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Differential protection by nicotinamide in a mouse model of glaucoma DBA/2J revealed by second-harmonic generation microscopy
Nicotinamide’s protection against glaucoma studied by second-harmonic generation microscopy
Boodram Vinessia Data curation Investigation
https://orcid.org/0000-0003-4936-2494
Lim Hyungsik Conceptualization Data curation Formal analysis Funding acquisition Investigation Supervision Writing – original draft Writing – review & editing ¤ *
Department of Physics and Astronomy, Hunter College of the City University of New York, New York, NY, United States of America
Anderson Michael G. Editor
University of Iowa, UNITED STATES OF AMERICA
Competing Interests: The authors have declared that no competing interests exist.

¤ Current address: School of Optometry, Indiana University, Bloomington, IN, United States of America

* E-mail: hl128@iu.edu
10 9 2024
2024
19 9 e030940017 4 2024
12 8 2024
© 2024 Boodram, Lim
2024
Boodram, Lim
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Glaucoma is a blinding disease where the retinal ganglion cells and their axons degenerate. Degradation of axonal microtubules is thought to play a critical role in the pathogenesis, but the mechanism is unknown. Here we investigate whether microtubule disruption in glaucoma can be alleviated by metabolic rescue. The integrity of axonal microtubules and the morphology of the retinal nerve fibers were evaluated by second-harmonic generation microscopy in a mouse model of glaucoma, DBA/2J, which received a dietary supplement of nicotinamide (NAM) for reducing metabolic stress. It was compared with control DBA/2J, which did not receive NAM, and non-glaucomatous DBA/2J-Gpnmb+. We found that the morphology of the retinal nerve fibers, but not axonal microtubules, are significantly protected by NAM. The decoupling is analogous to microtubule deficit, a glaucoma pathology in which axonal microtubules exhibit rapid degradation compared to the morphology of the retinal nerve fibers. Understanding microtubule deficit could provide insights into the divergent responses to NAM. From co-registered images of second-harmonic generation and immunofluorescence, it was determined that microtubule deficit was not due to a shortage of tubulins. Furthermore, microtubule deficit colocalized with the sectors in which the retinal ganglion cells were disconnected from the brain, suggesting that microtubule disruption is associated with axonal transport deficit in glaucoma. Together, our data suggests significant role axonal microtubules play in glaucomatous degeneration, offering a new opportunity for neuroprotection.

National Eye Institute EY033047 https://orcid.org/0000-0003-4936-2494
Lim Hyungsik http://dx.doi.org/10.13039/100000057 National Institute of General Medical Sciences GM140841 https://orcid.org/0000-0003-4936-2494
Lim Hyungsik This work was supported by funding from the National Institute of Health, EY033047 and GM140841 (H.L.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Data AvailabilityAll relevant data are within the paper and its supporting information files.
Data Availability

All relevant data are within the paper and its supporting information files.
==== Body
pmcIntroduction

Glaucoma is a leading cause of blindness worldwide [1, 2], where nearly half of the retinal ganglion cells (RGCs) and their axons are irreversibly lost by the time of diagnosis. The risk factors include aging and high intraocular pressure (IOP), but the pathogenic mechanism is poorly understood hampering the prevention of vision loss. Axonal microtubules have been postulated to play a crucial role in the disease [3–5]. It has been demonstrated that in DBA/2J (D2) mice, a well-characterized model of inherited glaucoma [6–9], microtubules in RGC axons (i.e., the retinal nerve fibers) decay with age more rapidly than the axons themselves [10]. The degradation of microtubules would impair axonal transport depriving the RGCs of essential tropic and metabolic supports. One of the possible drivers of microtubule loss is dysfunctional cellular metabolism, compromising active regulation that stabilizes the microtubule assembly. Microtubule stability and metabolic equilibrium are co-dependent: Conversely, disruption of microtubules, which enable intracellular transport of mitochondria, could induce local energetic shortfalls in the distal RGC compartments. Lowering metabolic stress with metabolites, e.g., nicotinamide adenine dinucleotide (NAD) or its precursor nicotinamide (NAM), has been shown to delay or halt axonal degeneration [11] and protect the RGCs against glaucoma [12, 13]. It is plausible that the mechanism of neuroprotection by NAD/NAM involves axonal microtubules.

Here, using NAM as a paradigm, we investigate how axonal microtubules of the RGCs respond to metabolic rescue. Second-harmonic generation (SHG) microscopy was employed for measuring microtubules in the retinal nerve fibers, which, by virtue of the sensitivity to uniformly polarized microtubules [14–16], provides distinctive information regarding the cytoskeleton’s integrity.

Results

An overview of the experiment is shown in Fig 1(A). A group of D2 mice (N = 41 eyes, 11 eyes from 8 males and 30 eyes from 16 females) received a dietary supplement of NAM prophylactically from 6 months of age at a low dose of 550 mg/kg body weight per day, as previously described (12, 13). As a control, another group of D2 mice received a normal diet without NAM (N = 33 eyes, 13 eyes from 8 males and 20 eyes from 12 females). The strain-matched, homozygous DBA/2J-Gpnmb+ mice (D2-Gpnmb+) were used as a non-glaucomatous control [17] (N = 19 eyes, 13 eyes from 8 males and 6 eyes from 4 females). At a desired age, the IOP was measured using a TonoLab tonometer [18] on three different dates prior to imaging, and the average was taken. Mosaics were acquired over a region around the optic nerve head (Fig 1(B)). Image processing was carried out to normalize the SHG intensity and evaluate the thickness of the retinal nerve fiber bundles [10] (Fig 1(C)). The normalized SHG intensity divided by thickness, namely SHG density, provides a measure of the density of axonal microtubules at a microscopic resolution. The mean SHG density was evaluated over the retinal nerve fibers in the mosaic. Also, the thickness was integrated over the region to obtain the volume of the retinal nerve fibers.

10.1371/journal.pone.0309400.g001 Fig 1 Overview of the experiment.

(a) Timeline of NAM diet, IOP recording, and SHG imaging. (b) Representative SHG images of degenerated D2 versus healthy D2-Gpnmb+ retinas. Scale bars, 300 μm. (c) Image processing for evaluating the mean SHG density ρ and the volume V, representing ‘microtubule density’ and ‘morphology’, respectively.

Morphology, but not microtubule density, of the retinal nerve fibers are protected by NAM

The effect of NAM diet was investigated on three properties, i.e., IOP, the volume and the mean SHG density of the retinal nerve fiber bundles (Fig 2 and S1 Table). D2 data contained the intrinsic variations of the strain and the experimental errors. The size of the latter can be estimated approximately from the variations of D2-Gpnmb+ data. Overall, the variations of D2 were greater than those of D2-Gpnmb+. The age-dependent changes of the D2 eyes were analyzed using a multiple linear regression model containing three main effects that are known to influence the pathology (i.e., age, sex, and diet) as well as two interactions (summarized in Table 1). While the IOPs of D2-Gpnmb+ mice remained stable, those of D2 without NAM increased with age, as expected. In D2 mice, the rate of IOP elevation with age varied depending on diet, which was significantly lower with NAM than without NAM (p = .046). Diminished IOP elevation has also been observed in the prior study but at a higher dose of NAM [12, 13]. Interestingly, while the volume of the retinal nerve fibers responded to NAM exhibiting significantly lower age-dependent loss (p = .049), the decay of the mean SHG density in D2 with NAM was not affected significantly (p = .43). Thus, the benefit of NAM supplement was outstanding only for protecting the morphology, but not the microtubule density, of the retinal nerve fibers. Sex difference was not significant.

10.1371/journal.pone.0309400.g002 Fig 2 Age-dependent effects of NAM dietary supplement on the D2 retinas.

IOP, the volume and the mean SHG density of the retinal nerve fibers versus age are shown for D2-Gpnmb+, D2 with and without NAM. Solid and open markers are male and female, respectively. Dashed lines are simple linear regressions with age and shaded bands are 95% confidence bounds.

