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Computer Science
Medicine

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Evaluating the Utility of EPIK in a Finger Tapping fMRI Experiment using BOLD Detection and Effective Connectivity

EPI with Keyhole (EPIK) is a hybrid imaging technique that overcomes many of the performance disadvantages associated with EPI. Previously, EPIK was shown to provide a higher temporal resolution and fewer image distortions than EPI whilst maintaining comparable performance for the detection of BOLD-based signals. This work carefully examines the putative enhanced sensitivity of EPIK in a typical fMRI setting by using a robust fMRI paradigm – visually guided finger tapping – to demonstrate the advantages of EPIK for fMRI at 3 T. The data acquired were directly compared to the community standard fMRI protocol using single-shot EPI to ascertain a clear comparison. Each sequence was optimised to offer its highest possible spatial resolution for a given set of imaging conditions, i.e., EPIK and EPI achieved an in-planar resolution of 2.08 × 2.08 mm2 with 32 slices and 3.13 × 3.13 mm2 with 36 slices, respectively. EPIK demonstrated a number of clear improvements, such as superior spatial resolution with favourable robustness against susceptibility artefacts. Both imaging sequences revealed robust activation within primary motor, premotor and visual regions, although significantly higher BOLD amplitudes were detected using EPIK within the primary and supplementary motor areas. Dynamic causal modelling, in combination with Bayesian model selection, identified identical winning models for EPIK and EPI data. Coupling parameters reflecting task-related modulations and the connectivity of fixed connections were comparably robust for both sequences. However, fixed connections from the left motor cortex to the right visual cortex were estimated as being significantly more robust for EPIK data.

In one of our previous studies 6 , it was shown that EPIK achieved comparable temporal stability and a similar BOLD detection performance to EPI (community standard technique) under the 'same' imaging parameters and the 'same' image resolution. In a recent study 7 , the capability of higher resolution imaging in EPIK was demonstrated with a high-resolution visual fMRI study. The study also showed that potential autocorrelation effects from the sharing of peripheral data in EPIK were too small to have an impact on the analysis of time-series data.
In contrast, the present work focuses on the investigation of the putative advantages of EPIK for fMRI. For this purpose, visually guided finger-tapping fMRI was performed to carefully examine brain activations in the visual and motor regions. Furthermore, in addition to the analyses of BOLD amplitudes, performance differences of EPIK and EPI were evaluated with regard to effective connectivity analyses between brain regions using dynamic causal modelling (DCM) 8 .
The EPIK sequence was optimised in a way that it achieved its highest possible image matrix size and number of slices whilst the other imaging parameters were kept identical (e.g. FOV, TR/TE, etc.). The optimised EPIK sequence was employed in a direct head-to-head comparison with single-shot EPI, which was also optimised in the same way. Consequently, the performance of EPIK was evaluated against single-shot EPI in the same functional experiment using a standard fMRI protocol. Previously, numerous studies have adopted acceleration techniques such as parallel imaging 9,10 , partial Fourier 11,12 or multi-band techniques 13 to enhance the spatial or temporal resolution in fMRI [13][14][15][16] . However, during the sequence optimisation in the present study, the use of acceleration techniques was excluded in order to avoid the deleterious effects thereof on the performance evaluation of each imaging method.
The parameters of interest were i) BOLD signal amplitude in motor and visual areas and ii) the modulation parameters, estimated by DCM, involved in the visually guided finger-tapping task. Based on the sequence characteristics of EPIK, we hypothesise that the performance of EPIK will be demonstrably better than standard EPI with regard to BOLD amplitude. That is to say, in a given TR, we expect EPIK to produce images with a higher spatial resolution and to be more immune to signal dropout. In particular, the higher spatial resolution achieved by EPIK is expected to result in a more accurate localisation of BOLD signals from activated voxels and to minimise the impact of non-activated neighbouring regions that would reduce the observed BOLD amplitude. Furthermore, given the higher spatial resolution, it might then be possible to measure the functional network dynamics within a given network of brain areas more reliably. Accordingly, DCM on EPIK and EPI are expected to identify the same winning models, since the observed network dynamics should per se be identical. However, we hypothesise that the coupling parameters estimated on the basis of EPIK data may be more robust across different scanning sessions.

