Lung microstructure in adolescent idiopathic scoliosis before and after posterior spinal fusion.

Adolescent idiopathic scoliosis (AIS) is associated with decreased respiratory quality of life and impaired diaphragm function. Recent hyperpolarized helium (HHe) MRI studies show alveolarization continues throughout adolescence, and mechanical forces are known to impact alveolarization. We therefore hypothesized that patients with AIS would have alterations in alveolar size, alveolar number, or alveolar septal dimensions compared to adolescents without AIS, and that posterior spinal fusion (PSF) might reverse these differences. We conducted a prospective observational trial using HHe MRI to test for changes in alveolar microstructure in control and AIS subjects at baseline and one year. After obtaining written informed consent from subjects' legal guardians and assent from the subjects, we performed HHe and proton MRI in 14 AIS and 16 control subjects aged 8-21 years. The mean age of control subjects (12.9 years) was significantly less than AIS (14.9 years, p = 0.003). At baseline, there were no significant differences in alveolar size, number, or alveolar duct morphometry between AIS and control subjects or between the concave (compressed) and convex (expanded) lungs of AIS subjects. At one year after PSF AIS subjects had an increase in alveolar density in the formerly convex lung (p = 0.05), likely reflecting a change in thoracic anatomy, but there were no other significant changes in lung microstructure. Modeling of alveolar size over time demonstrated similar rates of alveolar growth in control and AIS subjects in both right and left lungs pre- and post-PSF. Although this study suffered from poor age-matching, we found no evidence that AIS or PSF impacts lung microstructure. Trial registration: Clinical trial registration number NCT03539770.

Introduction

Adolescent idiopathic scoliosis (AIS) affects 2–3% of American adolescents, and approximately ten percent of these children will require posterior spinal fusion (PSF) [1]. There are conflicting studies as to whether PSF improves pulmonary function [2-4], and pulmonary MRI studies demonstrate increased lung dimensions and improved diaphragm function after PSF for AIS [5]. However, whether any improvements are due solely to improved pulmonary mechanics or whether AIS and PSF might impact alveolar size, number, or structure has not been investigated. The lung contains progressively dividing airway structures that lead to gas-exchanging respiratory bronchioles and terminate in clusters of alveoli termed the acinus. The adult human lung has a gas-exchange surface area of 24 to 69 m2 [6] with 274–790 million alveoli [7]. The majority of alveologenesis occurs postnatally, and it is controversial whether this processes ceases during childhood [8] or continues throughout adolescence [9]—the age at which spinal curvature in AIS increases.

Mechanical forces influence distal lung structure during the alveolar stage of lung development. Rodents [10], large animals [11], and humans [12] that undergo pneumonectomy experience a compensatory increase in the size and number of alveoli of the remaining lung, and in mice, ipsilateral phrenic nerve transection after pneumonectomy prevents compensatory lung growth [13]. A rabbit model of early onset scoliosis demonstrated impaired lung microvascular development and emphysema in the concave lung [14]. Studies in infantile-onset scoliosis demonstrated impaired alveolar development, impaired gas exchange and differential lung vascularization [15, 16]. We developed this study to investigate whether altered intrathoracic forces in AIS (e.g. impaired diaphragm function, compression of the concave lung, and expansion of the convex lung) cause impaired alveolarization as has been noted in earlier-onset scoliosis. Convex and concave refers to lateral spine geometry when observed in the anterior-posterior or posterior-anterior plane.