10.1371/journal.pone.0309400.t001 Table 1 Summary of statistical analysis.

D2 mice with and without NAM (N = 74 eyes) were analyzed using a multiple linear regression model y = α + βage ∙ Age + βsex ∙ Sex + βdiet ∙ Diet + βint1 ∙ Age ∙ Sex + βint2 ∙ Age ∙ Diet + ε.

	Dependent variables	
	IOP (mmHg)	Volume (x106 μm3)	Mean SHG density (a. u.)	
Age	0.60***	-1.50***	-25.04***	
(0.12)	(0.39)	(8.26)	
Sex	-1.98	-4.09	-87.27	
(1.56)	(5.08)	(106.52)	
Diet	2.14	-7.80	-66.32	
(1.48)	(4.83)	(101.30)	
Age∙Sex	0.17	0.58	9.04	
(0.15)	(0.49)	(10.33)	
Age∙Diet	-0.29**	0.94**	7.86	
(0.14)	(0.47)	(9.84)	
Constant	5.24***	20.76***	373.52***	
(1.22)	(3.98)	(83.49)	
Observations	74	74	74	
R2	0.47	0.25	0.18	
Adjusted R2	0.43	0.20	0.12	
F Statistic	12.03***	4.61***	2.93**	
(df = 5; 68)	
Note:

*p < .1

**p < .05

***p < .01

Mean SHG density and volume are negatively correlated with IOP

In addition to age-dependence, we investigated how the mean SHG density and the volume depended on IOP, another major risk factor of glaucoma (Fig 3). In D2 mice without NAM, we found modest negative correlations at the level of significance between the IOP versus the mean SHG density (Pearson correlation coefficient r = -0.42, p = .015) and versus the volume (r = -0.37, p = .033), implying that the loss of axonal microtubules and RGC axons could be downstream effects of IOP elevation. Then we examined how the dependence on IOP was modified by NAM diet. Specifically, we asked whether the protective effect of NAM, instead of being a direct consequence of bolstered metabolism, was mediated entirely by the reduced IOP elevation, which would preserve the correlation coefficient. On the other hand, if NAM had an additional mode of action to prevent the loss of RGC axons, the correlation was expected to decrease as the IOP explains less of the variation. Unfortunately, the correlation coefficient between the volume and the IOP could not be determined with confidence for the group of D2 mice with NAM diet (r = -0.19, p = .24), leaving the inquiry unresolved.

10.1371/journal.pone.0309400.g003 Fig 3 Correlation between morphology/microtubules and IOP.

The correlation coefficients for D2 with and without NAM are compared to examine the effect of NAM diet. Solid and open markers are male and female, respectively, overlaid with the best fits to the bivariate normal distribution.

Microtubule deficit evolves in a NAM-dependent manner during glaucoma progression

Disruption of axonal microtubules before the loss of RGC axons culminates in a pathological state of glaucoma called microtubule deficit, where the retinal nerve fiber bundles are morphologically intact but contain less than normal amount of microtubules [10]. Based on the differential protection of the volume and the microtubule density of the retinal nerve fibers, it was anticipated that NAM diet will alter the evolution of microtubule deficit in glaucoma. To illustrate this, the mean SHG density and the volume were analyzed jointly in the same samples. Provided that the rate coefficients of the mean SHG density ρ and the volume V are given by α and β, respectively, α=−1ρ⋅dρdt=−dlogρdt,β=−1V⋅dVdt=−dlogVdt

, then the ratio of coefficients is obtained from the slope of a log-log plot.

αβ=−dlogρ/dt−dlogV/dt=dlogρdlogV

The ratio greater than unity (α/β > 1) will be obtained in case of microtubule deficit, or if the volume is preserved better than microtubules. Overall, the D2 retinas without NAM had higher probabilities of microtubule deficit (Fig 4), reproducing the previous findings [10] with different experimental protocols and apparatus. It confirms microtubule deficit as a molecular marker of glaucoma. When the populations of D2 retinas with and without NAM diet were compared, the evolution of microtubule deficit was more appreciable in the D2 retinas with NAM than those without NAM, suggesting that the volume was protected in the progression of glaucoma better than axonal microtubules.

10.1371/journal.pone.0309400.g004 Fig 4 NAM-dependent evolution of microtubule deficit.

The relative course of degeneration of volume and mean SHG density are different for three groups. The parameters are normalized to the average values of D2-Gpnmb+ retinas. Solid and open markers are male and female, respectively. Dashed line, the unity slope. MT; microtubule.

Microtubule deficit is not due to paucity of tubulins

NAM exacerbates decoupling between axonal microtubules and morphology in glaucoma underlying microtubule deficit. Understanding microtubule deficit could allow an insight into the mechanism of divergent responses to NAM. The origin could be either a shortage of tubulin monomers, e.g., from inadequate expression or transport to the RGC axons, or a failure to stabilize the polymer, e.g., from dysfunctional regulation. To resolve these possibilities, we performed a co-registration of immunofluorescence and SHG images. The D2 retinas were fixed immediately after SHG imaging and double immunostained against two types of cytoskeletal protein, i.e., class III beta-tubulin (βIII) and phosphorylated neurofilament (pNF). βIII is a neuron-specific isoform of tubulin, and pNF is normally enriched in axons but accumulates in the soma of degenerating RGC’s. To examine the microtubule integrity and the abundance of tubulins in the same regions, βIII immunofluorescence was co-registered with SHG images. A total of twelve D2 retinas were evaluated, primarily focusing on the age range between 10 and 12 months. In the retinas exhibiting pronounced sectorial degeneration (N = 4), which is a characteristic in glaucoma pathology, the sectors of microtubule deficit always contained the morphologically intact retinal nerve fibers (Fig 5(A), between dashed lines). Also, it also showed high signals of βIII tubulins inside the sector, i.e., no substantial impairment in the expression or transport. It can be therefore concluded that microtubule deficit is not due to a lack of tubulins.

10.1371/journal.pone.0309400.g005 Fig 5 Microtubule deficit versus the distribution of cytoskeletal proteins.

(a) Mosaics of a D2 retina (22 months age, female) by SHG and immunofluorescence against the cytoskeletal elements, βIII tubulin and pNF. (b) Comparing two sectors with deficient and intact microtubules. Arrows, pNF+ RGC somas. Scale bars, 200 μm.

Loss of axonal microtubules colocalizes with the RGC’s disconnect from the brain

Axonal transport deficit is a milestone event in the progression of glaucoma, which is alleviated with NAM diet [12, 13]. The pathology could be caused by defects in the cytoskeleton or molecular motor proteins, whose functions rely on cellular energy. To elucidate the NAM’s effect on axonal transport, first the relationship between axonal microtubules and transport deficit must be elucidated. The somatic accumulation of pNF, which can be detected with monoclonal antibody lacking nonspecific staining of soma (2F11), is interpreted to suggest that the RGC is disconnected from the brain, as has been validated by retrograde tracing [19, 20]. To investigate the relationship between microtubule and transport deficits, we compared SHG density with the distribution of pNF. We found that pNF+ somas represented a small subset of the RGC population (in the order of dozens in an area of approximately 1.8 mm diameter around the optic nerve head) (Fig 5(B)) and were absent in highly degenerated areas (S1 Fig). It could be because not all RGCs undergo this pathological fate during glaucomatous degeneration, and/or the redistribution of pNF could be short-lived with pNF+ RGC somas disappearing altogether upon the cells’ death. Interestingly, pNF+ RGC somas were localized in sectors (Figs 5 and S1) and routinely coincided with that of low SHG density (Fig 5(B)). The result, together with intact βIII signals, suggests that the RGC’s connection to the brain is likely to be severed at the time of microtubule disruption while RGC axons are intact, i.e., at the stage of microtubule deficit.

