocytosis of CTB was nearing completion by the 4-week time point.

Bottom Line: RGCs were labeled by retrograde transport of fluorescently conjugated cholera toxin B (CTB) from the superior colliculus 48 hours prior to ONT or sham surgery.In ONT-CTB labeled fellow eyes, RNFLT increased by 18%, 52% and 36% at weeks 2, 3 and 4 (p<0.0001), but did not change in fellow ONT-eyes sans CTB.The strong fellow eye effect observed in eyes contralateral to ONT, only in the presence of CTB label, consisted of a dramatic increase in RNFLT associated with retinal microgliosis.

View Article: PubMed Central - PubMed

Affiliation: Discoveries in Sight Research Laboratories, Devers Eye Institute and Legacy Research Institute, Legacy Health, Portland, Oregon, United States of America.

ABSTRACT

Purpose: To determine the relationship between longitudinal in vivo measurements of retinal nerve fiber layer thickness (RNFLT) and retinal ganglion cell (RGC) density after unilateral optic nerve transection (ONT).

Methods: Nineteen adult Brown-Norway rats were studied; N = 10 ONT plus RGC label, N = 3 ONT plus vehicle only (sans label), N = 6 sham ONT plus RGC label. RNFLT was measured by spectral domain optical coherence tomography (SD-OCT) at baseline then weekly for 1 month. RGCs were labeled by retrograde transport of fluorescently conjugated cholera toxin B (CTB) from the superior colliculus 48 hours prior to ONT or sham surgery. RGC density measurements were obtained by confocal scanning laser ophthalmoscopy (CSLO) at baseline and weekly for 1 month. RGC density and reactivity of microglia (anti-Iba1) and astrocytes (anti-GFAP) were determined from post mortem fluorescence microscopy of whole-mount retinae.

Results: RNFLT decreased after ONT by 17% (p<0.05), 30% (p<0.0001) and 36% (p<0.0001) at weeks 2, 3 and 4. RGC density decreased after ONT by 18%, 69%, 85% and 92% at weeks 1, 2, 3 and 4 (p<0.0001 each). RGC density measured in vivo at week 4 and post mortem by microscopy were strongly correlated (R = 0.91, p<0.0001). In vivo measures of RNFLT and RGC density were strongly correlated (R = 0.81, p<0.0001). In ONT-CTB labeled fellow eyes, RNFLT increased by 18%, 52% and 36% at weeks 2, 3 and 4 (p<0.0001), but did not change in fellow ONT-eyes sans CTB. Microgliosis was evident in the RNFL of the ONT-CTB fellow eyes, exceeding that observed in other fellow eyes.

Conclusions: In vivo measurements of RNFLT and RGC density are strongly correlated and can be used to monitor longitudinal changes after optic nerve injury. The strong fellow eye effect observed in eyes contralateral to ONT, only in the presence of CTB label, consisted of a dramatic increase in RNFLT associated with retinal microgliosis.

Show MeSH
Related in: MedlinePlus