Hyperpolarized gas MRI is a well-established method for evaluation of distal lung architecture [17]. In this technique the nuclear particle spins of a noble gas (3He in this case) are aligned using an optical spin transfer device yielding a nuclear magnetization more than 100,000-fold greater than could achieved by magnetic alignment. The hyperpolarized helium (HHe) is inhaled and the airspaces imaged using a standard multi-slice diffusion MRI sequence during a ~15 second inspiratory breath hold. The intrinsic diffusion of the gas within the lung is restricted by the alveolar walls, and diffusion imaging provides maps of apparent diffusion coefficients (ADC), alveolar number density (N), mean linear intercept (L), and other morphometric properties of the distal airspaces [18, 19]. This technique has been used to assess lung structure in normal children [9], children with bronchopulmonary dysplasia [20, 21], and adults with emphysema [22-24]. To determine whether patients with AIS experience alterations in distal lung structure and whether PSF reverses these changes, we performed HHe and proton MRI in control subjects and in AIS subjects recommended for PSF with follow up imaging at 1 year.

Materials and methods

Human subjects

The Cincinnati Children’s Hospital Institutional Review Board (Approval 2013–8260) approved study procedures. Clinical trial registration number NCT03539770.

Screening

Patients with AIS being evaluated for PSF in the orthopedics clinic at Cincinnati Children’s Hospital were screened and approached for consent. Patients aged 8–21 years with AIS were considered eligible with a Cobb angle of >50° and recommendation for PSF. Cobb angles were measured using standard PA spine radiographs and accepted techniques [25]. Exclusion criteria included previous spinal surgery, history of any chronic lung disease or asthma, personal history of smoking, supplemental oxygen requirement at baseline, born at <35 weeks gestational age, mechanical ventilation in the first year of life, or room air oxyhemoglobin saturation of less than 95%. Control subjects were recruited through community advertising with the same inclusion and exclusion criteria except that AIS was an exclusion.

HHe MRI

All imaging was performed on a Philips Achieva 3.0T MRI with multinuclear capability. HHe was administered under FDA IND#122,670. 3He gas was polarized to approximately 50% polarization using a home-built rubidium optical spin transfer device [26]. Once polarized, HHe was diluted with nitrogen to a 50:50 volume ratio to ensure a consistent HHe intrinsic diffusion coefficient across subjects (D0 ≈ 1.2 cm2/s). The gas mixture was delivered to the subjects in the MR scanner and inhaled through a mouthpiece with two-way valve. Safety of this technique has been previously demonstrated [27, 28]. During the inspiratory breath-hold (FRC + 1 liter), 6–8 axial slices were acquired using a diffusion-weighted gradient echo MR sequence were acquired (b-values: 0, 2, 4, 6, 8 s/cm2, Δ = δ = 1.5 ms, TR = 25 ms, TE = 4.9 ms matrix size = (31–54) phase encodes (PE) × (36–76) read-outs (RO)).

Proton MRI

Ultrashort echo time (UTE) proton MRI images were obtained with multiple echo times (TE) using the ‘stack-of-stars’ radial acquisition scheme which was echo navigator gated at end expiration while breathing room air using the following parameters: repetition time (TR) = 5.8 ms, TE = 0.2/1.31/2.43 ms, flip angle (FA) = 5°, matrix size = 224×224, field of view (FOV) = 200×200 mm2, voxel size = 1.39×1.39×4 mm3 as previously reported [29].

Image analysis

HHe images were analyzed according to previously described methods [17]. In short, each voxel’s signal per b-value was fit to a multi-exponential model which provides7 quantitative, regional maps of HHe apparent diffusion coefficient (ADC [cm2/s]), alveolar number per unit volume (N [cm−3]), mean linear intercept (Lm [μm]), alveolar septal height (h, μm), and alveolar duct inner and outer radius (r and R respectively, μm). These maps were then segmented for separate left/right lung analysis. The UTE images with the longest echo-time (TE = 2.43 ms) were used to calculate right/left lung volumes since longer echo time images provide the greatest contrast between parenchyma and tissue. A threshold was applied to distinguish the lung interior from surrounding tissue, and the number of parenchymal voxels was counted for each lung; lung volume was calculated as the number of voxels multiplied by the voxel volume (7.73 mm3). For one case, UTE images were not acquired due to time restrictions. For this subject, lung volume was calculated using standard proton GRE sequences (voxel size = 1.95×1.95×10 mm3). Since lung volume increases with growth, we indexed lung volume measurements to the height-calculated functional residual capacity plus one liter [30].