Discussion

Taken together, our data supports a model in which NAM/NAD has several independent modes of action targeting distinct aspects of glaucoma pathogenesis. Considering that most cell types regulate diverse cellular processes in an energy-dependent manner, there could be non-RGC as well as RGC contributions in the protective effects of NAM/NAD. The IOP elevation has a component that responds to NAM, likely through the cells of the anterior segment, which is partially responsible for protecting the RGCs. It has been shown that NAM at a low dosage protects the RGCs without altering the IOP [12, 13], indicating that there is also IOP-independent protection. NAM reverses a variety of molecular changes in the RGCs that render them vulnerable to IOP elevation, e.g., mitochondrial dysfunction and synaptic elimination, which are likely to constitute the mechanism of IOP-independent protection. However, many aspects of IOP-independent protection are still obscure, e.g., how significant it is relative to IOP-dependent contribution. It is conceivable that the divergent responses to NAM are facilitated by an IOP-independent pathway. The morphology of RGC axons hinges on multiple conditions beside the integrity of microtubules. Contrary to axonal microtubules which might be primarily IOP-dependent, prevention of axon loss could be aided further by other IOP-independent factors that are bolstered metabolically by NAM. Detailed knowledge of distinct modes of action can improve the precision of NAM-based glaucoma therapy.

Differential protection by NAM also sheds critical insights into microtubule deficit. We hypothesized that metabolic stress, such as mitochondrial abnormalities or depletion of metabolites, might destabilize axonal microtubules to induce microtubule deficit. The hypothesis predicts that microtubule deficit will be mitigated by NAM supplement. However, our data do not support that microtubule disruption in glaucoma is strongly related to metabolic decline, leaving the pathogenic origin of microtubule deficit as a mystery. It is not a consequence of a shortage of tubulins in the RGC axon. Instead, microtubule deficit seems to be related to the elevated IOP, which exhibits a negative correlation with axonal microtubules. Regarding whether deteriorating axonal microtubules play a causative role in the eventual loss of RGCs, our multimodal data reveals spatially overlapping sectorial patterns of microtubule and axonal transport deficits. Correlated sectorial degenerations have allowed researchers to associate the loss of RGC axons with pathogenic insults at the optic nerve head [7, 21–23]. Similarly, the alignment of axonal transport and microtubule deficits suggests that the cytoskeletal breakdown might be an intermediate stage in the progressive RGC death. The proposed role of axonal microtubules offers a new opportunity for neuroprotection against glaucomatous degeneration. It also raises an important question regarding glaucoma therapy, i.e., whether the efficacy would be hampered without reversing the degradation of axonal microtubules.

Our discovery of persistent microtubule deficit with NAM seems inconsistent with a prior finding that anterograde axonal transport is recovered by NAM, as probed with cholera toxin β-subunit (CT-β) in the optic nerve head and the lateral geniculate nucleus [12]. It can be reconciled as follows: Although microtubule densities (i.e., mean SHG densities) are similarly low in NAM vs no-NAM D2 mice, the former has more abundant RGC axons thus could display more widespread CT-β signals. However, owing to persistent microtubule deficit, the CT-β signals are predicted to be much less intense in the individual cells of NAM D2 compared to those in the non-glaucomatous control (which has not been compared in the previous studies). Since microtubule deficit continues to worsen with age, axonal transport in NAM D2 mouse is likely to be compromised eventually at an age that microtubule density drops below a certain level, regardless of the integrity of RGC axons.

Our study has a few shortcomings. First, the protection of RGC axons by NAM/NAD could not be compared with that of RGC soma because RGC counts were not measured. The relative course of NAM/NAD effects in the distinct compartments of RGCs could provide additional insights into the mechanism of action. Second, our study did not include D2-Gpnmb+ on NAM diet as a control group, which could have informed us whether the IOP-independent effect of NAM is specific to the glaucoma process or present also in the non-glaucomatous eyes. Third, our IOP measurement was insufficient for unraveling the mechanism quantitatively. Specifically, our measured IOP values of D2 mice were lower than those in the literature [24, 25]. Although the exact reason for inaccuracy is unclear (especially since the TonoLab device permits the user only basic controls), it seems due to a scaling error in the rebound tonometer, possibly incorrect software setting or mechanical deviations, given that the IOPs were underestimated uniformly across the ages thus displaying the anticipated overall trends for D2 as well as D2-Gpnmb+ mice. Furthermore, the measurements were highly repeatable, yielding consistently close IOPs on three different dates (S1 Table). This type of error does not affect the overall conclusion of our study. Nonetheless, for achieving direct evidence of IOP elevation causing the pathology of RGCs, it would be highly desirable to improve the accuracy and the dynamic range of tonometer. It can be achieved by recruiting an independent IOP measurement. Cannulation and manometry is the gold standard of IOP measurement, which could provide the ground truths to calibrate TonoLab devices [26]. Although invasive, hence less suitable for repeat measurements, cannulation can avoid potential errors of TonoLab arising from age-dependent changes in the mechanical properties of the eye. Finally, quantitative analysis was confounded by the substantial unexplained variation of D2 mice. It could be beneficial to employ an alternative glaucoma model of IOP-induced rodents [27]. By isolating and regulating the IOP insult, it could resolve the relationship between the mitigation of IOP elevation and the metabolic rescue of RGCs by NAM/NAD. From the observed microtubule deficit’s negative correlation with IOP, it is anticipated that the pathology is likely to be present also in other glaucoma models.

We have demonstrated that SHG imaging is well-suited for interrogating microtubule deficit. The information conveyed by the SHG signal is much distinguished from immunofluorescence against tubulin monomers, which is necessary but not sufficient for the integrity of microtubule assembly. Characterizing microtubule deficit across the whole retina, which is nontrivial with electron microscopy, is straightforward with SHG imaging. Being sensitive to the functional form of axonal microtubules, SHG provides an ideal readout for dissecting their mechanistic role in glaucoma pathogenesis.

Materials and methods

Animals

All procedures were approved by the Hunter College Institutional Animal Care and Use Committee (IACUC). DBA/2J (# 000671) and DBA/2J-Gpnmb+ (# 007048) mice were purchased from The Jackson Laboratory and housed in the animal facility at Hunter College. DBA/2J mice were obtained at the ages of 8–12 weeks and housed until the desired age. Breeding pairs of DBA/2J-Gpnmb+ were obtained at the age of around 4 weeks and the offsprings were raised to the desired age.

Pharmacology

Nicotinamide was given at a low dose of 550 mg nicotinamide/kg body weight per day [12, 13] by adding to standard pelleted chow (2750 mg/kg in LabDiet 5001, based on an average 25-g mouse consuming 5 g of diet per day) (Bio-Serv).

IOP measurement

IOP was measured using a TonoLab tonometer (Icare) following the manufacturer’s manual. Mouse was awake (i.e., unanesthetized) and restricted during the measurement. Three measurements were performed on consecutive dates prior to SHG imaging. The time of measurement was standardized at a fixed hour during daytime to avoid the intraday fluctuations [28].

Tissue preparation

The animal was deeply anesthetized with isoflurane and the first eye was enucleated. After the enucleation of the second eye, the animal was euthanized by CO2 inhalation. The retinal flatmounts were prepared as previously described [10]. Briefly, an incision was made along the corneal limbus, the lens and sclera were removed, and radial cuts were made to relieve the curvature. The flat-mounted retina was transferred to a glass bottom dish (MatTek Corp.) and incubated at room temperature in the Ames’ medium (A1420, Sigma-Aldrich) oxygenated with 95%O2/5%CO2. Due to the lability of microtubules, SHG signals from the polymer last for a short period of time after enucleation (∼ 2–3 hours). Therefore, for the accuracy of analysis, the samples were discarded if the flatmounts were not of high quality for 3D mosaics or too much time was elapsed for the preparation.

