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The authors have declared that no competing interests exist.

Recent evidence suggests that the quantity and quality of conscious experience may be a function of the complexity of activity in the brain and that consciousness emerges in a critical zone between low and high-entropy states. We propose fractal shapes as a measure of proximity to this critical point, as fractal dimension encodes information about complexity beyond simple entropy or randomness, and fractal structures are known to emerge in systems nearing a critical point. To validate this, we tested several measures of fractal dimension on the brain activity from healthy volunteers and patients with disorders of consciousness of varying severity. We used a Compact Box Burning algorithm to compute the fractal dimension of cortical functional connectivity networks as well as computing the fractal dimension of the associated adjacency matrices using a 2D box-counting algorithm. To test whether brain activity is fractal in time as well as space, we used the Higuchi temporal fractal dimension on BOLD time-series. We found significant decreases in the fractal dimension between healthy volunteers (n = 15), patients in a minimally conscious state (n = 10), and patients in a vegetative state (n = 8), regardless of the mechanism of injury. We also found significant decreases in adjacency matrix fractal dimension and Higuchi temporal fractal dimension, which correlated with decreasing level of consciousness. These results suggest that cortical functional connectivity networks display fractal character and that this is associated with level of consciousness in a clinically relevant population, with higher fractal dimensions (i.e. more complex) networks being associated with higher levels of consciousness. This supports the hypothesis that level of consciousness and system complexity are positively associated, and is consistent with previous EEG, MEG, and fMRI studies.

Research into the neural origins of conscious experience has suggested that consciousness may be associated with the “complexity” of information processing in the brain [_{C}) [_{C} analyses a maximally random (or incompressible) signal would be indicated as having the highest “complexity.” This is, however, a somewhat counter-intuitive understanding of what we mean when we talk about the “complexity” of brain activity: the brain is complex not because it is highly random, but because it combines an incredible degree of order with a high degree of unpredictability. It is hard to imagine how a brain outputting algorithmically random noise could doing anything at all, let alone supporting consciousness and cognition. Not only is the brain both structured and unpredictable, it also shows one of the hallmarks of complex systems writ large: emergent dynamics over multiple scales. With this in mind, we strongly feel that consciousness science requires further discussion and refinement of what “complexity” means in the context of the brain. As a working definition, we propose that complexity should be understood as a fundamentally multi-scale phenomena, emerging in systems that display both a high degree of emergent structure and organization as well incompressible and unpredictable features.

One significant attempt to prose a more nuanced relationship between consciousness and complexity is the Entropic Brain Hypothesis (EBH). The EBH posits that consciousness emerges when the brain is near a critical zone between order and randomness, known as the critical regime, and that to move too far in either direction will result in a change in the quality of consciousness, and ultimately, loss of consciousness entirely [

A core feature of the EBH is that consciousness emerges, not where algorithmic complexity is maximal, but in the critical zone, on the boarder between low- and high-entropy states (“the edge of chaos”). Several publications suggest that the healthy brain operates at, or just below, this area of criticality [

One of the “fingerprints” of critical phenomena is the emergence of scale-free, or fractal, structures near the critical point [

Analyses of fractal dimension of brain activity has largely been done in the temporal domain (excluding the work by Ruiz de Miras et al.), primarily using analysis of one-dimensional EEG signals. There has been far less analysis of the fractal character functional connectivity networks, which encode higher-order relationships between elements in the system [

Based on these results, we made two hypotheses: (1) functional connectivity networks in the neocortex would have a measurable fractal character when all of the weaker edges had been thresholded out and (2) that in patients with reduced level of consciousness, fractal dimension would be reduced. To investigate this, we used resting-state fMRI data from healthy volunteers and patients suffering from reduced levels of consciousness associated with brain injury. We divided these patients into two subgroups based on clinical diagnosis: minimally-conscious state (MCS) and vegetative state (VS), based on accepted diagnostic criteria [

Since the fractal dimension of most real-world systems cannot be solved analytically, researchers commonly use a family of algorithms known as box-counting measures to determine the fractal dimension of a natural system. The box-counting dimension describes how the topology of a surface changes (or remains the same) at different scales. For any shape, two values are defined: _{B} which is the length of an _{B}), which is the minimum number of boxes necessary to ‘tile’ the surface in question. If the shape being tiled is a fractal, then:
_{B} is the box-counting dimension. Algebraic manipulation shows that _{B} can be extracted by linear regression as:

A similar logic is used when calculating the box-counting dimension of a graph. For a graph _{B} defines a set of nodes _{i} and _{j} the distance between them _{ij} < _{B}. To quantify the fractal dimension of the functional connectivity networks, a box counting method, the Compact Box Burning (CBB) algorithm [_{B}(_{B}) for a range of integer _{B} values 1..10. If _{B}(_{B})) vs. _{B}) should be roughly linear, with a slope of −_{B}. We chose the CBB algorithm because it is an easily-implemented algorithm that can handle smaller networks than alternatives such as the maximum-excluded mass-burning algorithm [

Due to the logarithmic relationship between box-size and fractal dimension, exponentially higher resolutions (in this case, numbers of nodes) are required to achieve modest increases in the accuracy of the measured fractal dimension. Computational explorations, where a box-counting method is used to approximate a fractal dimension that has already been solved analytically, show that the box-counting dimension converges to the true dimension with excruciating slowness [

We should note that we are not doing a truly rigorous power-law inference. The question of when an empirical distribution can be considered to follow a power-law is a rich field of research [

Our second test of fractal structure used a two-dimensional box-counting method to analyse the associated adjacency matrix representations of the functional brain networks. This analysis served two purposes: primarily, it was meant to replicate the results of the CBB analysis, however we also hoped that, if it did replicate the initial results, it could be a more computationally efficient method for estimating the fractal dimension of a brain network. By using a different representation of the network, we hoped to show that the quality of network fractal dimension is conserved across isomorphic representations. This would increase our confidence in the CBB results by showing that our findings are unlikely to be an artefact of that particular algorithm. For a given graph _{i} and _{j}, the corresponding adjacency matrix,

In the resulting matrix, every 1 represents an edge between two nodes _{i} and _{j}. If the distribution of edges _{B} at every value of _{B}. For the purposes of this analysis, we took the mean of those values and defined that average as the fractal dimension of each image. The adjacency matrices were exported as binary .jpg images for analysis, and the default values for FracLac’s batch image analysis were used.

We hypothesized that this method, while more accessible and less abstract than the Compact-Box-Burning algorithm, would be less sensitive to small changes in fractal dimension between conditions, as some information is lost when doing a two-dimensional box counting algorithm on a flat network representation, rather than operating directly on a graph.

We used the Higuchi temporal fractal dimension algorithm, widely used in EEG and MEG analysis, to calculate the fractal dimension of temporal brain activity [

For each time-series _{1}, _{2}, …_{max}, the length of that series, _{m}(

We then define the average length of the series 〈_{m}(

If our initial time-series ^{−D}. As with the procedure for calculating the network fractal dimension, the algorithm iterates through values of _{max} and calculates ^{−1}), extracting _{B} used to calculate the network fractal dimension. The Higuchi algorithm requires a pre-defined _{max} value as an input, along with the target time-series. This value is usually determined by sampling the results returned by different values of _{max} and selecting a value based on the range of _{max} where the fractal dimension is stable. For both DOC datasets, we chose _{max} = 64 as this was the largest value that our algorithm could handle.

The implementation we used was from the PyEEG toolbox [

Ethical approval for these studies was obtained from the Cambridgeshire 2 Regional Ethics Committee, and all subjects gave informed, written consent to participate in the study. Twenty five healthy volunteer subjects were recruited for scanning. The acquisition procedures are described in detail in [

Of the 25 healthy volunteer datasets, 10 were excluded, either because of missing scans (n = 2), or due of excessive motion in the scanner (n = 8, 5mm maximum motion threshold). For this study, we only used the awake, control condition described in the original Stamatakis study, ignoring the drug conditions.

