Limited changes in locomotor recovery and unaffected white matter sparing after spinal cord contusion at different times of day.

The circadian gene expression rhythmicity drives diurnal oscillations of physiological processes that may determine the injury response. While outcomes of various acute injuries are affected by the time of day at which the original insult occurred, such influences on recovery after spinal cord injury (SCI) are unknown. We report that mice receiving moderate, T9 contusive SCI at ZT0 (zeitgeber time 0, time of lights on) and ZT12 (time of lights off) showed similar hindlimb function recovery in the Basso mouse scale (BMS) over a 6 week post-injury period. In an independent study, no significant differences in BMS were observed after SCI at ZT18 vs. ZT6. However, the ladder walking test revealed modestly improved performance for ZT18 vs. ZT6 mice at week 6 after injury. Consistent with those minor effects on functional recovery, terminal histological analysis revealed no significant differences in white matter sparing at the injury epicenter. Likewise, blood-spinal cord barrier disruption and neuroinflammation appeared similar when analyzed at 1 week post injury at ZT6 or ZT18. Therefore, locomotor recovery after thoracic contusive SCI is not substantively modulated by the time of day at which the neurotrauma occurred.

Introduction

Time of day affects the incidence of various acute pathologies including myocardial infarct (MI) and ischemic stroke [1]. Moreover, the severity of those injuries may also be influenced by the time of day at which they occur [2, 3]. Such effects stem from circadian rhythmicity of biological processes that determine the risk of a blood vessel occlusion and/or rupture and/or modify the tissue injury response [1]. Thus, in morning hours when the human active period begins, circadian maxima (acrophases) of blood pressure, sympathetic tone, and hemostasis may explain the higher occurrence and greater severity of MI and stroke seen at that time [1]. In addition, circadian modulation of metabolism, pro-inflammatory potential, immunity, anti-oxidant defenses and blood-tissue barriers may directly affect the sensitivity to acute injuries [1, 4–6].

Circadian rhythmicity of biological processes is mediated by oscillations of gene expression produced by a set of conserved transcription factors (TFs) of the clock pathway including BMAL1, CLOCK and NPAS2 [7]. The clock pathway in hypothalamic suprachiasmatic nucleus (SCN) neurons synchronizes clock pathways in other cells of the body. The clock pathway TFs engage feedback loops that underlie oscillating expression of them and their regulators.

In rodents, the activity of the effector outputs of the clock pathway in most non-SCN tissues is low at ZT18-0 (late night/early morning) or high at ZT6-12 (afternoon/early evening), respectively [8-10]. Such oscillations coincide with differential responses to such challenges as MI, infection, endotoxic shock or autoantigen exposure while genetic disruption of clock signaling nullifies those time of day effects [11-15].

Time of day effects are also documented in several models of acute brain injury [16-21]. However, the maximum severity of brain damage peaked at distinctly different times depending on the model used [16-21]. Such variability suggests that unique, injury-specific pathogenic mechanisms may be differentially sensitive to circadian regulation. There are no reports of circadian effects on spinal cord injury (SCI).

BMAL1 is the principal non-redundant TF of the clock pathway output [7]. After moderate contusive SCI at the T9 level, Bmal1 mice showed enhanced locomotor recovery, increased white matter sparing as well as reduced inflammation and improved blood-spinal cord barrier function in the injury epicenter region [22]. Therefore, the pathogenesis of SCI may be regulated by circadian rhythms. The current work was initiated to test whether SCI outcomes differ if the injury occurs at different times of the day.

Methods

Animals

Six-week old C57Bl/6 wild-type female mice were obtained from the Jackson Laboratory (Bar Harbor, ME). Animals were maintained in a 12:12 light-dark cycle (6:00 light on, 18:00 light off) with food and water available ad libitum for 2 weeks. After five days of habituation to handling (performed in the same room where behavioral assessments were later performed) mice were randomly assigned to different experimental groups. All animal procedures were approved by the University of Louisville Institutional Animal Care and Use Committee and strictly adhered to NIH guidelines.

Spinal cord injury

Avertin anesthesia, T9 spinal cord contusion (50 kdyn, IH impactor, Infinite Horizons, Lexington, KY) and post-surgery care were performed as previously described [22, 23] (see S1 Methods for detailed information including anesthesia and post-surgery analgesia). The surgeries were performed in two separate studies by the same team of investigators 12 h apart. In study 1, surgeries were at ZT0-1.5 (6:00–7:30, n = 11) or ZT12-13.5 (18:00–19:30, n = 11); in study 2, surgeries were at ZT5.5–6.5 (11:30–12:30, n = 14) and ZT17.5–18.5 (23:30–00:30, n = 14). Both groups were given identical post-surgical care and maintained under the same conditions for six weeks. Three mice were lost (1 in ZT0 euthanized after accidental rupture of the bladder during bladder expression, 2 in ZT12 were found dead at dpi 8 and dpi 10). See S1 Table for detailed contusion parameters (recorded force, displacement, velocity).

Assessments of locomotor function

All behavioral assessments were performed at the same time (9:00–11:30) for both groups of mice by individuals without knowledge of group assignment. Hindlimb locomotor function was evaluated in an open field using the Basso Mouse Scale (BMS) by raters trained by Dr. Basso and colleagues at the Ohio State University [24]. Evaluations were performed weekly, first before the injury to determine baseline values, and then for six weeks starting at week 1 after SCI. The horizontal ladder test was performed as described previously using Columbus Instruments Sensor and RS-232 Mini Counter (Columbus Instruments; Columbus, OH, USA) with 2.5 mm rungs spaced 3.5 cm apart [25]. Briefly, each animal underwent five stepping trials per session and the total number of footfalls was quantified for the left and right limbs, respectively. A baseline session before SCI was followed by bi-weekly assessments starting at 2 weeks post-injury. To normalize the highly variable individual performance that was observed in this test as assessed by the number of errors at each post-injury testing time, the difference in the number of errors between the current and the previous testing session was also calculated for each animal. The positive or negative value of that parameter indicates worsened or improved ladder walking test performance as compared to the previous testing session, respectively. Gait was analyzed using the Treadscan Gait Analysis system (Cleversys, Reston, VA) after completion of all locomotor assessments at week 6 after SCI as described previously [26]. Briefly, mice were placed on a variable speed treadmill with a clear belt, and a high-speed digital video (ventral view) of the stepping animals was recorded. Each animal was placed on the treadmill, the speed of the belt increased until the animal could no longer maintain position, and then decreased slowly until stable walking was achieved. Ten+ consecutive step cycles were recorded at such individually optimized walking speed (if an animal was unable to execute 10 cycles, at least two 5+ step cycles were recorded). A minimum of 8 step cycles/animal were then analyzed using the Treadscan software.