SHG microscopy

An experimental setup for SHG microscopy was similar to the previous study [10, 15]. Briefly, 100-fs pulses at an 80-MHz repetition rate from a Ti:Sapphire laser (Chameleon Ultra, Coherent, Inc.) were used for the excitation. The output wavelength was 900 nm. The polarization state of excitation beam was controlled with half- and quarter-waveplates. A water-dipping microscope objective lens (HC FLUOTAR L 25x 0.95NA, Leica) was used to focus the excitation beam onto the sample. The average power was approximately 20 mW at the sample. The forward-propagating SHG from the sample was collected with an UV-transparent high-NA objective lens (UApo340 40× 1.35NA, Olympus), passed through a narrow-bandpass filter (<20-nm bandwidth) at a half of the excitation wavelength (400 nm), and then detected with a photomultiplier tube (PMT; H7422-40, Hamamatsu, Inc.). Images with 512×512 pixels were acquired, and the pixel dwell time was ∼3 μs. A region was imaged twice for orthogonal linear polarizations, which then were summed into a composite image. Z-stacks were acquired in a step of 2 μm. For creating mosaics, a total of 9 regions (742x742 μm2 each) were imaged on and around the optic nerve head at 1-mm radius.

Immunohistochemistry and confocal microscopy

Immunohistochemistry was performed similar to the prior studies [20, 23, 29]. After SHG imaging, the retinal flatmount was fixed with 4% paraformaldehyde for 20 minutes at room temperature. The sample was dehydrated sequentially in 25%, 50%, 75%, and 100% cold methanol for 15 min each and then permeabilized with dichloromethane for 2 hours. Then the retina was rehydrated in 75%, 50%, 25%, and 0% cold methanol for 15 min each. The sample was blocked in a buffer containing 5% normal serum and 0.3% Triton™ X-100 (Thermo Fisher Scientific, Inc.) for 3 hours. It was incubated in the primary antibody buffer at 4°C for 3 days. Rabbit and mouse monoclonal antibodies against βIII tubulin (EP1569Y, Abcam, Inc.) and pNF (2F11, EMD MilliporeSigma), respectively, were used at 1:300 dilution. The sample was incubated in the secondary antibody buffer at 4°C for 2 hours. Goat anti-rabbit and anti-mouse IgG antibodies conjugated with Alexa Fluor 594 and Alexa Fluor 647, respectively, were used (AB150080 and AB48389, respectively, Abcam, Inc.). The sample was mounted on a glass slide in Vectashield medium (Vector Laboratories, Inc.), and then imaged with a Leica TCS SP8 DLS confocal microscope using an oil-immersion objective lens (HC PL APO CS2 40× 1.3NA, Leica).

Image analysis

Image processing was done using ImageJ [30] and MATLAB (MathWorks, Inc.). Mosaics were created using an ImageJ stitching plugin [31]. SHG normalization and thickness estimation was done as previously described [10]. Briefly, the composite SHG intensity was corrected for topography and normalized by dividing with the Fano factor. The thickness of the retinal nerve fiber was evaluated by means of context-free image segmentation, which was done through edge detection and histogram-based thresholding. The thresholded z-stack images were sum-projected and then multiplied with the z-step (2 μm) to obtain the thickness of the retinal nerve fibers. The SHG density was obtained by dividing the normalized SHG intensity by thickness. The volume of the retinal nerve fibers was evaluated by integrating the thickness over the region.

Statistical analysis

Statistical analysis was performed using R [32]. The homogeneity of variance and normality were verified by inspecting the residual plots and the Q-Q plot. The collinearity of the explanatory variables was tested by the variance inflation factors. The IOP, the volume, and the mean SHG density were analyzed as the response variables with a multiple linear regression model. Three main effects of age, sex, and diet were considered. Of three possible interactions, the term between sex and diet was dropped on the ground that the effect of NAM is not known to be dependent on sex. Also, to aid model specification, the fit of multiple linear regression model was assessed by the adjusted R-squared. The alpha level for statistical significance was 0.05.

Supporting information

S1 Table The measurement values of D2 and D2-Gpnmb+.

(DOCX)

S1 Fig Microtubule deficit versus the distribution of cytoskeletal proteins.

(a) Mosaics of a D2 retina (12 months age, female) by SHG and immunofluorescence against the cytoskeletal elements, βIII tubulin and pNF. Scale bars, 200 μm. (b) Comparing two sectors in different stages of RGC degeneration. Arrows, pNF+ RGC somas. Scale bars, 100 μm.

(TIF)

10.1371/journal.pone.0309400.r001
Decision Letter 0
Anderson Michael G Academic Editor
© 2024 Michael G Anderson
2024
Michael G Anderson
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version0
27 May 2024

PONE-D-24-15544Protective effects of nicotinamide in a mouse model of glaucoma DBA/2 studied by second-harmonic generation microscopyPLOS ONE

Dear Dr. Lim,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We were able to obtain two reviews from people very respected in this kind of work. There were elements of excitement for the work, but as you'll see, they raise a significant number of issues with the current manuscript - all of which must be addressed. In keeping with the scope of PLoS One, I would emphasize that there is no weight applied regarding perceived impact. Thus, it is fine to point out caveats/weaknesses in the discussion, to refer to some aspects of the experiment as "negative" or as "interesting observations", to discuss aspects of the experimental design that might have been better designed knowing what is now known, to replace words like "indicates" with "suggests", and so forth. There is a requirement that "what is said matches what was done" and that nothing is over-stated. Thus, I believe with careful editing that a revised manuscript that addresses the Reviewers comments in the above context is possible (with no additional required experiments), but the editing will need to be extensive.  In addition to the comments of the two critiques, I would ask that a few sentences, or even a Figure of some sort, be added that help simply explain (and/or illustrate) how the measurements with second harmonics are related to the simplified terms of "morphology" vs "microtubules" that are used reiteratively. I think this would help make the paper more approachable to a broad readership.

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We look forward to receiving your revised manuscript.

Kind regards,

Michael G Anderson, PhD

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This manuscript by Boodram and Lim explores microtubule disruption following elevated intraocular pressure in a glaucoma model. The Authors then utilize nicotinamide (the amide of vitamin b3 and a precursor to NAD) to perform a metabolic rescue. The main findings of the study is that glaucoma in the DBA/2J mouse results in microtubule loss and loss of morphology which is partly rescued by nicotinamide treatment. Although the paper is of interest and supports the growing literature on metabolic dysfunction in glaucoma, the data presented it not sufficient to support the claims. The following should be considered:

1. Low n and lack of power:

D2 glaucoma is high heterogenous (a benefit and caveat in the model). This means that typically 30-60 eyes are used in any one experiment. The data presented here is thin and highly variable. In fact, the Authors claim that this might be due to experimental error (!).

How can the Authors, or the Readers be convinced of the data?! The n is also strangely presented - is it n = images, n = eyes, n = mice? For example: "(N=19, 13 from 8 males and 6 from 4 females)" - what does this mean? Does it refer to the number of eyes and if so what was the criteria for choosing that eye?

Although the slopes look convincing, they are highly overlapping and even if the stats support the slope, it might be a statistically relevant, but not biologically relevant, finding. It should also be noted that the D2 mice have very low IOPs in these slopes. This is likely due to the low n, but this Reviewer also questions the use of the TonoLab on D2 mice (which has been shown to give erroneous readings unless done frequently, repeatedly, and on large numbers of mice.

2. Proper use of D2 mice.

It would be helpful to refer to DBA/2J mice and their WT counterparts using the correct nomenclature and abbreviations (e.g. D2). This will help with searching for these studies and indexing.

How do the Authors justify their staging of D2 glaucoma? D2 glaucoma is highly variable across animal facilities based on climate, diet, pathogens etc. More information is required. Preferably some levels of RGC counts or axon counts should be given and not just IOP in the matrix (which has a high level of flux).