The resulting images were preprocessed using the CONN functional connectivity toolbox [

Data was acquired at Addenbrooke’s Hospital in Cambridge, UK, on a 3T Tim Trio Siemens system (Erlangen Germany). Ethical approval for testing patients was provided by the National Research Ethics Service (National Health Service, UK; LREC reference 99/391). A sample DOC patients with verifiable diagnosis were recruited from specialised long-term care centers. Written consent was obtained from the patient’s legal representatives. Medication prescribed to each patient was maintained during scanning. T1-weighted images were acquired with an MP-RAGE sequence (TR = 2300ms, TE = 2.47ms, 150 slices, 1 × 1 × 1mm^{2} resolution). Functional images, 32 slices each, were acquired using an echo planar sequence (TR = 2000 ms, TE = 30 ms, flip angle = 78 deg, 3 x 3 x 3.75mm^{2} resolution). Subjects were split into two groups: those who met the criteria for being in a minimally conscious state (MCS, n = 10), and those who were in a vegetative state (VS, n = 8).

Preprocessing was performed with SPM12, MATLAB, and CONN, as described above. The first five volumes were removed to eliminate saturation effects and achieve steady state magnetization. Slice-timing and movement correction (motion estimation and correction) were performed as above, including outlier detection, structural segmentation and normalization, 6 mm FWHM Gaussian smoothing, and denoising with a band-pass filter with range [0.008, 0.09] Hz. To reduce movement-related and physiological artefacts specific to DOC patients, data underwent further de-spiking with a hyperbolic tangent squashing function. Next the CompCor technique was used to remove the first 5 principal components of the signal from the white matter and cerebrospinal fluid masks, as well as 6 motion parameters and their first order temporal derivatives and a linear de-trending term [

After preprocessing, BOLD time-series data were extracted from each brain in CONN and the cerebral cortex was segmented into 1000 distinct ROIs, using the Schaefer Local/Global 1000 Parcellation [

For some DOC patients, there were ROI nodes which mapped to regions that had been so damaged that no detectable signal was recovered: these time-series were removed from analysis. For the MCS patients, the average number of removed nodes was 1.2 ± 1.53 nodes (≈ 0.12% of all nodes), while for the VS patients it was 5.38 ± 7.12 nodes (≈ 0.54% of all nodes). We expect that the removal of such a comparatively small number of nodes to have a negligible effect on our overall-analysis. For each brain region, it’s associated time-series _{i}(_{j}(

The correlation matrix has a series of ones that run down the diagonal, corresponding the correlation between each timeseries and itself which, if treated directly as a graph adjacency matrix, would produce a graph where each node had exactly one self-loop in addition to all it’s other connections. To correct for this, the matrices were filtered to remove self-loops by turning the diagonal of ones to zeros, ensuring simple graphs.

Finally, following the findings by Gallos et al. (2012), that fractal character was only present at high thresholds the matrices were binarized with a 95% threshold, such that:

All surviving values _{ij} < 0 ↦ 0. The result is a sparse, symmetric, binary matrix, _{i} and column _{j} corresponds to an ROI in the initial cortical parcellation, and the connectivity between all nodes is given by

Three 1000 × 1000 adjacency matrices, representing the three conditions.

A visualization of a healthy, control functional connectivity network. Node size and darkness indicate a higher degree. Shown here are coronal, axial, and sagittal projections of the network onto a two-dimensional plane. Image made using Gephi [

To contextualize our results in the broader space of possible graphs, we generated synthetic null networks from a variety of classes to compare our three groups of functional connectivity graphs to. Here, “null graph” refers to a network with the same number of nodes but a structure unrelated to brain connectivity. By creating these null networks, we can explore the general behaviour of the network fractal dimension algorithm and use this knowledge to inform our empirical findings. We generated three types of graph:

Lattices: a highly-ordered type of graph, where every node makes connections to it’s

Random Graphs: A highly disordered type of graph, where every instance of the graph is selected at random from the space of all possible graphs. A population of 50 random graphs was generated and the average fractal dimension calculated. Each graph had 1000 nodes, and an identical number of edges to the natural functional connectivity networks, thresholded at 95%.