Materials and Methods
pulse sequence: epiK. EPIK combines the acquisition of a central portion of k-space -the "keyhole" -with a continuous, interleaved update of the high spatial-frequency information from the periphery of the k-space. In stark contrast to the original keyhole method, the continuous update of the high spatial-frequencies ensures that only small parts of the k-space periphery are correlated and those only in a limited number of scans. Figure 1 shows the schematic representation of the k-space trajectory for three EPIK scans. Each scan comprises a central keyhole and an interleaved acquisition of the peripheral lines of k-space. Here, the central k-space region (k-space keyhole: K K ) is completely sampled with the Nyquist criterion (Δk y = 1/FOV), whilst the peripheral k-space regions (k-space sparse: K S ) are sparsely sampled with Δk y ' = 3/FOV. The first line number of the peripheral k-space at the 1st, 2nd and 3rd scan is 1, 2 and 3, respectively; the first line number becomes 1 again at the 4th scan. Through the sharing of the sparse region data originating from three consecutive scans, and with the keyhole region being updated for every measurement, complete k-space data can be obtained for every scan. Effectively, then, K K is updated after every scan and K S is updated every fourth scan. To ensure that the phase increases smoothly when sharing the sparse region data, correct echo time shifting (ETS) 17 is an integral part of the sequence. In this study, the keyhole region was configured as one-fourth of the k-space and thus, the total number of phase encoding lines to be sampled in each EPIK scan reduces to half of that for comparable EPI. When a fourth scan is acquired, the peripheral k-space data from the first scan are discarded and thus the periphery, K S , is updated by application of this sliding window.
For this work, the above configuration was employed on a Magnetom Tim ® Trio 3 T MRI scanner (Siemens Medical Solutions, Erlangen, Germany) with a 12-channel phased array coil from the manufacturer. point spread function (pSf). For each imaging method (i.e. EPI and EPIK), the signal decay trajectory was simulated as a function of the sampled k-space line and the corresponding PSF was obtained. Here, the T 2 * value used in this simulation was 66 ms, which is known to be the T 2 * value of the grey matter at 3 T 18,19 . From the PSF plot of each method, the full-width at half-maximum (FWHM) was examined and compared between the imaging methods.
The performance of EPIK in PSF was also evaluated under the conditions of fMRI. The very same simulation described above was carried out for the case when there were dynamic signal changes. Here, the rate of dynamic signal changes was set to 5% from the 1st to the 3rd scan. The 5% change is a typical ratio of BOLD signal increase relative to the baseline at 3 T. The purpose of using a rapid signal variation here (i.e. 5% change within 3 temporal scans) was to simulate the worst case in EPIK. Note that in reality, the dynamic signal changes from the 1st to 3rd scan is not as rapid as in the current simulation. fMRi experiments. A visually-guided finger-tapping paradigm was employed for in vivo fMRI experiments to elicit robust activations in visual and motor brain regions. Each experimental session started with a 20 s baseline period (fixation) followed by 7 periods of 1 Hz finger tapping with the right index finger (duration 32 s) www.nature.com/scientificreports www.nature.com/scientificreports/ triggered by a visual stimulus (two red lines centrally presented above and below the black fixation cross, which was displayed throughout the entire duration of the measurement). The finger tapping activation blocks were alternated with resting blocks of a duration of 32 s, during which the subject did not move, but continued to fixate on the black fixation cross. Scanning comprised 2 dummy scans in order to a reach steady state. Thereafter, 225 scans were acquired resulting in a total acquisition time of 8 min 29 s for each fMRI run. A single healthy male volunteer, screened to exclude neurological and psychiatric illnesses, participated in the study. After a complete description of the study, written informed consent was obtained prior to scanning. The local institutional review board (IRB; RWTH Aachen University, Germany) approved the study protocol, screening questionnaires and consent forms. All experiments were performed in accordance with relevant guidelines and regulations.
Functional data acquisition was carried out on a weekly basis on the same subject. For each session, fMRI data were acquired with the following parameters for both sequences: FOV = 200 × 200 mm 2 , flip angle = 90°, TR/ TE = 2200/30 ms and slice thickness = 3 mm with a distance factor of 10%. Under the given imaging conditions above, the voxel size/number of slices achieved by EPI and EPIK were 3.13 × 3.13 mm 2 (matrix: 64 × 64)/36 and 2.08 × 2.08 mm 2 (matrix: 96 × 96)/32, respectively. The fMRI runs were repeated twice using the two imaging methods, EPI and EPIK, in alternating order. Thus, of the 14 sessions acquired in total, 7 sessions started with EPI (followed by EPIK) and 7 started with EPIK (followed by EPI). This was to control for possible habituation effects (potentially leading to a decrease of the blood-oxygenated-level-dependent (BOLD) response) in this robust, but simple, finger-tapping paradigm. It is noted that EPIK employs data sharing and therefore it is true that a single EPIK volume contains data from three TRs whereby the keyhole, which has over 90% of the SNR, is unique to each of the three shots. The TRs required to reconstruct the EPIK volumes is three times TR (i.e. 6600 ms). However, since a sliding window reconstruction is applied for EPIK, the temporal throughput is also the same as EPI (i.e. one EPIK temporal volume every 2200 ms).
