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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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The relationship between RGC density measured in vivo by CSLO and RGC density measured post mortem by epifluorescence microscopy at week-4.
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pone-0113011-g005: The relationship between RGC density measured in vivo by CSLO and RGC density measured post mortem by epifluorescence microscopy at week-4.

Mentions: The data plotted in Figure 5 demonstrate that the RGC density derived in vivo from CSLO images acquired during the final follow-up time point (week-4) were well correlated with RGC density measurements derived ex vivo from epifluorescence microscopy images of flat-mount retinae (Pearson R = 0.91, p<0.0001). Since both estimates contain inherent error, Deming regression was applied to determine the slope and intercept of the relationship, which resulted in the following equation: RGC density by CSLO in vivo (per mm2) = 0.58*RGC density by microscopy ex vivo (per mm2)+103 (per mm2), similar to the findings in two of our previous studies [26], [39]. There is evidence in these data of “saturation” whereby a quadratic model provided a statistically better fit than a linear model (F = 13, p = 0.001), suggesting that the resolution of the in vivo CSLO method places an upper limit on density measurements. Thus the limitations of imaging in vivo (lower gain, contrast and resolution) result in an underestimate of RGC density as compared with post mortem microscopy, but there is a strong correlation between the two over most of their dynamic ranges. Note that one or two retinas from the Group-3 (sham) fellow eyes had lower densities despite adequate post mortem assessment of CTB fluorescence at the injection site in the superior colliculus. Their results are presumed to be due to limited uptake and/or transport of CTB from the superior colliculus and although they may be “outliers”, they do not affect the correlation between in vivo CSLO and ex vivo microscopy counts.


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)

The relationship between RGC density measured in vivo by CSLO and RGC density measured post mortem by epifluorescence microscopy at week-4.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0113011-g005: The relationship between RGC density measured in vivo by CSLO and RGC density measured post mortem by epifluorescence microscopy at week-4.
Mentions: The data plotted in Figure 5 demonstrate that the RGC density derived in vivo from CSLO images acquired during the final follow-up time point (week-4) were well correlated with RGC density measurements derived ex vivo from epifluorescence microscopy images of flat-mount retinae (Pearson R = 0.91, p<0.0001). Since both estimates contain inherent error, Deming regression was applied to determine the slope and intercept of the relationship, which resulted in the following equation: RGC density by CSLO in vivo (per mm2) = 0.58*RGC density by microscopy ex vivo (per mm2)+103 (per mm2), similar to the findings in two of our previous studies [26], [39]. There is evidence in these data of “saturation” whereby a quadratic model provided a statistically better fit than a linear model (F = 13, p = 0.001), suggesting that the resolution of the in vivo CSLO method places an upper limit on density measurements. Thus the limitations of imaging in vivo (lower gain, contrast and resolution) result in an underestimate of RGC density as compared with post mortem microscopy, but there is a strong correlation between the two over most of their dynamic ranges. Note that one or two retinas from the Group-3 (sham) fellow eyes had lower densities despite adequate post mortem assessment of CTB fluorescence at the injection site in the superior colliculus. Their results are presumed to be due to limited uptake and/or transport of CTB from the superior colliculus and although they may be “outliers”, they do not affect the correlation between in vivo CSLO and ex vivo microscopy counts.

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