Statistical analysis

The average value for all measurements from right, left, and bilateral lungs were used in analysis. Statistical comparisons were made using R version 6.2, ggplot2 and dply packages [31-33]. As many values were not parametric, we used Mann Whitney U-test to analyze all data with Wilcoxon Signed Rank test for sequential measurements in the same subject. For modeling of L with age, the generalized linear modeling (glm) function of ggplot2 was utilized giving standard error ranges for modeled functions. p-values of less than 0.05 were considered significant.

Results

Demographics and enrollment

From April 1, 2015 to April 30, 2019 we enrolled 14 AIS and 16 control subjects. Data loss occurred in 1 AIS subject after the first scan and in 3 control subjects after the second scan. One AIS and 2 control subjects were lost to follow-up. One AIS subject had diffusion but not proton data available. Demographic data is show in Table 1.

Table 1

Demographics.

		Control (n = 16)	AIS (n = 14)	p-value
Age (years)		12.9	14.9	0.003
Sex (% Female)		63	92	0.46
Height (cm)		150.7	161.2	0.02
Race	Caucasian (%)	94	93	1
	African American (%)	0	7
	Asian (%)	6	0

Imaging and data processing

A schematic depicting how the gradient echo HHe MR imaging quantifies airspace size is shown in Fig 1A and what derived measures represent in Fig 1B. Fig 1C shows maps of several different measurements from a single control subject’s first imaging session.

Fig 1

HHe MRI overview.

(A) Schematic of a single HHe atom that is measured at to sequential time points. In a normal alveolar duct (top), although the atom diffuses the same total distance (dashed line) measurement is limited to the measured distance (Δx) and is relatively small. In dilated ducts, diffusion is less restricted and Δxis larger. (B) Schematic representation of the outer alveolar duct radius (R), the inner duct radius (r), and the septal height (h). (C) Representative axial images of the raw HHe data (top), directly measured apparent diffusion coefficients (ADC), calculated mean linear intercept (L) values, derived alveolar density (N) values. Manual segmentation of right and left lung values was performed to obtain unilateral values for right-left comparisons.

No difference in distal lung structure in AIS versus control

With the exception of expected anatomic differences between right and left lungs, proton and HHe image analysis at the first time point revealed no significant differences between control and AIS subjects (Table 2) or between left/concave or right/convex lungs (all AIS subjects had levoscoliosis Fig 2, Table 3) with regards to normalized lung volumes, mean linear intercept, alveolar density, septal height, inner or outer duct diameters, or surface to volume ratios.

Table 2

Baseline differences between AIS and control.

		Control (n = 16)	AIS (n = 13)	p-value
Lung Volume (cm3)*	Right	735	833	0.53
	Left	609	702	0.86
	Bilateral	1350	1502	0.49
Normalized Lung Volume (to Predicted FRC+1L)#		0.66	0.62	0.57
Lm (μm)*	Right	173.7	172.1	0.9
	Left	175.6	172.4	0.98
	Bilateral	171.6	172.9	0.82
Alveolar Number (104)#	Right	12.34	12.07	0.74
	Left	9.66	10.09	0.9
	Bilateral	22.11	21.52	0.9
Alveolar Density (cm−3)*	Right	156	150	0.46
	Left	153	153	0.78
Septal Height (μm)*	Right	142.5	157.1	0.35
	Left	146.1	154.4	0.56
Inner Duct Diameter (μm)*	Right	138.8	134.4	0.9
	Left	132.9	135.2	0.9
Outer Duct Diameter (μm)*	Right	281.8	283.9	0.86
	Left	289.1	282.7	0.86
Surface to Volume Ratio (cm−1)*	Right	232.4	233.9	0.86
	Left	238.3	233.3	0.86

*Directly measured values #Calculated values.

Fig 2

Impact of AIS on distal lung architecture.