Analysis of fMRi data. For all 14 sessions, the two fMRI runs based on EPI and EPIK were separately analysed. The statistical parametric mapping software SPM12 (Wellcome Department of Imaging Neuroscience, London; http://www.fil.ion.ucl.ac.uk/spm/software/spm12/) was used for preprocessing and analysis of the functional imaging data. Functional images were spatially realigned to correct for interscan movements. Then, the mean EPI/EPIK image of each session was computed and spatially normalised to the MNI single-subject template using the normalisation function as implemented in SPM12. Finally, data were smoothed using a Gaussian kernel of 4 mm full-width half-maximum.
On the first level, an onset regressor defined the onsets of finger tapping blocks, and block length was set to 32 s for each session. The haemodynamic response was modelled using a canonical haemodynamic response function (HRF) and its time derivative. The six head movement parameters, as estimated during the realignment procedure, were included as explanatory regressors in the model. To specify the first-level contrasts, each regressor of interest was compared with the implicit baseline by setting this regressor to 1 and all other regressors to zero. The resulting contrast images were then entered into a second level analysis.
On the second level, model estimation was performed using the ANOVA flexible factorial design as implemented in SPM12, with sessions as a covariate to remove between-session variance. To test the conjunction null hypothesis, a conjunction analysis was performed on the baseline contrasts to identify voxels that were significantly activated in both the EPI as well as in the EPIK sessions. Furthermore, BOLD amplitudes, as measured by EPIK and EPI, were compared within these voxels. www.nature.com/scientificreports www.nature.com/scientificreports/ Dynamic causal modelling (DcM). In order to compare the effects of EPIK relative to EPI on effective connectivity, dynamic causal modelling (DCM) 8 was implemented separately for both sets of data. DCM demands slice-time corrected data. Accordingly, for the DCM analysis, data pre-processing was repeated but including slice-time correction as implemented in SPM12. All other pre-processing parameters and steps were identical, as described above.
The task performed in the present study was a visually guided finger-tapping task that requires an information exchange between the visual and the motor systems. Hence, the effective connectivity pattern between early visual areas and the primary motor area was particularly relevant. Three volumes of interest (VOIs) were defined based on the MNI coordinates obtained in the standard GLM second-level analyses and the participant's individual anatomy. Visual information was presented centrally in the visual field and was hence represented bilaterally in visual cortex. Therefore, two VOIs were selected, one in each hemisphere. Centre coordinates for each visual VOI were chosen based on the activation pattern observed in the second-level conjunction analysis. In particular, in each hemisphere, the coordinates of the maximum activated voxel within the largest cluster found in the occipital cortex were set as the centre of the VOI. Accordingly, MNI coordinates for the left visual VOI were MNI [−32 −88 6] and those for the right visual VOI were MNI [36 −78 −2]. In order to derive the coordinates for the VOI representing the primary motor area M1, a two-step procedure was implemented. First, based on the subject's anatomical image, the hand knob contralateral to the response hand was identified. In a second step, the activation pattern as observed in the second-level conjunction analysis was taken into account. The nearest local maximum activation with regard to the hand knob was defined as the centre VOI and was located at MNI [−30 −26 58].
For each session, the first eigenvariate was extracted from voxels within 4 mm sphere centred at the respective MNI coordinate (exceeding a threshold of 0.8). Accordingly, three signal time courses were extracted for each session and for each sequence.
Four different DCM models ( Fig. 2) were defined, taking into account signal time courses from these three regions, and hence constituted the model space for the subsequent Bayesian model selection 20,21 , which allows the most likely DCM to be identified. All models involved bidirectional fixed connections between both visual areas. Two models involved bidirectional fixed connections between both the visual areas and the motor area (models 1 and 2). In one of these models (model 1), connections between the visual and the hand areas were modulated by the experimental task. Two additional models (models 3 and 4) comprised unidirectional connections from both visual areas to the hand area, rather than bidirectional connections. In model 3, the connection from the visual area to the motor area was modulated by the experimental task, while no such modulation was implemented in model 4. For all models, the experimental task was set as the driving input for the given DCMs via both visual regions. www.nature.com/scientificreports www.nature.com/scientificreports/ These four DCM models were estimated separately for the EPIK and the EPI sessions using DCM12 as implemented in SPM12. A random-effects Bayesian model selection was performed separately for both data sets to identify the most likely DCM 21 .
Furthermore, the variability of coupling parameters of the winning model was compared between EPIK and EPI. Therefore, the variance of the estimated DCM parameters reflecting directional intrinsic connections in the model (A-matrix) was estimated separately for each imaging sequence and then compared using an F-test. Similarly, the variance of parameters reflecting task-induced connectivity modulations (B-matrix) were estimated and compared via F-tests.