White matter sparing was performed as described previously [23]. Briefly, after completion of behavioral analyses (day 42 post-injury), mice were deeply anaesthetized and perfused transcardially with ice cold phosphate buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in PBS. Twenty μm serial transverse cryosections from a 4 mm spinal cord segment centered at the injury epicenter were stained for myelin with iron- eriochrome cyanine (EC). For each animal, the section with the least amount of myelinated white matter was identified as the injury epicenter. White matter sparing was defined as % relative white matter area (per total section area) at the epicenter as compared to the relative white matter area 2 mm rostral from the injury epicenter.

Immunofluorescence staining for markers of blood-spinal cord barrier permeabilization

Some mice from study 2 (SCI at ZT6 and ZT18) were deeply anesthetized and transcardially perfused with PBS and then 4%PFA in PBS at 1 week post injury. Tissue processing, immunofluorescence staining, imaging and image analysis followed previously described methods [22]. Briefly, fibrin/fibrinogen, hemoglobin, CD36 and CD45 were detected using the following primary antibodies: anti-fibrinogen (α chain) (rabbit, 1:150, Bioss Antibodies, bs-7548R), anti-hemoglobin α (HBA1+2, rabbit, 1:150 LifeSpan BioSciences, LS-C409143), anti-CD36 (mouse, 1:100, BD Pharmingen, 552544), and anti-CD45 (rat, 1:150, Millipore, CBL1326). Every fifth longitudinal section from each cord (5x 20 μm sections/animal) was immunostained for the indicated markers and photographed with a 10× objective (Nikon Eclipse Ti epifluorescent microscope) and stitched using Nikon Elements software during acquisition. Elements software was used to threshold baseline brightness and contrast identically for each image for all quantitative object and field measurements. The lesion site was defined as 1,500 μm region that was centered in the injury epicenter and included the injury penumbra. It corresponds to a heterodomain that is a pathology-affected region exhibiting extravascular deposition and disorganization of vascular laminin as well as disrupted GFAP immunoreactivity [27, 28]. The total area of the lesion site positive for each marker was quantified by digital image analysis using the basic densitometric thresholding features of Elements software, similar to methods previously reported [28]. Threshold values were obtained and set for each marker and held constant for each image quantified. The percentage of the lesion site area positive for each marker was quantified. For each animal, at least 3 sections spanning the injury epicenter were analyzed and marker signal area was averaged and normalized to WT control values.

qRT-PCR analysis of circadian oscillations of gene expression

Naïve mice (coming from the same batch of animals as that used for SCI study 1 and undergoing same handling habituation) were deeply anesthetized and transcardially perfused with ice cold PBS at ZT1 or ZT12 to collect a 5 mm segment of the thoracic spinal cord and the liver. Total RNA extraction, synthesis of cDNA and SYBR Green-based qPCR analysis using the ΔΔCt quantification method and Gapdh as a normalizer followed previously described methodology [22]. See S2 Table for primer information. The results were compared to publicly available data on oscillations of the clock pathway mRNAs in various tissues of 7–8 week old C57Bl6 mice including the brain stem, the cerebellum, and the liver (http://circadb.hogeneschlab.org/mouse) [8].

Statistical analyses

Repeated measures ANOVA (RM ANOVA) followed by Bonferroni post hoc t-test for multiple comparisons was used for analyzing BMS and horizontal ladder locomotor recovery data. Gene expression, white matter sparing, and immunofluorescence data were analyzed using the non-parametric Mann-Whitney U test.

Results

In various tissues including intact male or female rat spinal cord as well as male C57Bl6 mouse brain stem, cerebellum or liver transcript levels for most clock pathway mRNAs oscillate with a maximum amplitude at or around ZT0 and ZT12 [8, 29] (http://circadb.hogeneschlab.org/mouse, S1 and S2 Figs). Such oscillations indicate activity of the clock pathway, as its core components are also clock pathway-regulated at the transcriptional level [7]. Therefore, levels of selected clock pathway transcripts were analyzed at ZT1 and ZT12 in the intact spinal cord and the liver of naïve mice taken from the same cohort that was used for the SCI study 1. At ZT12, Bmal1 decreased by 42.5% in comparison to ZT1 (Fig 1A). Consistent with increasing BMAL1 TF activity during the rodent inactive period, expression of several BMAL1 target genes including Nr1d1, Nr1d2, Cry1, Per1, Per2, Per3, and Dbp was higher by 25–65% at ZT12. At that time, Bmal1 showed a 98% decline in the liver with several of its target genes showing strong increases by 50–90% (Fig 1B). These data validate natural modulation of the clock pathway in the spinal cord and liver tissues of mice that were used for SCI experiments. The findings are consistent with greater circadian regulation of the transcriptome in the liver as compared to the CNS [8] and the reported maximum amplitude for many clock pathway genes at the start and the end of the mouse active period (S1 and S2 Figs).

Fig 1

Time of day effects on expression of clock pathway mRNAs in the intact mouse spinal cord.