(A) All AIS subjects had levoscoliosis. The mean linear intercept (L) values were no different in left/concave vs right/convex lungs. (B) Alveolar surface to volume ratio decreased with age as expected since alveolar size increases and neoalveolarization slows during adolescence. There was no discernible difference between control and AIS groups or between right and left lungs with regard to this derived value.

Table 3

Right vs left lung comparisons in AIS vs. control.

	Control (n = 16)			AIS (n = 13)
	Right	Left	p-value	Right	Left	p-value
Lung Volume (cm3)*	735	609	0.08	833	702	0.05
Lm (μm)*	173.7	175.6	0.83	173.1	172.4	1
Alveolar Number (104)#	12.34	9.66	0.07	12.07	10.09	0.24
Alveolar Density (cm-3)*	156	153	0.78	150	153	0.69
Septal Height (μm)*	142.5	146.1	0.96	157.1	154.4	0.45
Inner Duct Diameter (μm)*	138	132.9	0.96	134.4	135.2	1
Outer Duct Diameter (μm)*	281.8	289.1	0.78	283.9	282.7	0.76
Surface to Volume Ratio (cm-1)*	232.4	238.3	0.93	233.9	233.3	1

*Directly measured values #Calculated values.

No meaningful changes in distal lung structure after PSF

Since alveolar size and number both increase during childhood [9], we evaluated how lung volumes, alveolar number, and microstructural measurements changed 1 year after PSF in AIS subjects and at one year after initial imaging in control subjects. In both control and AIS, there was an expected increase in lung volume and increase in alveolar size (Fig 3, Table 4). Post-PSF, reduced volume of the left/convex lung and increased alveolar density in the right/concave lung neared statistical significance when considered individually. In comparing the change in right vs. left lung measures, none were statistically significant though the relative lack of post-PSF convex lung growth is notable (Table 5).

Fig 3

Change in alveolar size at one year.

(A) In the left/concave and (B) right/convex lungs of control and AIS subjects, there was the expected increase in alveolar size over time without significant differences between them. Lines connect subjects and the shaded regions indicate standard error for both the first and 1-year HHe images as determined using generalized linear modeling.

Table 4

One year post-PSF changes.

		Control (n = 10)	AIS/PSF (n = 10)	p-value
Lung Volume (cm3)*	Right	45	46	0.24
	Left	52	8	0.05
	Bilateral	64	87	0.12
Normalized Lung Volume (to Predicted FRC+1L)#		0.07	0.11	0.88
Lm (μm)*	Right	9.6	6.7	0.34
	Left	8.2	12.7	0.79
	Bilateral	8.5	9.5	0.38
Alveolar Number (104)#	Right	0.05	-0.04	0.96
	Left	0.08	-0.21	0.36
	Bilateral	0.14	-0.45	0.83
Alveolar Density (cm−3)*	Right	-22	2	0.05
	Left	-15	-5	0.26
Septal Height (μm)*	Right	-5.3	-3.9	0.57
	Left	-8.8	-3.7	0.17
Inner Duct Diameter (μm)*	Right	9.8	11	0.38
	Left	9.2	16.7	0.46
Outer Duct Diameter (μm)*	Right	11.8	0.1	0.38
	Left	8.9	4.3	0.79
Surface to Volume Ratio (cm−1)*	Right	-14.1	-9.6	0.13
	Left	-9.9	-17.4	0.27

*Directly measured values #Calculated values.

Table 5

Change in right vs. left lung measurements in AIS vs. control.

	Control (n = 10)			AIS (n = 10)
	Right	Left	p-value	Right	Left	p-value
Lung Volume (cm3)*	45	52	0.66	46	8	0.7
Lm (μm)*	9.6	8.2	0.91	6.7	12.7	0.91
Alveolar Number (104)#	0.05	0.08	0.76	-0.04	-0.21	0.7
Alveolar Density (cm-3)*	-22	-15	0.08	2	-5	0.62
Septal Height (μm)*	-5.3	-8.8	0.09	-3.9	-3.7	0.79
Inner Duct Diameter (μm)*	9.8	9.2	1	11	16.7	0.62
Outer Duct Diameter (μm)*	11.8	8.9	0.08	0.1	4.3	0.57
Surface to Volume Ratio (cm−1)*	-14.1	-9.9	0.62	-9.6	-17.4	0.97

*Directly measured values #Calculated values.