All null graphs were generated using the already-implemented graph generators in NetworkX [

All statistical analysis was carried out using Python 3.6 using the Anaconda Python environment (

The Kruskal-Wallis test found significant differences between the fractal dimension of functional connectivity networks for all three conditions (H(19.91), p-value ≤ 0.0001). The median value _{B} for the healthy control condition was 3.513 (IQR: 3.472-3.555), for MCS patients it was 3.309 (IQR: 3.21-3.438), and for VS patients it was 3.102 (IQR: 2.922-3.281). Post-hoc analysis with the Mann-Whitney U test found significant differences between each condition: control vs. MCS (U(13), p-value = 0.0003), control vs. VS (U(3), p-value = 0.0001), and MCS vs. VS (U(20), p-value = 0.042). For a visualization of these results see

_{B} for the healthy control condition: 3.49 ± 0.1 (n = 15), for MCS patients: 3.29 ± 0.16 (n = 10), and for VS patients: 3.07 ± 0.29 (n = 8). Box length must always take integer values and does not have a regular metric unit. Post-hoc analysis with the Mann-Whitney U test found significant differences between each condition: control vs. MCS (H(41), p-value = 0.032), control vs. VS (H(23), p-value = 0.009), and MCS vs. VS (H(20), p-value = 0.042). _{B} and _{B}) in all three conditions.

Metric | Healthy Control | MCS | VS |
---|---|---|---|

Network Fractal Dimension |
3.513 (IQR: 3.472-3.555) | 3.309 (IQR: 3.21-3.438) | 3.102 (IQR: 2.922-3.281) |

Adj. Matrix Fractal Dimension |
1.731 (IQR: 1.716-1.742) | 1.706 (IQR: 1.697-1.717) | 1.693 (IQR: 1.67-1.7) |

Temporal Fractal Dimension |
1.21 (IQR: 1.2–1.23) | 0.946 (IQR: 0.927- 0.963) | 0.912 (IQR: 0.893-0.952) |

Table of results describing the how different conditions behaved under each measures of fractal dimension. Data reported are median (IQR: 25%-75%). Significance calculated using the Kruskal-Wallis analysis of variance.

** < 0.01

*** < 0.001

These results are consistent with our hypothesis that level of consciousness is positively associated with network complexity, as measured by the fractal dimension. This also shows that the direct network fractal dimension measure is sensitive enough to discriminate between different clinically useful diagnoses of grey states of consciousness, rather than simply it’s binary presence or absence.

The Kruskal-Wallis test found significant differences between the fractal dimensions of the adjacency matrices for the three conditions (H(10.24), p-value = 0.006). The median value for the healthy controls was 1.731 (IQR: 1.716-1.742), the median value for MCS patients was 1.706 (IQR: 1.697-1.717), and for VS patients it was 1.693 (IQR: 1.67-1.7). Post-hoc analysis with the Mann-Whitney U test found significant differences between the control and MCS conditions (U(33), p-value = 0.01), and the control and VS conditions (U(18), p-value = 0.0036), but not the VS and MCS conditions (U(25), p-value = 0.099). To ensure that our measures of network fractal dimension and adjacency matrix fractal dimension were associated, we correlated these values against each other and found a significant positive correlation (r = 0.58, p-value = 0.0005). All the significant p-values survived Benjamini-Hochberg FDR correction. For a visualization of these results, see

As with the direct measure of network fractal dimension, these results show that complexity is associated with level of consciousness. While this method is sensitive enough to differentiate between healthy controls and patients with disorders of consciousness, unlike the direct measure, it was not able to discriminate between disorders of consciousness of varying severity.

Kruskal-Wallis test found significant differences between all three conditions (H(25.1), p-value = 3.5 × 10^{−6}). The median value for the Awake patients was 1.21 (IQR: 1.2—1.23). The median value for the MCS patients was 0.946 (IQR: 0.927- 0.963), and for VS patients it was 0.912 (IQR: 0.893-0.952). Testing with the Mann-Whitney U test found a significant difference between the Awake and MCS conditions (U(0), p-value = 1.8 × 10^{−5}), the Awake and VS conditions (U(0), p-value = 6.13 × 10^{−5}) and the MCS and VS conditions (U(17), p-value = 0.023). For visualization of these results, see

Visualization of the difference in Higuchi temporal fractal dimension between the Awake, MCS and VS conditions. The Awake condition had the highest FD with a median value of 1.21 (IQR: 1.2–1.23). As expected, the MCS condition had the next highest dimension, with a median value 0.946 (IQR: 0.927-0.963) followed by the VS condition, with a median of 0.912 (IQR: 0.894-0.952). The Wilcoxon signed-rank test found a significant difference between the MCS and VS conditions (U(17), p-value = 0.023) and between both conditions and the Awake condition (U(0), p-value = 1.8 × 10^{−5}, MCS) and (U(0), p-value = 6.13 × 10^{−5}).