Levels of mRNAs were determined at ZT1 and ZT12 by qPCR using total RNA from the lower thoracic segment of the spinal cord (A) and the liver (B). Gapdh was used as a normalizer for expression level determinations, B2m was also included as an additional normalizing transcript. Note that lower levels of Bmal1 expression at ZT12 coincide with increased levels of several BMAL1 target genes suggesting increased activity of the clock pathway output. The observed differences in the spinal cord are consistent with reported maximal amplitudes of clock pathways mRNA in other non-SCN regions of mouse brain at the beginning and the end of the active period (S1 and S2 Figs). For each transcript, data represent average fold change of a time point with maximal expression ± SD; *, p<0.05, ns>0.05, U test; n = 3 mice/time point.

To determine whether oscillations of the clock pathway activity at the time of injury correlate with a long-term locomotor recovery, moderate T9 SCI was first performed at ZT0 or ZT12 (study 1). Mean displacement was similar for both groups (567.3 ± 88.7 at ZT0 or 575.0 ± 83.1 μm at ZT12, p>0.05, t-test, S1 Table) suggesting no difference in severity of the primary injury. Similar recovery of hindlimb function was revealed with terminal BMS scores of 4.90 ± 0.61 or 4.83 ± 0.32 (Fig 2A). While mean number of errors in the ladder walking test was similar for both groups throughout the recovery, mean difference in error number between the current and the previous testing session suggested minor differences in performance trajectories (Fig 2B and 2B’). Thus, after initial improvement at week 4, ZT0 mice modestly declined at week 6 (error number difference -6.9±3.6 vs. 2.5±3.0, respectively; p<0.001, Bonferroni post-hoc t-test, Fig 2B’). In contrast, no significant changes were observed in the ZT12 group (error number difference -3.7±4.7 vs. -2.5±4.0, respectively; p>0.05, Bonferroni post-hoc t-test, Fig 2B’).

Fig 2

Moderate effects on locomotor recovery and unaffected white matter sparing after SCI at ZT0 or ZT12.

, Locomotor recovery after T9 50 kdyn IH contusion was quantified using the BMS (A and A’) or the horizontal ladder test (mean number of errors is shown in B and mean difference in number of errors between the current and the previous testing session is shown in B’; for week 2 post-SCI the pre-injury baseline served as a normalization reference). The surgeries were done 12 h apart and all the assessments were done at the same time. For either parameter, RM ANOVA showed significant effects of time after injury; time of injury only affected the ladder test performance but not the BMS (see S3 Table for more results). , WMS was analyzed after completion of behavioral assessments 6 weeks after SCI. , Representative images of EC myelin staining in spinal cord sections cut through the injury epicenter. , Similar % spared white matter in the injury epicenter of ZT0 SCI and ZT12 SCI mice (ns, p>0.05, U test). All data represent means ± SD; in B, ns, *, **, *** represent p>0.05, <0.05, <0.01, <0.001, Bonferroni post-hoc t-test, respectively.

In rodents with low thoracic level contusive SCI, white matter loss at the injury epicenter is the primary correlate of hindlimb functional deficits [24, 30]. Therefore, the observed limited effects on functional recovery correlated well with the lack of significant differences in % spared white matter between the groups (Fig 2C and 2D).

Similar results were obtained in study 2 which compared effects of SCI at ZT6 and ZT18 (Fig 3). The only difference was a significantly higher number of ladder crossing errors in ZT6 vs. ZT18 mice at week 6 (18.68±4.71 vs. 12.52±3.07, respectively, p>0.01, Bonferroni post-hoc t-test, Fig 3B). Noteworthy, a minor yet significant difference at the baseline is unlikely to explain those week 6 effects as ZT18 mice were slightly worse performers before the injury (ZT6 vs. ZT18, 1.6±0.68 vs. 2.47±.57, respectively, p>0.01, Bonferroni post-hoc t-test Fig 3B). Analyzing individual error number difference from the previous session normalizes ladder test performance data against any potential baseline bias. Such analysis confirmed worsened performance of ZT6 mice at week 6 with no such worsening in ZT18 animals (Fig 3B’). The terminal kinematic analysis at week 6 revealed minor, yet significant, differences suggesting that ZT18 animals recovered more function as compared to ZT6 (S3 Fig). That improvement included slightly reduced compensatory usage of forelimbs and improved coordination of stepping without direct evidence for better hindlimb function (S3 Fig). Consistent with such a modest impact on functional recovery, similar % white matter was spared in ZT18 and ZT6 groups.

Fig 3

Moderate effects on locomotor recovery and unaffected white matter sparing, after SCI at ZT6 or ZT18.

In study 2, SCI as well as functional and histological analyses were performed as described for study 1 (Fig 2). , Despite the significant main effect in RMANOVA for time of injury, no significant group differences in BMS were observed with post hoc testing at any time point; BMS subscore (A’) was also unaffected. , horizontal ladder walking test revealed higher number of errors in ZT6 vs. ZT18 mice at week 6 which and a minor opposite difference at the baseline; analyzing mean difference in number of errors between the current and the previous testing session as depicted in B’ removed any potential baseline bias and still showed similar ZT6 group worsening at week 6 (B’, see also S3 Table). , Representative images of EC myelin staining in spinal cord sections cut through the injury epicenter. , Similar % spared white matter in the injury epicenter of ZT6 SCI and ZT18 SCI mice (ns, p>0.05, u-test). All data are means ± SD; in B, ns, *, **, *** represent p>0.05, <0.05, <0.01, <0.001, Bonferroni post hoc t-test, respectively.

As it has been previously reported that in Bmal1 mice, acute/subacute (i.e.1-7 days post-injury) reductions in hemorrhage, blood spinal cord barrier (BSCB) disruption and neuroinflammation correlate well with enhanced recovery and terminal white matter sparing [22], study 2 also included several animals to evaluate those processes at 1 week post-injury. However, immunofluorescence analysis for markers of hemorrhage (hemoglobin), BSCB disruption (fibrinogen) or neuroinflammation (VCAM1, CD36, CD45) did not reveal any significant differences in their expression in the injury region of ZT6 vs. ZT18 mice (Fig 4). Therefore, even at the ZTs when the overall moderate effects on locomotor recovery appeared to be maximized, unaffected white matter sparing at week 6 correlated well with seemingly similar disruption of vascular integrity and the neuroinflammatory response 1 week after SCI.