Discussion

In this small study of alveolar size, distal lung microstructure, and alveolar number in AIS, we found neither significant differences nor meaningful trends (a) between control and AIS subjects at baseline, (b) between concave and convex lungs of AIS subjects at baseline, or (c) in change of alveolar microstructure at 1 year following PSF in AIS subjects. We found expected changes in lung volume and alveolar density in the formerly convex lung of AIS subjects following PSF. Our failure to find any microstructural changes in concave vs. convex lungs in AIS suggest that the previously reported changes in intrathoracic dynamics in AIS [5] do not impact alveolar development. Although the number of subjects is small, our findings do not support the hypothesis lung microstructure is changed in AIS.

Several study limitations should be noted. (a) Our use of a fixed inspired volume of HHe could have biased our findings to larger alveolar size in younger subjects. Using height and age normalized HHe volumes may have modestly reduced alveolar sizes in younger subjects, but this change would be predicted to reduce the already non-significant difference between AIS and control MLI. (b) The number of subjects enrolled was low and matching was poor. We had difficulty recruiting healthy females in the 15-18-year-old age range, and the age discrepancy between control and AIS groups make direct comparison difficult—particularly since increases in lung function measures are known to lag somatic growth in adolescence [34]. Subject matching and comparison of right and left lungs in the same subject somewhat alleviate the concern with poor age matching. (c) Follow-up time may have been insufficient. The one-year follow-up time was chosen based on normal post-operative clinic scheduling after PSF (the latest routine follow-up in the orthopedics clinic). It is possible that one year was insufficient follow-up to discover structural changes following PSF and to see whether the alveolar density in post-PSF right/convex lung normalizes. (d) We did not evaluate the impact of kyphosis. (e) We did not perform pulmonary function testing because we felt that we were unlikely to see significant differences in this small cohort, and we did not wish to impose additional time burdens on families already committing two additional hours for our study after spending several hours in clinic. (e) We used non-parametric tests for some parametric data. Within most measures, some groups of data were parametric and others non-parametric. While applying a parametric test to some marginally significant results would have permitted crossing the significance threshold, we chose the above route for consistency and rigor.

Conclusions

In summary, we found no evidence that AIS appreciably impacts alveolar size, alveolar duct morphometry, or alveolar number. The only changes that neared significance post-PSF were ones expected from spinal alignment.

12 Sep 2020

PONE-D-20-21934

Lung Microstructure in Adolescent Idiopathic Scoliosis Before and After Posterior Spinal Fusion

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Reviewer #1: Lung growth and adaptive change is an interesting topic and worthy of general readership.

Major comments

The premise of these experiments is a bit conflicted. The authors propose to investigate “whether any improvements (after surgery) are due solely to improved pulmonary mechanics or whether (surgery) impacts lung structure.” Further, the authors make a convincing argument for potential adaptive changes in alveolar structure. The experimental problem is that mechanical forces in the chest and alveolar microstructure are confounded in human studies. Since the alveoli are agnostic with respect to the spine--they only respond to intrathoracic forces--it is not at all surprising that the alveoli have adapted normally to their post-surgical mechanical environment. A positive result would have been surprising indeed! The authors should probably acknowledge this limitation in greater depth.

Minor comments

Table 1 is showing demographics. Why are p-values reported for some quantities in this table and not others? Presumably, they are trying to show to what extent the distribution of a particular parameter (i.e. age, sex, etc) is different between the two groups.