While preliminary, these results are nicely consistent with our initial hypothesis, that level of consciousness is positively associated with the fractal dimension of brain activity. Furthermore, these results complement the findings from the network fractal dimension by showing that the fractal dimension of brain activity’s relationship to consciousness is measurable in temporal, as well as spatial, dimensions.

As predicted, all the classes of null graphs had much lower fractal dimensions than any of our brain networks, as calculated by the CBB algorithm. The 2-dimensional lattice graph with 1000 nodes had a fractal dimension of ≈ 0.15. The 3-dimensional lattice with the same number of nodes had a fractal dimension of ≈ 0.184. The set of random networks had a higher fractal dimension, although it was still far lower than any of the real functional connectivity networks, with a median value of 0.279 (IQR: 0.279, 0.2792).

These results show that the fractal dimension measure is distinct from a measure of order/randomness, as both highly ordered networks and highly random networks return similarly low values as compared to the functional connectivity networks.

In this study we found that the complexity of functional connectivity networks, as measured by the fractal dimension, was significantly associated with level of consciousness in healthy volunteers and patients with DOC of varying severity. When calculated using the Compact Box-Burning (CBB) Algorithm [

Emergent scale-freeness is one of the hallmarks of critical dynamics [

The results presented here are consistent with previous work of ours using similar techniques on fMRI data from adult volunteers under the influence of LSD and psilocybin [

One difficulty of much of this work is creating an intuitive understanding of what it means for a system to have a “lower” fractal dimension than another. Considerable previous research has shown that loss of consciousness is associated with lower fractal dimensions [_{B} ∝ −_{B}(_{B}))/_{B}), a higher fractal dimension corresponds to a steeper slope: small changes in _{B} correspond to comparatively more dramatic changes in _{B}). This may indicate a “rougher” topology, with a more heterogenous distribution of high- and low-density regions. Ruiz de Mira et al., (2019) discuss fractal dimension in terms of both integration and differentiation, suggesting that alterations to the fractal dimension may represent differences in the ability of a system to balance these competing properties.

To discuss the aetiology of the changes in network fractal dimension, we turn to previous studies, which have shown that the cerebral cortex has fractal characteristics and that changes to the fractal dimension of both the grey matter and white matter are associated with changes in cognition and the presence of clinically relevant conditions [

There are several limitations for this study that are worth considering and suggest a need for further validation. We acknowledge the comparatively small sample size, particularly in the VS condition. As previously mentioned, the requirements of fMRI image processing demand images of brains from individuals with reduced levels of consciousness, but are not so geometrically distorted as to make registration into MNI space impossible. This puts a limit on the number of brains eligible for inclusion in this kind of study. There is also the issue of parcellation resolution: we tried several different parcellations of various sizes, but only the parcellation with 1000 ROIs had a high enough resolution to return a meaningful result, and even that was still too small to permit more than 10 integer values for _{B}. There is also a question about the Higuchi fractal dimension results: HFD typically ranges between 1-2 and so is abnormally low in both the MCS and VS conditions. This result is reasonably robust to the

These results point to several possible subsequent investigations. First, how do temporal and spatial fractal dimensions relate to other temporal (e.g. Lempel-Ziv complexity, sample entropy, etc) and network domains (scale-freeness, small-worldness, etc)? Previous studies have used power-law, or heavy-tailed degree distributions as an indicator of fractal structure [

Going forward, we hope that this kind of analysis may one day be useful in a clinical context for estimating whether consciousness is present in patients who may be unable to give a voluntary behavioural affirmation of awareness. The fractal dimension measure encodes significant information about the complexity of a system into a single, easily digestible measure that seems to be relevant in a clinically meaningful population. As, at least in larger hospitals, MRI scans are already a routine part of clinical assessments in cases of brain damage, this measure could be incorporated into the normal course of treatment.