Fig 4

Similar expression of markers of hemorrhage, BSCB disruption and neuroinflammation at 1 week after SCI at ZT6 and ZT18.

Some mice from study 2 were euthanized at 1 week post injury and markers of hemorrhage (hemoglobin), BSCB disruption (fibrinogen) and neuroinflammation (VCAM1, CD45 and CD36) were evaluated by immunofluorescence using longitudinal sections through the injury epicenter region. , Representative images depicting marker stainings. , Quantification of marker staining immunofluorescence (IF) signal area in a 1.5 mm lesion region spanning the injury epicenter (% total lesion area occupied by the positive IF signal). All data are means ± SD; ns, p>0.05, U test).

Discussion

Our results suggest that after contusive SCI, impairment of hindlimb function and its subsequent recovery are only modestly affected by the time of day at which the injury occurred. In addition, consistent with published clock pathway gene expression studies of rodent CNS tissue, we report relatively higher or lower activity of the clock pathway at the beginning and the end of the mouse active period, respectively [8, 9]. As reliable determination of the nadir and the zenith for a circadian-regulated mRNA requires probing at a minimum of 6 different timepoints/day across at least two days, we do not have sufficient data to determine the spinal cord rhythm of clock pathway transcripts [31]. However, we can rely on published reports and/or publicly available data sets from other areas of the mouse CNS as well as other organs that, apart from SCN, show similar phase of oscillations for all major mediators of the clock pathway with a zenith of the clock pathway activity by the start of the active period and a nadir at its end (S1 and S2 Figs). In addition, our spinal cord or liver data show that clock pathway mRNA changes at ZT1 and ZT12 are of similar magnitude as maximal amplitudes reported in the mouse CNS or liver, respectively (Fig 1, S1 and S2 Figs). One notable exception is Nr1d1/Nr1d2 which peaks in the middle of the inactive period (S1 Fig). Therefore, our analysis likely underestimates its maximal circadian oscillations in the spinal cord and the liver. However, the presented findings confirm that in the cohorts of mice that were used for SCI studies, natural circadian oscillations of gene expression occurred in the spinal cord and the liver and that their phase was likely similar to that reported in rodents. Thus, circadian rhythmicity of gene expression and its effector biological processes could be considered as a potential variable that determines outcome of SCI.

While no significant changes in locomotor recovery were observed when using BMS to compare injuries at ZT0 vs ZT12 or ZT6 vs. ZT18, modest yet significant effects emerged in ladder walking test performance. At week 6, lower number of errors was observed in animals receiving SCI at ZT18 as compared to ZT6. Modest differences in recovery trajectories also emerged when comparing ZT0 vs. ZT12 mice. Overall, the relatively greater sensitivity of the ladder walking to detect time of day effects may be related to its dependence on additional components of locomotion such as hindlimb-forelimb co-ordination, sensory feedback, forelimb function, fine motor control of the digits and trunk stability [32]. Indeed, locomotion on a flat surface such as that assessed for hindlimbs in BMS is often unaffected when ladder test deficits are evident in various types of rodent CNS lesions [32]. Noteworthy, terminal kinematics analysis showed small, but significant, differences with lesser compensatory forelimb usage and improved coordination without improvement in direct measures of hindlimb function in ZT18 mice. Thus, more coordinated stepping and reduced need for compensatory activity of forelimbs could have contributed to the improved ladder test performance in that group. While mechanisms of those minor effects are unclear, they correspond well to unaffected terminal white matter sparing as that parameter most closely correlates with BMS [24, 30].

An intriguing possibility exists that the relatively better ladder test performance at week 6 in animals with night time vs. day time SCI may be due to reduced spasticity. The latter phenomenon, which is expected to have profound negative effects on ability to execute precise paw placements and grasp the ladder rungs, develops several weeks after rodent SCI reaching a plateau at around week 8 [33-35]. Of note, mice that were injured during the inactive period showed initial improvement in test performance from week 2 to week 4 that was followed by significant worsening at week 6 (Figs 2B and 3B). Such a time course would fit well with appearance of spasticity after rat or mouse SCI [33-35]. Interestingly, no significant week 6 worsening was observed in animals that were injured during the active period. Thus, different time of day at which injury occurs may affect development of spasticity during the chronic phase of recovery. Future studies would be needed to directly address such an interesting possibility.

While this report is the first to address the question of time of day effects on pathogenesis of SCI, others have documented existence of circadian modulation in other acute CNS injury models. Thus, in a middle cerebral artery occlusion (MCAO) model of rat stroke, acute infarct volume was three times larger with a stroke at ZT22 than at ZT10 [16]. However, relatively moderate time of day effects were reported in a murine MCAO model [21]. Global brain ischemia at ZT14 resulted in maximal hippocampal apoptosis in a rat, while the greatest hippocampal damage followed ZT6 ischemia in mice [17, 19]. In a mouse subarachnoid hemorrhage model, less apoptosis was observed in the hippocampus and the cortex when the insults occurred at ZT12 as compared to ZT2 [20]. Closed skull traumatic brain injury (TBI) in rats at ZT17 reduced brain damage area and acute mortality with a transient improvement in locomotor function as compared to ZT5 TBI [18]. These results indicate that dependent on the CNS injury model used, injuries that occurred in the second half of the active phase through the start of the inactive phase, when clock pathway activity reaches a nadir, produced maximal or minimal pathology at acute/subacute phases. While the currently presented SCI time of day studies did not reveal major differences in long term recovery or tissue sparing, the aforementioned reports of circadian effects focused on acute/subacute pathological changes with long term functional recovery or lesion healing either not examined or unaffected. Hence, in several types of acute CNS injury in rodents, including mouse SCI, the time of insult may have, at most, only transient effects on secondary damage with moderate long lasting functional impact.