Not sure what they mean on page 9, line 118 where they differentiate between left/concave and right/convex lungs. My understanding is that both lungs have a convex (near the diaphragm) and concave (costal) surfaces and that these curvatures are not specific to a particular lung. I think they need to define what they mean by left/concave and right/convex.

Table 3: There seems to be an extra line in the table with “8” entered in one of the columns.

Figure 1. The last sentence in the figure caption has the text: “…and manual masking of right and left lungs (bottom).” I have no idea what to what the authors are referring here.

Figure 3. The label on this figure is misleading. It says “Change in Alveolar Size after PSF”. This can only be true for the AIS subjects undergoing a posterior spinal fusion (PSF) procedure. But they also show control values for the two scans separated by one year and certainly the controls did NOT undergo a PSF. Also, the figure inset shows the “control” data to be a black circle and the AIS data, a green circle. The figure only has pink and green symbols. Also, the shaded regions need to be defined as the uncertainty in the fit vs. age of the mean linear intercept. And they need to state what type of analytical expression was used to fit the data…was it a linear fit?

Reviewer #2: The authors set out to examine alveolar-airspace size in children with AIS, comparing them with healthy controls and to determine whether a specific surgical procedure (PSF) alters airspace characteristics.

The concept and methodology of the study is good. Authors make a good job of explaining complex concepts, and the various comparisons (control vs AIS baseline, left vs right, AIS pre and post PSF).

However, the various methodological flaws which have been acknowledged by the authors in the discussion prevent any meaningful conclusion.

1. The first major problem is the numbers of subject studied. It is acknowledged to be a ‘pilot’ study at various places. However, the authors attempt to draw conclusions in discussion section : e.g. ‘ While the study is not powered to prove the absence of an effect, the lack of any discernible trends in control vs AIS, concave vs. convex lung, or pre- and post-PSF imaging in AIS subjects argues against the presence of any such effect.’ (page 13, line 140-142). In my opinion (and from a statistical standpoint) there should not be any conclusion drawn from presence or absence of trends, where the differences do not reach a predefined (usually p<0.05) level of statistical significance.

2. The second problem is the difference in age and size of the control subjects (smaller and younger than AIS subjects.

a. This needs to be mentioned in the abstract section.

b. There has been an attempt to normalise using lung volumes derived from UTE proton MRI at TE = 2.43ms (page 7, line 79-81). If this technique has been described before (and compared to standard techniques), this should be referenced here.

c. Following this, normalisation has been attempted using NHANES III FVC values. I believe it is more appropriate to normalise by Functional residual capacity - FRC (or possibly FRC plus one liter, as this is the value at which measurements are taken)

d. Were any of the 3HeMRI derived parameters (Lm, alveolar number, etc.) normalised by volume? The values do not seem to be defined (don’t see any subscript for tables either).

3. The third problem is the relatively short interval between the primary and follow-up measurements. Authors do mention this in the discussion

a. Also, Adolescence is the period of growth spurt and rapid changes in height (and thoracic volume). It is well known that lung function (% predicted by height) lags behind physical growth. It is possible that alveolar dimensions also lag behind. This is especially important to consider because the control group are probably at a different stage of growth spurt compared to AIS group.

4. There is one conclusion that can be drawn from the fact that alveolar dimensions (alveolar density, septal height, ID diameter, OD diameter, S/V ratio) are similar between left and right lungs in the AIS group – that alveolar development should have continued after development of scoliosis (otherwise, the right side should have higher alveolar density).

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18 Sep 2020

September 18, 2020

To the Editor and Reviewers:

Thank you very much for your helpful critiques and suggestions on our manuscript “Lung Microstructure in Adolescent Idiopathic Scoliosis Before and After Posterior Spinal Fusion” (PONE-D-20-21934)

Below is a point-by-point response to critiques with reviewer text.

We hope that you are satisfied with these changes and look forward to a positive response.

Sincerely, Brian Varisco.

Associate Editor:

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We have updated the format of the manuscript.

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The informed consent and minors’ assent is now stated.