How was the NAM diet made and administered? It is not sufficient to reference a paper from another group (it is barely acceptable to reference yourself rather than putting the methods down). Was NAM chow provided ad libitum? Did the Authors control the volume of the food or follow how much the mice were eating to make sure it was 550 mg/kg/d?

In the 'Animals' section it said that mice were bought in. Were they bought in and bred? Or bought in at the right ages? If so, how long were they acclimatized for? What was their diet before they were brought into the colony?

3. Other general comments:

PLOSONE is not a specialized journal. Both the intro and discussion are lacking in information. Why did you do these experiments? What is the background? What is the meaning? What is the literature that led you to your hypotheses?

Figure legends are too brief and should more accurately explain their subject matter. Figures would also benefit from legends.

Following NAM treatment microtubule dysfunction is not fully recovered. The Authors discuss axon transport loss, however, 2 independent groups have shown that NAM prevents loss of axon transport and recovers PERG (PMID: 32605122, 28209901), so what is the mechanism or the Author's understanding of these results?

Reviewer #2: The authors evaluate the effects of nicotinamide on microtubules in DBA/2 glaucoma. Samples sizes are good (maybe a little on the low side) and timepoints evaluated are appropriate for this model. The results are modest with small effects that appear independent of a reduction in IOP elevation making drawing conclusions of the effects of NAM on microtubules directly challenging. Overall, the authors need to more carefully consider the results and not be afraid to state negative finding as an important contribution to the field. Specific issues that also need to be addressed are:

Major comments:

Table 1: Individual measurements (e.g. IOP readings) should be shown, not just summary statistics.

Lines 80-85: IOP by tonolab versus IOP by cannulation. Worth stating differences in accuracy given differences in this study to previous reports. Also, no relationship between IOP and RGC loss has been observed before, likely due to the fact that a single measurement of IOP is not reflective of IOP insult in DBA mice. Please include discussion.

Lines 151-152: Conclusion is overstated. Evidence shown is that there is no impairment in expression or transport. Please modify.

Results do not always include consideration of effect of NAM treatment, particularly in the last couple of sections. Given the title states “Protective effects of nicotinamide…” – these sections should be modified.

Overall, the Discussion could do with a more thoughtful interpretation of the data particularly in relation to previous studies in multiple models – beyond the DBA model.

Related to the limited discussion – figure 6 is too simplistic with little consideration of other complexities in human glaucoma and animal models, including the DBA/2J model. E.g., compartmentalized RGC stress as a result of IOP elevation, aging, mitochondrial deficits, glial cell activation to name a few.

Minor comments:

If mice came from The Jackson Lab, please refer to them as DBA/2J.

Line 59-60: Experimental description talks about N – make sure this refers to eyes and not mice

Line 177: What does “NAM promotes the morphology of the retinal nerve fibers more significantly than microtubules…” Please clarify.

Although not powered necessarily, are there any trends in sex differences?

**********

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Reviewer #1: No

Reviewer #2: Yes: Gareth Howell

**********

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10.1371/journal.pone.0309400.r002
Author response to Decision Letter 0
Submission Version1
16 Jun 2024

We are grateful for the thoughtful, expertly comments provided by the editor and reviewers. They have allowed us to identify areas of weakness in the previous version where our findings weren’t described adequately for the general readership. We revised the manuscript extensively with more detailed technical information to improve the rigor. We believe all the raised concerns are addressed. We received the following common criticisms from both reviewers:

1. IOP measurements with TonoLab

Both reviewers raised concerns about the IOP measurements. The IOP values of D2 mice we measured with TonoLab were lower than those in the literature. We used TonoLab according to the manufacturer’s instructions. Since the machine does not allow the user any control over calibration, other than choosing mouse or rat in the settings, the reason for the difference is unclear to us. Nonetheless, we concluded the measurements were reliable because 1) the measured IOPs on three different dates were consistently very close and 2) they displayed anticipated overall trends, such as D2 mice with age and NAM diet, as well as vs. D2-Gpnmb+. Individual IOP readings, which reviewer #2 asked about, are included as a Supplementary information in the revised version (S1 Table). Given the high repeatability/reproducibility, it seems unlikely that the underestimation was due to the low n (either number of samples or measurements), as reviewer #1 suspects. Rather, it could be a result of incorrect software setting or mechanical deviations of the rebound tonometer (which might be also behind the wide variability found in the literature). Conceivably, it is possible to determine, for a particular instrument that we used, the relationship between the actual and measured IOPs by performing a linear regression with the ground truths (e.g., by manometer). However, while the presumed scaling error does not affect the overall conclusion of our study, new errors that are harder to correct might arise from the extra steps of calibration. Considering these factors, the IOP values are presented as originally obtained with our TonoLab device, but a new paragraph is added to the Discussion regarding the accuracy of IOP measurement, as reviewer #2 recommended.

2. N, the number of eyes

Both reviewers pointed out the ambiguity of what N means. All N in the manuscript indicates eye samples, which we clarified in the revision. Reviewer #1 asked why the number of eyes was less than twice the number of animals and whether it was due to some selection. It is not the case. Not all eye samples yielded data because of the stringent conditions of SHG imaging. SHG signal from axonal microtubules lasts only for a few hours after enucleation, then it gradually decreases (presumably as the cytoskeleton depolymerizes in the absence of active regulation). No fixation protocol is yet known to preserve the signal. Consequently, there is a limited time window within which the imaging experiment must be completed. Furthermore, the quality of fresh, unfixed flatmount preparation is crucial for acquiring stacks of microscopic images across the large region. For the rigor of data, we discarded the retina samples that either took too long to prepare the flatmount or the full 3D volume could not be captured. Because of these demanding requirements, the success rate of SHG imaging was less than 100%. This information is added to the Methods.

3. DBA/2J (D2): Nomenclature and Animal section

DBA/2J (or D2) are used to denote the strain, as both reviewers suggested. Reviewer #1 asked about the acclimation. DBA/2J mice were brought in from The Jackson Lab at the ages of 8-12 weeks and housed until the desired age. Breeding pairs of DBA/2J-Gpnmb+ were obtained at the age of around 4 weeks and the offsprings were raised to the desired age. The information is now included in the Methods.

4. Figure legends

Both reviewers commented that figure captions are insufficient. We made substantial improvements in the revision.

Other comments:

5. (Reviewer #1) D2 variability and experimental error

Our description of the experimental errors was misunderstood, i.e., “Overall, the variations of D2 data were greater than those of Gpnmb+ representing primarily the experimental errors.”. The sentence is rewritten to clarify this.

6. (Reviewer #1) Microtubule deficit vs. PERG and anterograde axonal transport of CT-β

Reviewer #1 noted that other labs have shown that NAM prevents loss of axon transport and recovers PERG (PMID: 32605122, 28209901). Assuming that PERG is more correlated with the integrity of RGC axons (i.e., morphology) than microtubule cytoskeleton, it is understandable that NAM diet improves PERG signal. We can also reconcile the recovered anterograde axonal transport with our finding, i.e., that the mean SHG density, which is proportional to the density of microtubules per axon caliber, is not significantly protected by NAM as follows: Between NAM vs. no-NAM D2 retinas, as compared in the previous studies (Williams et al.), there are more total microtubules in the former, despite similarly low densities of microtubules, just because there are more RGC axons. Provided that the spatial extent of CT-β signals (both in the ONH and LGN) is correlated with the total amount of microtubules rather than with the density, it is expected that the NAM D2 retina will display more widespread CT-β signals than the no-NAM D2 retina. Based on our data, however, individual RGCs of the NAM D2 retina still suffer microtubule deficit. So, the CT-β signals are predicted to be much less intense in individual cells of the NAM D2 retina than those of the non-glaucomatous control (which Williams et al. did not compare). Furthermore, our results show that microtubule deficit (i.e., low mean SHG density) continues to worsen with age, even with NAM diet. Consequently, we predict that axonal transport in the NAM D2 retina will be compromised eventually at an age when the microtubule density drops below a certain level, regardless of the integrity of RGC axons. We included this as a paragraph in the Discussion.