One could argue that the severity of the SCI paradigm used for this work may have been too high to detect time of injury effects. This is unlikely as this moderate IH contusion has been used in several mouse studies in which locomotor recovery and terminal white matter sparing were increased or decreased by various genetic or pharmacological manipulations that targeted the secondary injury cascades [22, 23, 26, 28, 36, 37]. Hence, excessive primary damage is unlikely to explain our negative findings.

Why is post-SCI recovery only slightly affected by the time of injury despite our prior findings that Bmal-/- mice have improved functional outcome [22]? While there are many possible explanations to reconcile those observations, one should note that BMAL1 has been implicated in gene expression regulation beyond circadian rhythms [38, 39]. Loss of such a regulatory activity was proposed to contribute to a neurological phenotype of BMAL1-deficient mice and may have also played a role in SCI outcome [38]. Moreover, altered clock pathway/BMAL1 activity after, but not before, and/or at the time of SCI may be a critical contributor to secondary injury and long-term recovery after SCI. Interestingly, acute increases of at least some clock pathway components were observed in the injured mouse spinal cord tissue [22]. Those findings may indicate that after SCI, the clock pathway is reset to a new, post-injury time. Hence, modulation of the secondary damage by the injury-regulated activity of the clock pathway may override any earlier influences from natural circadian oscillations. Lastly, a confounding factor that may alter the significance of natural clock pathway oscillations in experimental SCI may be pre- and post-surgery care including such potential clock-resetting stimuli as anesthesia and analgesia [40, 41].

Noteworthy, a recent study using various rodent brain ischemia models showed that an acute CNS insult may engage different pathogenic mechanisms dependent on the time of day [42]. Specifically, active but not inactive period ischemia was shown to be associated with reduced penumbra and resistance to several established neuroprotective interventions suggesting differential diurnal engagement of potential therapeutic targets including reactive oxygen species or the NMDA receptor. Thus, while the final outcome of an acute CNS injury may be only moderately affected by the time of injury, the latter parameter may be an important determinant of responsiveness to neuroprotective interventions. Future studies are needed to examine such an interesting concept in the context of SCI.

Conclusions

Current work using moderate contusive thoracic SCI indicates no major injury time of day effects on long term locomotor recovery or white matter integrity in mice. However, slightly improved terminal performance in ladder walking after active period SCI suggests subtle, but significant, effects that may involve structural and/or functional plasticity of spinal cord circuitries. Obtaining clinical data to test if time of injury affects SCI outcome in humans will be extremely difficult as confounding injury co-morbidities and variable extents of spontaneous recovery will restrict necessary samples sizes [43, 44]. Future preclinical analyses to address potential effects on additional outcomes such as immune system dysregulation [45] or differential involvement of distinct pathogenic mechanisms of secondary injury [42] may provide insight into potential novel therapeutic avenues that could be acutely initiated depending on time of injury.

Spinal cord injury and post-surgery animal care.

(DOCX)

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ARRIVE compliance questionnaire.

(PDF)

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Contusion parameters for each individual animal.

(DOCX)

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qPCR primers.

(DOCX)

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Results of RM ANOVA for BMS and ladder test.

(DOCX)

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Circadian oscillations of selected clock pathway mRNAs in the brain stem, the cerebellum and the liver of C57Bl6 mice.

The data are from the publicly available circadian transcriptome database (http://circadb.hogeneschlab.org/mouse). All presented mRNAs show significant circadian oscillations in all three tissues (JTK p<0.05) except Cry1 (non-significant cycling in the brain stem /JTK p = 0.058/) and B2m (no cycling in any tissue /JTK p = 1/). Note that the presented default output graphs from the circadb database show one full 24 h period from ZT24 though ZT48. Therefore, ZT1 or ZT12 in Fig 1 corresponds to ZT25 or 36 in S1 and S2 Figs, respectively.

(PDF)

Click here for additional data file.

The data are from the publicly available circadian transcriptome database (http://circadb.hogeneschlab.org/mouse). All presented mRNAs show significant circadian oscillations in all three tissues (JTK p<0.05) except Cry1 (non-significant cycling in the brain stem /JTK p = 0.058/) and B2m (no cycling in any tissue /JTK p = 1/). Note that the presented default output graphs from the circadb database show one full 24 h period from ZT24 though ZT48. Therefore, ZT1 or ZT12 in Fig 1 corresponds to ZT25 or 36 in S1 and S2 Figs, respectively.

(PDF)

Click here for additional data file.

Minor differences in gait between mice injured at ZT6 vs. ZT18.

After completion of BMS and ladder walking at week 6 after SCI, gait analysis was performed using the Treadscan system. , Although the average coordinated plantar index (CPI, a ratio between numbers of plantar step cycles with correct sequence of limb placement to all step cycles) was not different between the groups (A), significantly more ZT18 animals achieved high level of coordination (B, CPI>0.6). C-F, Stride analysis revealed no significant differences in hindlimb function with consistent effects on forelimbs suggesting reduced compensatory usage in the ZT18 group. Those include longer and less frequent strides (C), as well as longer swings (D) and shorter stance (E). While the BMS-correlated rear track width was unaffected (Beare et al. 2009, PMID: 19886808), forelimb-hindlimb foot base of support was shorter in the ZT18 group (F). The latter parameter was the only direct indication of potentially improved hindlimb function in ZT18 mice (less hindlimb dragging); additional indirect support for moderate improvement in hindlimb function is provided by the aforementioned lower compensatory usage of forelimbs in that group. Data represent means ± SD; Binominal Proportion test, CPI distribution (z = 2.0, p<0.05, *); RM ANOVA with left vs. right side and time-of-injury used as two factors, effect of time-of-injury: swing (F1,36 = 8.9, p<0.01**), % of swing (F1,36 = 6.3, p<0.05*), % of stance (F1,36 = 6.3, p<0.05*), stride (F1,35 = 6.5, p<0.05*), stride frequency (F1,36 = 6.3, p<0.05*), foot base of support (F1,36 = 14, p<0.01**); ns, p>0.05. Overall, gait parameters suggest only minor effects of time-of-injury on locomotor recovery as compared at ZT6 vs. ZT18.