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Reviewer #1:

Lung growth and adaptive change is an interesting topic and worthy of general readership.

Major comments

The premise of these experiments is a bit conflicted. The authors propose to investigate “whether any improvements (after surgery) are due solely to improved pulmonary mechanics or whether (surgery) impacts lung structure.” Further, the authors make a convincing argument for potential adaptive changes in alveolar structure. The experimental problem is that mechanical forces in the chest and alveolar microstructure are confounded in human studies. Since the alveoli are agnostic with respect to the spine--they only respond to intrathoracic forces--it is not at all surprising that the alveoli have adapted normally to their post-surgical mechanical environment. A positive result would have been surprising indeed! The authors should probably acknowledge this limitation in greater depth.

It seems that we did not clearly articulate the linkage between spine curvature and intrathoracic forces that is the underlying premise of the study. The linkage between the spine curvature and pulmonary mechanics is based on a proton MRI study that found impaired diaphragm function in AIS and improvement post-PSF (Chu study). We made this clearer in the abstract and re-arranged and re-written the middle part of the introduction to make this premise more clear.

Minor comments

Table 1 is showing demographics. Why are p-values reported for some quantities in this table and not others? Presumably, they are trying to show to what extent the distribution of a particular parameter (i.e. age, sex, etc) is different between the two groups.

With only one non-white subject per group, we did not report the fact that differences in race between the groups were not statistically significant, but now we have done so.

Not sure what they mean on page 9, line 118 where they differentiate between left/concave and right/convex lungs. My understanding is that both lungs have a convex (near the diaphragm) and concave (costal) surfaces and that these curvatures are not specific to a particular lung. I think they need to define what they mean by left/concave and right/convex.

Thank you. Convex and concave lungs are orthopedic terminology with regards to spinal curvature. If the curvature. In the AP or PA plane, the lung that is being compressed is the convex lung and the lung that is being expanded is the concave lung. This has been clarified in the abstract and in the introduction.

Table 3: There seems to be an extra line in the table with “8” entered in one of the columns.

This was a formatting problem. All of the tables have been reformatted to fit journal specifications.

Figure 1. The last sentence in the figure caption has the text: “…and manual masking of right and left lungs (bottom).” I have no idea what to what the authors are referring here.

We have re-written this sentence to read that manual segmentation of right and left lung values was performed to obtain the values used in right-left comparisons.

Figure 3. The label on this figure is misleading. (a) It says “Change in Alveolar Size after PSF”. This can only be true for the AIS subjects undergoing a posterior spinal fusion (PSF) procedure. But they also show control values for the two scans separated by one year and certainly the controls did NOT undergo a PSF. (b) Also, the figure inset shows the “control” data to be a black circle and the AIS data, a green circle. The figure only has pink and green symbols. (c) Also, the shaded regions need to be defined as the uncertainty in the fit vs. age of the mean linear intercept. And they need to state what type of analytical expression was used to fit the data…was it a linear fit?

(a) The figure header was changed to read “Change in Alveolar Size at One Year.”

(b) With regard to the figure inset control data being a black circle and AIS data being a green (or aqua) circle), we think the reviewer is referring to the figure legend that is inset within panel A and that perhaps the black circle was a rendering issue. The downloaded Figure 3 TIF from the PLoS One submission portal has a dark pink square for control and an aqua square for AIS. When we double checked the submission PDF, the colors and shapes looked appropriate.

(c) We agree with the reviewer that an explicit statement that stating the use of generalized linear modeling is appropriate.

Reviewer #2: The authors set out to examine alveolar-airspace size in children with AIS, comparing them with healthy controls and to determine whether a specific surgical procedure (PSF) alters airspace characteristics.

The concept and methodology of the study is good. Authors make a good job of explaining complex concepts, and the various comparisons (control vs AIS baseline, left vs right, AIS pre and post PSF).

However, the various methodological flaws which have been acknowledged by the authors in the discussion prevent any meaningful conclusion.