In this study, we show that high-resolution, cortical functional connectivity networks have fractal characteristics and that, in patients with disorders of consciousness induced by traumatic brain injury or anoxic brain injury, reduction in the fractal dimension is associated with more severe disorders of consciousness. This is consistent with theories that associate the content, and quality, of consciousness with the complexity of activity in the brain. Furthermore, we believe that, with refinement, this measure may inform diagnosis and stratification in a clinical setting where physicians need to make judgements about a patients consciousness in the absence of behaviourally unambiguous indicators.

These are the results for the network fractal dimension, adjacency matrix fractal dimension, and higuchi fractal dimension for each of the three conditions (Awake, MCS, VS).

(CSV)

These are the results for the network fractal dimension analysis of the lattice networks and the ER random networks.

(CSV)

This work was supported by grants from the Wellcome Trust Clinical Research Training Fellowship to RMA (Contract grant number: 083660/Z/07/Z); the UK Medical Research Council [U.1055.01.002.00001.01 to JDP; the James S. McDonnell Foundation to JDP; the Evelyn Trust, Cambridge to JA, the National Institute for Health Research (NIHR, UK), Cambridge Biomedical Research Centre and NIHR Senior Investigator Awards to JDP and DKM; The Canadian Institute for Advanced Research (CIFAR) to DKM and EAS; the Stephen Erskine Fellowship (Queens’ College, Cambridge) to EAS; the British Oxygen Professorship of the Royal College of Anaesthetists to DKM. MC was supported by the Cambridge International Trust and the Howard Sidney Sussex Research Studentship. TFV is supported by NSF-NRT grant 1735095, Interdisciplinary Training in Complex Networks and Systems. The Evelyn Trust, Cambridge and the EoE CLAHRC fellowship to J.A; this research was also supported by the NIHR Brain Injury Healthcare Technology Co-operative based at Cambridge University Hospitals NHS Foundation Trust and University of Cambridge. We would like to thank Victoria Lupson and the staff in the Wolfson Brain Imaging Centre (WBIC) at Addenbrooke’s Hospital for their assistance in scanning. We would like to thank Dian Lu and Andrea Luppi for useful discussions, and all the participants for their contribution to this study.

PONE-D-19-27002

Fractal Dimension of Cortical Functional Connectivity Networks Predicts Severity in Disorders of Consciousness

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Reviewer #1: It is not mathematically elegant the way in which the matrix M is defined on p. 11. The value of M_{ij} is defined in terms of itself. It makes sense in a programming setting where M is a variable whose value changes. If the authors want to explain successive filters that were applied to M, they can introduce M’, M’’ and then M, or something like that. Finally, “All surviving values M_{ij} < 0 ...”. Is it correct? If I understood the procedure, at that moment all values were 0 or 1.

With respect to the use of “null graphs” or “null networks”, it can be ambiguous at it can refer to graphs with no edges. Please indicate that you refer to a “null model” or graphs not representing brain connectivity.

Some typos:

- There is a missing reference on p. 3 “including sleep (?)”

- Unbalanced parenthesis on Eq. (6)

- In the 7th line of p. 10 there is a quotation mark that can be removed: during scanning.” T1-weighted images

- Also at the bottom of p. 10, the quotation marks in ”Schaefer Local/Global 1000 Parcellation” can be removed.

- In the caption of Fig. 1, I think “volunteers” may be changed to “volunteer”

- p. 15: “Due to the large different in scan-lengths between”. Difference?

Reviewer #2: This paper presents a study on the analysis of FD of cortical functional networks comparing three groups: healthy controls, MCS patients and VS patients. Three different FD analyses were performed: HFD, box-counting and CBB. Significant differences in FD were found between groups, with FD decreasing as the level of consciousness decreases.

The paper is clear and well written and it was a pleasure for me to read it. Conclusions are based on the results obtained. The topic of the paper has high interest.

Nevertheless, I have some comments and suggestions for the authors:

1. More relevant and related studies should be cited and compared in the paper regarding fractal analysis applied to consciousness, and fractal analysis of brain networks. For example, in the second paragraph of page 4, references Ieva 2014 and Ieva 2015 are very generic. HFD studies on consciousness at the end of Introduction (page 5) are relative old. Please, cite, comment and compare current studies on FD and consciousness such as Ruiz de Miras et al. 2019 and other references in that study: “Fractal dimension analysis of states of consciousness and unconsciousness using transcranial magnetic stimulation”. Computer Methods and Programs in Biomedicine 175, pp. 129-137. 2019.