(PDF)

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13 Apr 2021

PONE-D-21-09445

Unaffected functional recovery after spinal cord contusions at different circadian times

PLOS ONE

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Reviewer #1: Yes

Reviewer #2: Yes

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

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**********

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Reviewer #2: Yes

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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: In this manuscript Slomnicki et al investigate whether a moderate T9 contusion SCI performed in mice at Zeitgeber time 1 or 12, when lights are turned on or off, effects the functional recovery and histological outcome. They confirm that several key clock regulators are differentially expressed at these times in uninjured spinal tissue. They then go on to perform a contusion injury at these two time points and demonstrate no difference in hindlimb recovery based on the BMS and ladder test as well as no difference in white matter sparing at the lesion epicentre.

The manuscript is well written and of interest to the field. The experiments are conducted to a high quality with clear aims and conclusions. I have only minor comments that need to be addressed before publication.

Minor concerns:

1. In the title the authors should to be careful when stating circadian time; this manuscript only investigates the effects of Zeitgeber time at two timepoints based around the light cycle.

2. There is often a lag before the circadian rhythm responds to changes in the expression of clock regulatory genes, it would be of interest to perform a SCI at other times points. However the reviewer appreciates that this may be out of the scope of the current manuscript.

3. In Supp. 1 and 2 the Zeitgeber time starts at 18, it would be more relevant to see the differences at 0 to 12 as these are the times used in the study.

4. It would be of interest to determine whether the injury resets the clock and induces changes in the expression of clock regulatory genes at the chronic time point.

5. In the authors previous publication using Bmal1-/- mice they demonstrate an effect on the integrity of the BSCB and inflammatory response acutely after SCI. It may be interesting to also examine some of these histological markers in this chronic SCI tissue.

6. In the methods please state at what time of day behavioural assessments were performed.

7. Supp.2 the Dbp plot needs to be re-aligned.

Reviewer #2: In the manuscript Slomnicki et al studied how the time of day affects functional recovery and anatomical adaptation after moderate contusive SCI at T9 level in mice, performing injuries at Zeitgeber time (ZT)1 and ZT12, i.e. when lights are on and off, respectively.

The authors confirmed differential expression of clock genes in uninjured spinal cord between ZT1 and ZT12. Then they performed SCI at these 2 time points and found no difference in hindlimb recovery as measured by BMS and ladder tests. Furthermore, they found no difference in sparing at the injury site in the white matter.

The manuscript is well written and the research question is of interest for the field. While the manuscript is experimentally of very good quality, the reviewer has a number of concerns/comments that need to be addressed before publication:

Major concerns:

1. Clock genes and their downstream pathways usually present a lag in the response to environmental stimuli (Zeitgebers such as light) as also shown by the clock gene expression in Suppl Fig 1 and 2. Indeed, while no difference was found between ZT0 and ZT12, increasing the number of injury time points, i.e. increased temporal resolution, is required to claim that time of day does not play a role, as the authors themselves point out in the discussion.

2. Since in their previous publication the authors found an effect on the integrity of the BBB and acute inflammatory response in mice with Bmal1 deletion, it would be relevant to examine these aspects in the present study. In fact, here there is no mention of the different cell types present in the intact and injured spinal tissue, what is the clock in the different cell types? And importantly, what happens to them before and after injuries performed at different ZTs?

3. Although no difference in functional recovery was found, circadian changes may be subtle and the analysis performed by authors not powerful enough to fully appreciate them, it may be relevant to analyse anatomical changes such as plasticity, regeneration and sprouting of different tracts/neurons affected by injury.

Minor comments

1. The title states “circadian times”, however the injuries are performed on mice kept in a normal LD cycle which makes it impossible to determine circadian time but only zeitgeber time or time of the day

2. In Suppl Fig 1 and 2 the graphs show a time starting at ZT18, it would be ideal to have them all starting at ZT0, especially considering that the injuries have been performed at ZT0 and ZT12

3. The time at which the lights are on is usually termed ZT0 and not ZT1. It is not clear why the author performed injury at ZT0 (Fig 2) but show qPCR analysis at ZT1 (Fig 1)

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Reviewer #2: No

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20 Sep 2021

REVIEW RESPONSE:

Reviewer #1, Point # 1: In the title the authors should to be careful when stating circadian time; this manuscript only investigates the effects of Zeitgeber time at two timepoints based around the light cycle.

Response: We replaced the term with either or . Also, additional injury timepoints were added for the revision (study 2 examines ZT6 vs. ZT18).

Reviewer #1, Point # 2: There is often a lag before the circadian rhythm responds to changes in the expression of clock regulatory genes, it would be of interest to perform a SCI at other times points. However, the reviewer appreciates that this may be out of the scope of the current manuscript.

Response: We fully agree that comprehensive analysis of time of injury effects should include more timepoints. Therefore, additional injury timepoints were added for the revision (the newly added study 2 examines ZT6 vs. ZT18, Revised Figs. 3 and 4).

Reviewer #1, Point # 3: In Supp. 1 and 2 the Zeitgeber time starts at 18, it would be more relevant to see the differences at 0 to 12 as these are the times useTd in the study.

Response: the graphs in Figs. S1 and S2 were generated as a default output of the circadb database. Each graph covers one full period (ZT24-ZT48). Hence, ZT0 or ZT12 in our data (Fig. 1) corresponds to ZT24 or ZT36, respectively. To avoid confusion, we modified the legend for Fig S1 and S2 to explain that shift.

Reviewer #1, Point # 4: It would be of interest to determine whether the injury resets the clock and induces changes in the expression of clock regulatory genes at the chronic time point.