7. (Reviewer #1) Background for the hypothesis of the study

Reviewer #1 asked for an appropriate context leading to the present study. The question, in our opinion, is related to the editor’s comment about our insufficient explanation of "morphology" vs "microtubules". We addressed this by including Fig. 1(c), as suggested, and clarifying the concepts in the revised text.

8. (Reviewer #1) Staging of D2 glaucoma

Reviewer #1 asked about the staging of D2 glaucoma. Unfortunately, our experimental design did not include the measurement of RGC counts. Only IOP and the properties of RGC axons, i.e., volume and mean SHG density, were measured as continuous independent variables, and their relationships were examined with age. The volume of the retinal nerve fibers can be considered an optical surrogate of RGC axon counts. We discuss this as one of the limitations of our study in the revision.

9. (Reviewer #1) NAM administration

Reviewer #1 questioned about NAM administration. NAM was added to the standard Rodent LabDiet 5001, the same diet the D2 mice received before switching to NAM-containing diet. For a dosage of 550 mg nicotinamide/kg body weight per day, we included 2750 mg nicotinamide/kg diet based on an average 25-g mouse consuming 5 g of diet per day. This information is added to the Methods.

10. (Reviewer #2) Last few sections of Results are not related to NAM treatment.

Reviewer #2 noted that the last two sections are not related to NAM treatment, which, considering that the title is “Protective effects of nicotinamide…”, should be modified. We agree with this critique. Those sections seek to investigate the details of microtubule deficit, or decoupling of morphology and microtubules, which was also evident in the response to NAM treatment. To address this comment, we changed the title to “Differential protection by nicotinamide…” and the sections are revised accordingly.

11. (Reviewer #2) Limited discussion

There were two related comments by Reviewer #2 regarding the limited discussion, i.e., that figure 6 is too simplistic and that the sentence “NAM promotes the morphology of the retinal nerve fibers more significantly than microtubules.” should be clarified. In response, we revised Fig 6 and the caption, and rewrote the first paragraph of Discussion entirely to enhance clarity.

Reviewer #2 encouraged us to include “a more thoughtful interpretation of the data particularly in relation to previous studies in multiple models – beyond the DBA model.”. In response, we added a new discussion comparing our finding of persistent microtubule deficit with the previously reported recovery of axonal transport by NAM. We also included a paragraph in the Discussion on how other glaucoma models beyond DBA/2 model could be beneficial for mechanistic studies of the NAM neuroprotection.

12. (Reviewer #2) Lines 151-152: Overstated conclusion regarding the origin of microtubule deficit.

The overstated conclusion is corrected.

Attachment Submitted filename: Response to Reviewers_Boodram and Lim.docx

10.1371/journal.pone.0309400.r003
Decision Letter 1
Anderson Michael G Academic Editor
© 2024 Michael G Anderson
2024
Michael G Anderson
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version1
21 Jun 2024

PONE-D-24-15544R1Differential protection by nicotinamide in a mouse model of glaucoma DBA/2J revealed by second-harmonic generation microscopyPLOS ONE

Dear Dr. Lim,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

As you will see below, there is a request to improve readability / clarity / cohesiveness prior to sending out for full re-review.

Please submit your revised manuscript by Aug 05 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Michael G Anderson, PhD

Academic Editor

PLOS ONE

Additional Editor Comments:

Thank you for resubmitting your revised manuscript, which seems to have addressed several of the comments that were raised in initial review. However, prior to sending it back to the reviewers, I am asking that you first address a few important text issues that remain required. Between issues which appear to have been introduced during the extensive editing, and others remaining to be addressed, there is concern with readability that must be addressed. I hope handling these issues in this way might save time in the overall review process, and that they could be addressed with a modest amount of work – but they must be addressed to make the manuscript more readable and coherent.

1. The manuscript recurrently uses “morphology” to refer to the volume of the nerve fibers and “integrity” to refer to the SHG density. However, this distinction is not crisply defined and sometimes used in slightly different iterations that add confusion.

Please revisit how this central concept of the manuscript is addressed and strive to make it uniform and more readily appreciated. If the specific finding is that the NFL thickness is maintained by NAM, while the SHG density signal is not – it’s unclear why the additional terms are needed at all? (“Morphology” can refer to many things with respect to a retina or an axon).

Please revisit the descriptions for how volume was measured – it’s unclear if it’s based on IHC or inferred from the microscopy in general, if its synonymous with NFL thickness; or is a second measurement coming from the SHG imaging?

Throughout the manuscript (preferably at least in the Introduction and again in the Discussion) please integrate some language generic to an ophthalmic researcher describing what the two main measures likely relate to. Is “volume / morphology” the NFL thickness? If it comes from SHG, is the signal from the entire depth of the NFL, such that its decay would indicate NFL thinning? Is “density / integrity” from a single plane, or an average of each plane, or something else? Does it’s decay indicate that microtubules are lost or disorganized? Is the overall finding that NAM failing to protect SHG density an indication that NAM doesn’t protect the qualities of microtubules giving rise to SHG, perhaps that microtubules still become somehow structurally disorganized? If so, please use a sentence or two to restate the main finding and main implication plainly.

2. The term “microtubule deficit” is not defined until midway through the manuscript. If this phrase is going to be used as a noun to name a phenomenon, please define it early in the Introduction and again in the Methods.

3. In different places, citations 12 and 13 are used to support that NAM induces a change in IOP, and that it does not cause a change in IOP – please double check your intended meanings.

4. The phrase “…lower quantities than normal for the caliber” (L29, L128) is confusing – does this mean caliper of the individual axons or caliper of some other metric?

5. Issues 1-4 are present in the current iteration of the Abstract – with lines 26-30 particularly confusing. Please revisit to make sure that this portion of the manuscript can “stand alone” and does not use abbreviations or references to phenomenon that aren’t defined.

6. The Discussion raises several interesting points, but they don’t coalesce to make it clear what the primary model being proposed for glaucoma and NAM are with respect to microtubules, what the caveats/discordant data are, and which portions might best be called “speculative”.

[Note: HTML markup is below. Please do not edit.]

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While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

10.1371/journal.pone.0309400.r004
Author response to Decision Letter 1
Submission Version2
2 Jul 2024

We are grateful for the thoughtful, expertly comments provided by the editor and reviewers. They have allowed us to identify areas of weakness in the previous version where our findings weren’t described adequately for the general readership. We revised the manuscript extensively with more detailed technical information to improve the rigor. We believe all the raised concerns are addressed. We received the following common criticisms from both reviewers:

1. IOP measurements with TonoLab

Both reviewers raised concerns about the IOP measurements. The IOP values of D2 mice we measured with TonoLab were lower than those in the literature. We used TonoLab according to the manufacturer’s instructions. Since the machine does not allow the user any control over calibration, other than choosing mouse or rat in the settings, the reason for the difference is unclear to us. Nonetheless, we concluded the measurements were reliable because 1) the measured IOPs on three different dates were consistently very close and 2) they displayed anticipated overall trends, such as D2 mice with age and NAM diet, as well as vs. D2-Gpnmb+. Individual IOP readings, which reviewer #2 asked about, are included as a Supplementary information in the revised version (S1 Table). Given the high repeatability/reproducibility, it seems unlikely that the underestimation was due to the low n (either number of samples or measurements), as reviewer #1 suspects. Rather, it could be a result of incorrect software setting or mechanical deviations of the rebound tonometer (which might be also behind the wide variability found in the literature). Conceivably, it is possible to determine, for a particular instrument that we used, the relationship between the actual and measured IOPs by performing a linear regression with the ground truths (e.g., by manometer). However, while the presumed scaling error does not affect the overall conclusion of our study, new errors that are harder to correct might arise from the extra steps of calibration. Considering these factors, the IOP values are presented as originally obtained with our TonoLab device, but a new paragraph is added to the Discussion regarding the accuracy of IOP measurement, as reviewer #2 recommended.