2. Page 6. “To quantify the fractal dimension …, the Compact Box Burning (CBB) algorithm was used…”. Please, explain the reasons why that method for computing the FD of networks was selected, and include an appropriate reference to the paper describing the method. Please, clarify whether the author used any third-party software for computing the CBB or a home-made program.

3. Page 11. Definitions of F(t) and Hi(t) are missing.

4. Page 14. Table 1 would provide more information if it includes which comparisons have a p-value < 0.05

5. Page 13. Statistical analysis. It is a bit strange for me that authors have used Python for statistical analysis instead of using SPSS or MATLAB as usual in the field. Please, explain.

6. Page 15. 3.3. Higuchi temporal fractal dimension. Authors claim that they did not include the HFD analysis on awake subjects because of the different and smaller number of samples regarding DOC subjects. However, this reviewer thinks that this analysis should be included, explaining the possible limiting factors.

7. Page 15. “Surprisingly, we found no significant correlation between temporal fractal dimension and network fractal dimension …”. It is not such a surprise, since related studies, such as Ruiz de Miras et al. 2019, are in the same line. HFD needs be complemented with other measures to adequately characterize the signal.

Other minor comments are:

- Abstract, page 5, 8, 9 and 10. Even though BOLD is a common term in the field, it would be good to provide the formal noun at the first occurrence.

- Page 3: “… who have had their level of consciousness reduced by a range of mechanisms, including sleep (?), sedation …”. Missing reference.

- Page 6: “… the surface in question. the shape …”. Revise punctuation.

- Page 11: “… following the findings by Gallos et al., that …”. Provide complete reference.

- Page 11. “All surviving values Mij …”. Revise punctuation.

- Page 12. “All null graphs … in NetworkX”. Provide reference.

- Page 16. “… the fractal dimension of a these networks …”. Revise the sentence.

- Page 16. “… in the nervous system, which is turn may explain …”. Revise the sentence.

Reviewer #3: In this research report entitled “Fractal dimension of cortical functional connectivity networks predicts severity in disorders of consciousness” the authors explored the FD of functional connectivity networks and time series of BOLD signals from healthy participants and patients with disorders of consciousness (MCS, VS). They found that in all measures the FD for healthy participants was higher than for patients; and it was higher for MCS than VS patients. These results might indicate that the FD of functional structures of the brain is sensitive to abnormal states of consciousness.

General comment

The structure of the paper is convenient and it is clearly developed along the manuscript. The theoretical background is also clear and simple to explain but in my opinion some confusion exists on the use of terms as fractal dimension, fractal character and complexity. I will raise this issue in the next paragraph section.

The study is well described and its predictions are straightforward. However, I do not think it is the best experimental design. Although the results are very easy to interpret, I believe additional analyses would provide more information about the nature of the differences between the FD in the experimental groups. I will comment this point in the section about the results.

Comments on theoretical concepts

1- One of the more repeated concepts in the manuscript is ‘complexity’. I believe that in general we need to take care when use it because in most studies it is a synonym of ‘randomness’. In this study complexity is identified with a system between order and randomness, with being more or less fractal or with the level of FD that a given signal exhibits. From my point of view, complexity is not a well defined concept in science. Even in the so called science of complexity there is no agreement about the exact meaning of it. Hence, the authors need to define what complexity is in the framework of this particular investigation. I encourage the authors to give a specific working definition for complexity in the introduction section and follow it in the rest of the manuscript.