Response: We agree that this would be an interesting question to answer. However, we feel that this resource-intensive experiment is out of scope of our current paper that is addressing the effects of time of injury on post-injury recovery rather than consequences of the injury for the rhythmicity (to do this correctly with injured animals one would have to study at least 2 periods with 4 probing times/period- 2x4= 8 timepoints per arm). In the context of our data, the most interesting time window for such an experiment would be during acute/subacute phases of recovery when white matter is still being lost and a clock reset could nullify any potential effects of time of injury on lesion size. At least with thoracic level injuries in humans or rats, rhythmicity of various physiological rhythms appears normal at chronic timepoints (PMIDs: 22474242, 21231876, 30627655). Thus, limited chronic outcome could be expected with T9 contusion in mice.

Reviewer #1, Point # 5: In the authors previous publication using Bmal1-/- mice they demonstrate an effect on the integrity of the BSCB and inflammatory response acutely after SCI. It may be interesting to also examine some of these histological markers in this chronic SCI tissue.

Response: We agree that this would be an interesting question to answer. Unfortunately, we did not have any tissue left from our original study (study 1 with SCI at ZT0 and ZT12). However, we generated extra tissue in the new study that probed effects of SCI at ZT6 and ZT18. As in study 2, effects of injury time, while still limited, were greater than those in study 1 (ZT18 group showed small but significant improvement in BMS and better ladder test performance as compared to ZT6), we focused our BSCB/neuroinflammation analyses on that experiment. We have chosen day 7 post injury because at that timepoint BSCB leak is still present, neuroinflammation is well developed, and, Bmal1 deficiency strongly reduced both those processes (Slomnicki et al. Scientific Reports 2020). However, we did not observe any differences between SCI at ZT6 vs. ZT18. Those new data are shown in the revised Fig. 4.

Reviewer #1, Point # 6: In the methods please state at what time of day behavioural assessments were performed.

Response: Modified as requested (all testing was done between 9:00-11:30).

Reviewer #1, Point # 7: Supp.2 the Dbp plot needs to be re-aligned.

Response: Modified as requested.

Reviewer #2, Major concern #1: Clock genes and their downstream pathways usually present a lag in the response to environmental stimuli (Zeitgebers such as light) as also shown by the clock gene expression in Suppl Fig 1 and 2. Indeed, while no difference was found between ZT0 and ZT12, increasing the number of injury time points, i.e. increased temporal resolution, is required to claim that time of day does not play a role, as the authors themselves point out in the discussion.

Response: We conducted another study that compared effects of SCI at ZT6 vs. ZT18. That experiment resulted in similar conclusions as the original experiment with SCI at ZT0 and ZT12. The new data are presented in the revised manuscript as Fig. 3 and Fig. 4.

Reviewer #2, Major concern #2: Since in their previous publication the authors found an effect on the integrity of the BBB and acute inflammatory response in mice with Bmal1 deletion, it would be relevant to examine these aspects in the present study. In fact, here there is no mention of the different cell types present in the intact and injured spinal tissue, what is the clock in the different cell types? And importantly, what happens to them before and after injuries performed at different ZTs?

Response: We agree that defining injury time effects on acute/subacute dysfunction of BBB/BSCB and the neuroinflammatory response would be an interesting question to answer. Therefore, we generated extra tissue in the new study that probed effects of SCI at ZT6 and ZT18. As in that study (study #2), effects of injury time of day on recovery, while still limited, were greater than those in study #1 (ZT18 group showed small but significant improvement in BMS and terminal better ladder test performance as compared to ZT6), we focused our BSCB/neuroinflammation marker analyses on that experiment. We have chosen day 7 post injury 7 because at that timepoint BSCB leak is still present, neuroinflammation is well developed, and, Bmal1 deficiency strongly attenuates those processes (Slomnicki et al. Scientific Reports 2020). However, we did not observe any differences between ZT6 vs. ZT18. Those new data are shown in the revised Fig. 4.

While we agree that defining cell type specific regulation of the clock pathway in the intact and injured spinal cord would be of great interest, performing such a resource and time intensive analysis is beyond the scope of the current manuscript.

Reviewer #2, Major concern #3. Although no difference in functional recovery was found, circadian changes may be subtle and the analysis performed by authors not powerful enough to fully appreciate them, it may be relevant to analyse anatomical changes such as plasticity, regeneration and sprouting of different tracts/neurons affected by injury.

Response: We agree that those additional analyses could provide more insight into subtle time of injury effects on the pathogenesis of SCI. However, such additional experiments would require significant resources and relatively long time to complete. Therefore, their addition would greatly delay timely publication of the current data. To acknowledge a possibility that such additional effects may exist, we added the following statement to the final conclusions paragraph of the discussion:

Reviewer #2, minor concern #1: The title states “circadian times”, however the injuries are performed on mice kept in a normal LD cycle which makes it impossible to determine circadian time but only zeitgeber time or time of the day

Response: We replaced the term with or .

Reviewer #2, minor concern #2: In Suppl Fig 1 and 2 the graphs show a time starting at ZT18, it would be ideal to have them all starting at ZT0, especially considering that the injuries have been performed at ZT0 and ZT12

Response: The graphs in Figs. S1 and S2 were generated as a default output of the circadb database. Each graph covers one full period (ZT24-ZT48). Hence, ZT0 or ZT12 in our data (Fig. 1) corresponds to ZT24 or ZT36, respectively. To avoid confusion, we modified the legend for Fig S1 and S2 to explain that shift.

Reviewer #2, minor concern #3: The time at which the lights are on is usually termed ZT0 and not ZT1. It is not clear why the author performed injury at ZT0 (Fig 2) but show qPCR analysis at ZT1 (Fig 1).

Response: We define ZT0 as the time of lights on (see the Abstract). The difference between the RNA analysis and the SCI study 1 was due to adjustments for staff availability. The rationale of the RNA analysis was to confirm oscillating activity of the clock pathway. That goal was met with the executed experimental design. Also, densely probed mRNA data from the circadb database (Figs. S1, S2), suggest that one hour difference between the experiments would unlikely change the overall conclusions as to the likely status of the clock pathway activity in the ZT0 SCI group.