1. The first major problem is the numbers of subject studied. It is acknowledged to be a ‘pilot’ study at various places. However, the authors attempt to draw conclusions in discussion section : e.g. ‘ While the study is not powered to prove the absence of an effect, the lack of any discernible trends in control vs AIS, concave vs. convex lung, or pre- and post-PSF imaging in AIS subjects argues against the presence of any such effect.’ (page 13, line 140-142). In my opinion (and from a statistical standpoint) there should not be any conclusion drawn from presence or absence of trends, where the differences do not reach a predefined (usually p<0.05) level of statistical significance.

As the reviewer likely appreciates, the study was accomplished stringing together several small grants and that without even the hint of a meaningful signal, our plans to leverage this preliminary data into a more definitive study were abandoned, but the data is still worth disseminating. However, the study wasn’t powered or designed to show absence of difference as the reviewer notes. We have modified the verbiage from “pilot” to “small” and modified our discussion to state that although limited by small numbers, our findings do not support the hypothesis AIS causes lung microstructural changes.

2. The second problem is the difference in age and size of the control subjects (smaller and younger than AIS subjects.

a. This needs to be mentioned in the abstract section.

We highlighted this shortcoming in the abstract

b. There has been an attempt to normalise using lung volumes derived from UTE proton MRI at TE = 2.43ms (page 7, line 79-81). If this technique has been described before (and compared to standard techniques), this should be referenced here.

This is the same technique that was reported in the Roach paper referenced in the sentence that the reviewer refers to. To make it more clear that this is the same validated technique that was previously reported, we moved the reference to the end and included verbiage to that effect.

c. Following this, normalisation has been attempted using NHANES III FVC values. I believe it is more appropriate to normalise by Functional residual capacity - FRC (or possibly FRC plus one liter, as this is the value at which measurements are taken)

This is a very good point and FRC plus one liter would be the ideal normalization value. That data is not available in NHANES, but a 1980 study using helium dilution provides FRC values for children (Buist, AJRCCM) and a 1990 study (Thorsteinsson, Anesthesiology) provide such values. We used the Thorsteinsson study as it contained a greater number of subjects and had tighter confidence intervals.

d. Were any of the 3HeMRI derived parameters (Lm, alveolar number, etc.) normalised by volume? The values do not seem to be defined (don’t see any subscript for tables either).

No, the raw measurements of these values were compared (for example cm^3 for lung volume, microns for Lm, cm^-1 for surface to volume ratio, all in parentheses). The reviewer’s point seems to be that it should be easier to look at the table and determine which parameters were directly measured vs. derived. We have added * and # with subscripts to make this more clear.

3. The third problem is the relatively short interval between the primary and follow-up measurements. Authors do mention this in the discussion

We agree that a 3-5 year follow-up would have been better, but since AIS patients are discharged from orthopedics clinic after 1 year follow-up, this was impracticable.

a. Also, Adolescence is the period of growth spurt and rapid changes in height (and thoracic volume). It is well known that lung function (% predicted by height) lags behind physical growth. It is possible that alveolar dimensions also lag behind. This is especially important to consider because the control group are probably at a different stage of growth spurt compared to AIS group.

This is s a good point and statement to this effect has been added to discussion of weaknesses with a reference to Mahmoud et al 2018.

4. There is one conclusion that can be drawn from the fact that alveolar dimensions (alveolar density, septal height, ID diameter, OD diameter, S/V ratio) are similar between left and right lungs in the AIS group – that alveolar development should have continued after development of scoliosis (otherwise, the right side should have higher alveolar density).

This is true and has been more explicitly stated in discussion.

Submitted filename: PLoS One Reviewer Response.docx

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23 Sep 2020

Lung Microstructure in Adolescent Idiopathic Scoliosis Before and After Posterior Spinal Fusion

PONE-D-20-21934R1

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29 Sep 2020

PONE-D-20-21934R1

Lung microstructure in adolescent idiopathic scoliosis before and after posterior spinal fusion

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