2. N, the number of eyes

Both reviewers pointed out the ambiguity of what N means. All N in the manuscript indicates eye samples, which we clarified in the revision. Reviewer #1 asked why the number of eyes was less than twice the number of animals and whether it was due to some selection. It is not the case. Not all eye samples yielded data because of the stringent conditions of SHG imaging. SHG signal from axonal microtubules lasts only for a few hours after enucleation, then it gradually decreases (presumably as the cytoskeleton depolymerizes in the absence of active regulation). No fixation protocol is yet known to preserve the signal. Consequently, there is a limited time window within which the imaging experiment must be completed. Furthermore, the quality of fresh, unfixed flatmount preparation is crucial for acquiring stacks of microscopic images across the large region. For the rigor of data, we discarded the retina samples that either took too long to prepare the flatmount or the full 3D volume could not be captured. Because of these demanding requirements, the success rate of SHG imaging was less than 100%. This information is added to the Methods.

3. DBA/2J (D2): Nomenclature and Animal section

DBA/2J (or D2) are used to denote the strain, as both reviewers suggested. Reviewer #1 asked about the acclimation. DBA/2J mice were brought in from The Jackson Lab at the ages of 8-12 weeks and housed until the desired age. Breeding pairs of DBA/2J-Gpnmb+ were obtained at the age of around 4 weeks and the offsprings were raised to the desired age. The information is now included in the Methods.

4. Figure legends

Both reviewers commented that figure captions are insufficient. We made substantial improvements in the revision.

Other comments:

5. (Reviewer #1) D2 variability and experimental error

Our description of the experimental errors was misunderstood, i.e., “Overall, the variations of D2 data were greater than those of Gpnmb+ representing primarily the experimental errors.”. The sentence is rewritten to clarify this.

6. (Reviewer #1) Microtubule deficit vs. PERG and anterograde axonal transport of CT-β

Reviewer #1 noted that other labs have shown that NAM prevents loss of axon transport and recovers PERG (PMID: 32605122, 28209901). Assuming that PERG is more correlated with the integrity of RGC axons (i.e., morphology) than microtubule cytoskeleton, it is understandable that NAM diet improves PERG signal. We can also reconcile the recovered anterograde axonal transport with our finding, i.e., that the mean SHG density, which is proportional to the density of microtubules per axon caliber, is not significantly protected by NAM as follows: Between NAM vs. no-NAM D2 retinas, as compared in the previous studies (Williams et al.), there are more total microtubules in the former, despite similarly low densities of microtubules, just because there are more RGC axons. Provided that the spatial extent of CT-β signals (both in the ONH and LGN) is correlated with the total amount of microtubules rather than with the density, it is expected that the NAM D2 retina will display more widespread CT-β signals than the no-NAM D2 retina. Based on our data, however, individual RGCs of the NAM D2 retina still suffer microtubule deficit. So, the CT-β signals are predicted to be much less intense in individual cells of the NAM D2 retina than those of the non-glaucomatous control (which Williams et al. did not compare). Furthermore, our results show that microtubule deficit (i.e., low mean SHG density) continues to worsen with age, even with NAM diet. Consequently, we predict that axonal transport in the NAM D2 retina will be compromised eventually at an age when the microtubule density drops below a certain level, regardless of the integrity of RGC axons. We included this as a paragraph in the Discussion.

7. (Reviewer #1) Background for the hypothesis of the study

Reviewer #1 asked for an appropriate context leading to the present study. The question, in our opinion, is related to the editor’s comment about our insufficient explanation of "morphology" vs "microtubules". We addressed this by including Fig. 1(c), as suggested, and clarifying the concepts in the revised text.

8. (Reviewer #1) Staging of D2 glaucoma

Reviewer #1 asked about the staging of D2 glaucoma. Unfortunately, our experimental design did not include the measurement of RGC counts. Only IOP and the properties of RGC axons, i.e., volume and mean SHG density, were measured as continuous independent variables, and their relationships were examined with age. The volume of the retinal nerve fibers can be considered an optical surrogate of RGC axon counts. We discuss this as one of the limitations of our study in the revision.

9. (Reviewer #1) NAM administration

Reviewer #1 questioned about NAM administration. NAM was added to the standard Rodent LabDiet 5001, the same diet the D2 mice received before switching to NAM-containing diet. For a dosage of 550 mg nicotinamide/kg body weight per day, we included 2750 mg nicotinamide/kg diet based on an average 25-g mouse consuming 5 g of diet per day. This information is added to the Methods.

10. (Reviewer #2) Last few sections of Results are not related to NAM treatment.

Reviewer #2 noted that the last two sections are not related to NAM treatment, which, considering that the title is “Protective effects of nicotinamide…”, should be modified. We agree with this critique. Those sections seek to investigate the details of microtubule deficit, or decoupling of morphology and microtubules, which was also evident in the response to NAM treatment. To address this comment, we changed the title to “Differential protection by nicotinamide…” and the sections are revised accordingly.

11. (Reviewer #2) Limited discussion

There were two related comments by Reviewer #2 regarding the limited discussion, i.e., that figure 6 is too simplistic and that the sentence “NAM promotes the morphology of the retinal nerve fibers more significantly than microtubules.” should be clarified. In response, we revised Fig 6 and the caption, and rewrote the first paragraph of Discussion entirely to enhance clarity.

Reviewer #2 encouraged us to include “a more thoughtful interpretation of the data particularly in relation to previous studies in multiple models – beyond the DBA model.”. In response, we added a new discussion comparing our finding of persistent microtubule deficit with the previously reported recovery of axonal transport by NAM. We also included a paragraph in the Discussion on how other glaucoma models beyond DBA/2 model could be beneficial for mechanistic studies of the NAM neuroprotection.

12. (Reviewer #2) Lines 151-152: Overstated conclusion regarding the origin of microtubule deficit.

The overstated conclusion is corrected.

Additional Editor comments for improving the readability:

13. NFL thickness versus morphology

The Editor asks why the term ‘morphology’ is needed for indicating the volume of the retinal nerve fibers if it is synonymous with the ‘NFL thickness’. It would be a misrepresentation to replace morphology/volume with NFL thickness. The latter is commonly used in the ophthalmic field because ophthalmic imaging modalities (e.g., OCT) have inadequate lateral resolutions due to the low NA of the human eye. As a result, they cannot quantify precisely the retinal nerve fibers in the later dimensions; thus, NFL thickness is used as a surrogate measure of morphology. A major difference is that the volume, as we evaluated here, measures the lateral loss of the retinal nerve fibers as well as the axial loss, i.e., the NFL thinning.

The volume was estimated from SHG images, as Fig 1(c) shows. The morphology of the retinal nerve fibers cannot be measured accurately by IHC due to tissue distortions (such as shrinking) occurring during the preparation.

As SHG intensity per thickness, SHG density probes specifically the changes in the microtubules regardless of the thickness.

The manuscript is revised to clarify these points. Whenever applicable, we tried to refer to the volume and SHG density, rather than morphology and microtubules, respectively.

14. The definition of “microtubule deficit”

The Editor asks to define the term early on. When the study was conceived, we were agnostic about the differential protection of NAM. In fact, our hypothesis was that both microtubules and morphology would be rescued. The connection to an unrelated phenomenon of microtubule deficit became relevant only after the data showed that the two properties responded differently to NAM treatment. So, we think it is important for the logical progression that the first mention of microtubule deficit appears after the result of divergence is presented. However, we agree that the term must be defined appropriately in Abstract so that it can stand alone. The manuscript is revised accordingly.