Submitted filename: ReviewResponse.docx

Click here for additional data file.

5 Oct 2021

PONE-D-21-09445R1Limited changes in locomotor recovery and unaffected white matter sparing after spinal cord contusion at different times of dayPLOS ONE

Dear Dr. Hetman,

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.

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Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. 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: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. 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: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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6. 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: Point 1: The new results for the normalised horizontal ladder in both study 1 and 2 appear confusing. The mice injured at ZT12 or ZT18 do not recover significantly more but rather that the mice injured at ZT0 or ZT6 get significantly worse at 6 weeks, do the authors have any suggestions in why these groups would deteriorate at week 6?

I can appreciate why the authors tried to normalized the horizontal ladder data due to high variability. However, this is an unusual and confusing way to analyse this data and I believe sufficient number of animals were used to observe real behavioural differences between the groups. I would recommend to show the unnormalized analysis using number of errors, as was provided in the first submission.

Point 2: On Line 264 the authors state “Both BMS (A) and normalized error score in the horizontal ladder walking test (B) revealed minor yet significant improvement in locomotor recovery between ZT18 and ZT6.” But the authors go on to say that “For BMS, no significant group differences were observed with post hoc testing at any time point” Please clarify and correct the statement and results of the BMS. The data appears to show an improvement with time, due to spontaneous recovery but no differences between the groups.

Point 3: Investigating the BSCB and neuroinflammation markers is a welcomed addition to the paper. It may also be interesting to assess fluorescence intensity of these markers at the lesion site. For instance, in the image in Fig.4 CD45 staining appears to be more intense at ZT18.

Point 4: The figure legend and methods for Fig.4 states that coronal sections were used for staining and image analysis yet the images in Fig.4 appear to be longitudinal images, please clarify this.

Reviewer #2: The reviewer appreciates the effort the authors made and thinks that the reviewers' comments have been sufficiently addressed and the data presented are sound and relevant for the field. The reviewer stull thinks that a refined analysis of the anatomical and molecular response to injury, in the form of neuronal plasticity/sprouting and cell-specific changes would add substantial value to a very interesting body of data, however acknowledges that this may be out of the scope of this particular study

**********

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Francesco De Virgiliis

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

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.

13 Oct 2021

RE: Resubmission of the manuscript PONE-D-21-09445R1

REVIEW RESPONSE:

REVIEWER #1: POINT 1: The new results for the normalised horizontal ladder in both study 1 and 2 appear confusing. The mice injured at ZT12 or ZT18 do not recover significantly more but rather that the mice injured at ZT0 or ZT6 get significantly worse at 6 weeks, do the authors have any suggestions in why these groups would deteriorate at week 6?

I can appreciate why the authors tried to normalized the horizontal ladder data due to high variability. However, this is an unusual and confusing way to analyse this data and I believe sufficient number of animals were used to observe real behavioural differences between the groups. I would recommend to show the unnormalized analysis using number of errors, as was provided in the first submission.

RESPONSE: We included the “raw” error number data for both study 1 (Fig. 2B) and study 2 (Fig. 3B) as requested. The normalized data are shown as well (Fig. 2B’ and 3B’). Results description and figure legends were modified accordingly (highlighted in the track change version of the manuscript). Edits were introduced to those sections to clearly define what the normalized error score is (in the revised manuscript we referrer to that parameter as a change in error number from the previous testing). The discussion section contains a paragraph that presents the case for post-injury spasticity as a potential driver for week 6 worsening in ladder test performance of animals with daytime injuries (lines 342-353).

REVIEWER #1, POINT 2: On Line 264 the authors state “Both BMS (A) and normalized error score in the horizontal ladder walking test (B) revealed minor yet significant improvement in locomotor recovery between ZT18 and ZT6.” But the authors go on to say that “For BMS, no significant group differences were observed with post hoc testing at any time point” Please clarify and correct the statement and results of the BMS. The data appears to show an improvement with time, due to spontaneous recovery but no differences between the groups.

RESPONSE: we apologize for those confusing statements. No significant effects of time of day on BMS were observed in study 1 or study 2. The sentences in question were all modified accordingly.

REVIEWER #1, POINT 3: Investigating the BSCB and neuroinflammation markers is a welcomed addition to the paper. It may also be interesting to assess fluorescence intensity of these markers at the lesion site. For instance, in the image in Fig.4 CD45 staining appears to be more intense at ZT18.

RESPONSE: we agree that quantifying the BSCB/neuroinflammation markers in a region- specific manner could be potentially interesting. However, given the overall negative nature of our findings and preferable use of signal area quantifications rather than signal intensity we do not expect that such analyses would affect our conclusions in a major way. To avoid confounding effects of staining to staining variability that can affect intensity we prefer signal area analyses. Thus, the apparent difference in CD45 signal intensity is unlikely to change results of the area analyses as shown in Fig. 4B.

REVIEWER 1, POINT 4: The figure legend and methods for Fig.4 states that coronal sections were used for staining and image analysis yet the images in Fig.4 appear to be longitudinal images, please clarify this.

RESPONSE: We use the term “coronal” to describe the sections that are cut in the coronal plane of the human body that in the spinal cord would include its long axis. We recognize that this may be confusing as the term “longitudinal” is usually used throughout the literature to describe such sections. To avoid such confusion, we replaced the term coronal with longitudinal.

Submitted filename: Review_Response.docx

Click here for additional data file.

21 Oct 2021

Limited changes in locomotor recovery and unaffected white matter sparing after spinal cord contusion at different times of day

PONE-D-21-09445R2

Dear Dr. Hetman,

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PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

9 Nov 2021

PONE-D-21-09445R2

Limited changes in locomotor recovery and unaffected white matter sparing after spinal cord contusion at different times of day.

Dear Dr. Hetman:

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

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