2- Another confusing point, at least for me, is that in this work it is suggested that one of the goals is to study if the functional networks are fractal-like (goal 1 in page 5). One might investigate this particular question by looking the small world properties or power law characteristics, etc.; but I believe that the FD alone does not indicate if a given object or network is fractal. It is suggested here that if the FD is higher for a given network it would show more fractal properties than other network that exhibits less FD. This is not necessarily true. The definition of a fractal is any structure made of copies of itself (self- similar) and depending of the nature of its self-similarity the FD will be high or low. For example, The Koch curve has a FD of 1.26, the Sierpinski triangle has a FD of 1.58, and I would not say that the latter is more fractal than the former. We might also use an empirical example: it is very likely to obtain a very high Higuchi’s fractal dimension for a random signal, and it does not mean that this is self affine or fractal. In general, FD indicates the density of the structure being measured, or the roughness of the object. It would be possible that in a given context a high FD indicate that it is a fractal and I believe that this is what it needs to be clarified in the manuscript. It would be needed to justify why a higher FD for a functional network means that it is a fractal. I know that in theory any topological dimension that is fractional belongs to a fractal structure but this is just for mathematical objects. Natural objects can show fractional dimensions calculated with algorithms of approximation without being fractal-like. For example, if you use the Higuchi’s fractal dimension with a simple sinusoidal curve, the FD would be slightly higher than 1. And this signal is not a fractal indeed.

3- I believe that the relationship between consciousness and complexity is not very clear in the manuscript. Complexity is related in this work with criticality as well as with the concept of complexity developed in Tononi’s theory. This is quite confusing and difficult to understand by a reader who is not familiar with these two perspectives. I would define the theoretical framework with more precision: A) Is this study testing differentiation and integration as a marker of consciousness? In this case, complexity is very well defined and one might discuss the results in this context. B) Is this study directed to find fractal structures and relate them with conscious states? Then I would try to link complexity with fractal dimension, as I suggested above, and give a solid argument to conceive FD as an indicator of self-affinity.

Comments on the methodology and the FD measures used in the study

1. A specific question I would like to raise here is Do authors really address the goal one of the study introduced in page 5? In order to state that networks constructed suppressing weak edges, have a fractal character, it would be convenient to obtain a different type of functional network and show that it is not fractal (or it is less fractal). Authors could have obtained networks with weak edges and use them for comparison. Moreover, this design would be also convenient to show if the FD decreases only in the networks with fractal characteristics vs networks with less fractal structure.

2. In this section I also want to point out that I am a bit confused about the values of the measures obtained in the study. There are three different measures. The first one is the FD of the Networks (NFD); the second one is the FD of the Adjacency matrix (AdFD); and the third one is the Higuchi’s FD (HFD).

In principle one might expect that a network maximum value of NFD would be 3 just because the maximal density of an object in a three dimensional space is 3. However, obtained values are higher than 3. Because I am not an expert in the box counting algorithm presented here, I might be wrong in this point.

The values of the AdFD measures are between one and two and this is reasonable because one might expect that FD=2 will be the more compact object in this space. But the values of HFD are a bit weird because they should range between 1 (straight line) and 2 (a very dense signal filling the entire 2 dimensional space). The means reported here are below 1. I believe it might be due to the selection of the k parameter but the authors need to check if there is an error in the estimation.

3. It would be useful to include the characteristics of the BOLD signals used in the study. At least authors should report the length of the segments, and the AD rate at which they were registered. Does ‘samples’ in section 3.3 refer to number of points in a segment? Is there only one segment per participant?

Comments on the results

The non parametric statistical approach introduced by the authors is adequate. The results showed that the FD was different for each group and measure. However, I would not say that one might predict (as suggested in the title of the manuscript) the severity of the disorder of consciousness. I think that in order to predict it would be necessary to include a larger sample of participants as well as a mathematical model of prediction (ROC curves, linear or non linear multiple regression, or a more sophisticated classifier). Hence I would say that the discussion should consider this fact and a different verb should be used in the title.

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Fractal Dimension of Cortical Functional Connectivity Networks & Severity of Disorders of Consciousness

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Reviewer #3: I have read the review sent by the authors and I would like to indicate that all potential issues I commented were adequately addressed. Terms as fractality, complexity or fractal dimension are clearer now throughout the manuscript.

I see that some technical aspects have been improved as well.

I appreciate the chage in the title, I think it is more precise now

Because I do not have further questions I recommend this work for publication

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Reviewer #3: Yes: Antonio J. Ibáñez-Molina

PONE-D-19-27002R1

Fractal Dimension of Cortical Functional Connectivity Networks & Severity of Disorders of Consciousness

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