15. “…lower quantities than normal for the caliber” (L29, L128) is confusing.

Microtubule deficit is explained/defined with more clarity.

16. Citations 12 and 13 on NAM vs. IOP

NAM can induce changes of IOP or not depending on the dosage, according to the work cited in 12 and 13. The manuscript is revised to explain this prior art.

17. Unclear model in the Discussion

The model is to explain how IOP-independent pathway of RGC protection, which has been shown previously, might underlie the divergent response to NAM that we observed. The manuscript is revised to make this point lucid.

Attachment Submitted filename: Response to Reviewers_Boodram and Lim.docx

10.1371/journal.pone.0309400.r005
Decision Letter 2
Anderson Michael G Academic Editor
© 2024 Michael G Anderson
2024
Michael G Anderson
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version2
24 Jul 2024

PONE-D-24-15544R2Differential protection by nicotinamide in a mouse model of glaucoma DBA/2J revealed by second-harmonic generation microscopyPLOS ONE

Dear Dr. Lim,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. 

Your revised manuscript has addressed most points raised in initial review.  However, neither Reviewer yet recommended “Accept”. In considering their remaining concerns (expressed partially in the comments to the Authors, and partially in comments to the Editor) and balancing them with the scope of PLoS One, I ask you to address a few lingering minor points. Note that most of these are readily achievable by modest text changes in the Discussion.

From Reviewer 1. Note that the Reviewer indicated concerns with the technical soundness and statistical analyses of the manuscript. In keeping with the scope of PLoS One, two minor changes would likely address these concerns:

In the Discussion of caveats, I suggest that you add text pointing out that Gpnmb+ mice on NAM diet were not included as a control group but could have been informative.

In the Results and/or Discussion (and Methods), please add a post hoc power analysis for at least one of the phenotypes and discuss the power briefly in the Discussion. Or, if the analyses are still in a discovery phase in which meaningful power analyses might be premature, please expand on what was learned from the current experiment that might guide future quantitative studies and point out the uncertain power of the current experiments in the caveats.

From Reviewer 2:

Regarding sufficiency of the presentation on caveats of rebound tonometry. I suggest that in the Discussion of IOP and the caveats associated with the rebound tonometer, please also note that cannulation (which some labs experienced with D2 mice consider a gold-standard for this strain) might also have helped achieve direct (or additional) evidence of IOP elevation across multiple ages.

Regarding Figure 6. I agree with the Reviewer that the simplistic nature of this Figure has a modest impact on the manuscript, but as an online journal space is not particularly limiting and leave it to your discretion whether you prefer to keep or drop this Figure.

Regarding IOP-independent mechanisms. Despite uncertainty in the mechanism, Williams has published on this topic and it would be appropriate to add a sentence or two and some citations.

==============================

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Michael G Anderson, PhD

Academic Editor

PLOS ONE

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Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

In addition to my suggestions found above, please note that the Figures use "DBA" as a label - which should be corrected to either the full strain name (DBA/2J) or its standard abbreviation (D2).

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

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Reviewer #1: Partly

Reviewer #2: Yes

**********

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Reviewer #1: No

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Please see comments to the Editor. There are still outstanding issues that need to be addressed. Otherwise the manuscript has been significantly improved.

Reviewer #2: The authors have done a good job of responding the majority of the comments. However, a couple still remain.

1. Please state that IOP by cannulation is the gold standard for the DBA/2J model and errors in tonolab can arise due to changes to the eyes with age.

1. I do not think Figure 6 is necessary and should be removed.

2. The discussion about the IOP-independent mechanisms should fleshed out a little further, possibly simply with further reference to the Williams et al Science paper indicating protection at the level of mitochondrial abnormalities.

**********

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Reviewer #1: Yes: Pete A Williams

Reviewer #2: Yes: Gareth Howell

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10.1371/journal.pone.0309400.r006
Author response to Decision Letter 2
Submission Version3
1 Aug 2024

We thank the reviewers for their thoughtful comments. We made every effort to understand the concerns and revised the manuscript to address all of them as summarized below.

(Reviewer 1) I suggest that you add text pointing out that Gpnmb+ mice on NAM diet were not included as a control group but could have been informative.

The text is included in the Discussion, as suggested. In hindsight, the control group could have informed us whether the IOP-independent effect of NAM is specific to the glaucoma process or also present in the non-glaucomatous eyes.

(Reviewer 1) Please add a post hoc power analysis for at least one of the phenotypes and discuss the power briefly in the Discussion. Or, if the analyses are still in a discovery phase in which meaningful power analyses might be premature, please expand on what was learned from the current experiment that might guide future quantitative studies and point out the uncertain power of the current experiments in the caveats.

Admittedly, we were not aware of the concept of post hoc power analysis prior to this comment. We found a fair amount of literature and online discussions on the topic, especially whether it is a meaningful analysis. Focusing on our study, we understand the question in hand is this: Given that we cannot reject the assumption that NAM diet does not have an effect on the mean SHG density (i.e., the null hypothesis), is there a sample size that we can calculate with which the effect could have been observed? The underlying reasoning is that the effect was not detected probably because it was too small and will be detectable with a larger sample size (or statistical power). The answer to the question is, yes, there can be such a sample size and, no, it cannot be calculated based on the current data. The explanation why such an estimation is flawed/misleading can be found in numerous papers (e.g., PMID: 11310512, 31552383, 29994928). In a nutshell, the estimated post hoc power is based on the variance of sample whereas the prospective power is on the variance of population. Because the estimated effect size is noisy, the power necessary to detect it (i.e., the post hoc power) is not informative. It has been proposed to present confidence intervals as an alternative (PMID: 11310512). This information is already in Fig.2, where the 95% confidence intervals are depicted around the slope of age-dependence for the two cases of diet.

(Reviewer 2) Please state that IOP by cannulation is the gold standard for the DBA/2J model and errors in tonolab can arise due to changes to the eyes with age.

The statement is added to the Discussion, as suggested.

(Reviewer 2) The discussion about the IOP-independent mechanisms should be fleshed out a little further, possibly simply with further reference to the Williams et al Science paper indicating protection at the level of mitochondrial abnormalities.

In response, we included a description of molecular changes reversed by NAM treatment, as shown in Williams et al., and the implications in the IOP-independent mechanism.

(Reviewer 2) I do not think Figure 6 is necessary and should be removed.

Figure 6 is removed, as suggested.

Attachment Submitted filename: Response to Reviewers_Boodram and Lim.docx

10.1371/journal.pone.0309400.r007
Decision Letter 3
Anderson Michael G Academic Editor
© 2024 Michael G Anderson
2024
Michael G Anderson
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version3
13 Aug 2024

Differential protection by nicotinamide in a mouse model of glaucoma DBA/2J revealed by second-harmonic generation microscopy

PONE-D-24-15544R3

Dear Dr. Lim,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Michael G Anderson, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Thank you for the final round of revisions. Sorry for the delay in accepting them as I was out of the country with no accessibility the past week.

I think the current manuscript has much solid data that the field will find meaningful. I would share that one of the reviewers was recurrently concerned with statistical power, which was minimally resolved, but might be helpful for your team to bear in mind as a concern for future work. For example, you might start incorporating and reporting pre-emptive power analyses. You could also report a calculation for a meaningful sample size if another group wanted to attempt to replicate a study - which the variability data from your study should be able to inform.

Reviewers' comments:

10.1371/journal.pone.0309400.r008
Acceptance letter
Anderson Michael G Academic Editor
© 2024 Michael G Anderson
2024
Michael G Anderson
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
30 Aug 2024

PONE-D-24-15544R3

PLOS ONE

Dear Dr. Lim,

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team.

At this stage, our production department will prepare your paper for publication. This includes ensuring the following:

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on behalf of

Dr. Michael G Anderson

Academic Editor

PLOS ONE
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