Results
Reconstructed images. Figure 3 shows reconstructed in vivo images from a representative fMRI session.
Two slices were chosen out of the multi-slice data set to best demonstrate the reduced susceptibility artefacts in the data obtained with EPIK. For comparison between EPI and EPIK, each slice was taken at a nearly identical slice position. Visual inspection of the figure suggests that the EPIK image (matrix size: 96 × 96; in-plane pixel size: 2.08 × 2.08 mm 2 ; see Fig. 3d,e) were reconstructed without any significant loss of signal or any severe degradation of image quality and, furthermore, the slices exhibit a noticeably enhanced spatial resolution, when compared with the EPI images (matrix size: 64 × 64; in-plane pixel size: 3.13 × 3.13 mm 2 ; see Fig. 3a,b). Moreover, due to the reduced number of lines per shot in EPIK and the use of an effectively shorter readout per phase encoding line in EPIK (0.445 ms) than in EPI (0.510 ms), a substantial reduction of geometric distortions was observed in the EPIK images. Signal drop-out due to susceptibility differences is easily visible in the EPI images, particularly in the region of the sinuses (marked by arrow 1 in Fig. 3a,c) and in the region of fourth ventricle (marked by arrow 2 in Fig. 3b,c); this signal dropout is more circumscribed in the EPIK images. To further aid comparison, the reconstructed axial slices are also presented in sagittal orientation (see Fig. 3c,f), which clearly reveals the reduction of geometric distortions in EPIK for the regions marked by both arrows. point spread function (pSf). Figures 4a and b depict the signal decay trajectory and the corresponding PSF for the stationary case (i.e. no dynamic signal changes). The FWHM of EPI (0.0170) was larger than that of EPIK (0.0121), which can be also visually verified from the enlarged plot of the central region (Fig. 4c). The smaller FWHM in EPIK implies that image blurring caused by the long echo train is less pronounced in EPIK than in EPI, i.e. EPIK has a better spatial resolution than EPI. For the present study, where the applied FOV was 200 mm, the FWHM in a mm-scale was 3.40 mm and 2.41 mm for EPI and EPIK, respectively, that is, an improvement of almost 41% for EPIK. Figures 4d and e show the results obtained on the setting with the dynamic signal changes. As shown in Fig. 4d, signal fluctuations are presented for the peripheral k-space regions, which were caused by the dynamic signal changes from the 1st to the 3rd scan. These signal fluctuations created two small peaks in the PSF, each of which is located in the tails of each side (see the arrows in Fig. 4e). However, the magnitude of these peaks is Sequence-specific differences in brain activation patterns. In order to assess both the common and differential activation patterns elicited by the two imaging sequences, the first level baseline contrasts were calculated for each session and were then taken to the second level. On the second level, a flexible factorial design, as implemented in SPM12, was set up, with "sessions" as covariates to remove between-session variance. A conjunction analysis was performed testing the conjunction null hypothesis for significant effects in both imaging sequences. The identified regions are listed in Table 1 with the respective statistical quantities such as cluster size, MNI-coordinates and t-scores. The statistical threshold for activated regions was set to a corrected p-value < 0.05 (FWE at the cluster-level based on an uncorrected threshold of 0.001 at the voxel-level).
For the conjunction analysis "visually-guided finger tapping" versus "baseline", both imaging sequences consistently revealed significant activations within large networks comprising both motor and visual brain regions. In particular, strong activation was found in the left primary motor cortex (M1), including the hand representation www.nature.com/scientificreports www.nature.com/scientificreports/ contralateral to the moving right hand. In addition, significant BOLD signals were observed in the medial (and lateral) frontal regions including the supplementary motor area (SMA) and the premotor cortex (PM). Bilateral cerebellar activations were also observed and were clearly stronger in the right cerebellar hemisphere, i.e., ipsilateral to the right moving hand. Motor activation was complemented by significant signal changes in early and higher visual areas (Table 1).
Within a subset of these brain regions, EPIK yielded significantly stronger BOLD amplitudes in a number voxels as compared to EPI (p < 0.05, FWE at the cluster-level based on an uncorrected threshold of 0.001 at the voxel-level). Stronger activation was found with EPIK within the core regions of our finger-tapping network. These activations were located in the left postcentral gyrus, extending to the precentral regions involving primary motor regions such as areas 4 a and p 22 . In addition, EPIK revealed stronger BOLD amplitudes in supplementary motor areas (SMA), the right cerebellum as well as in right visual cortex (Fig. 5, Table 2). The reverse contrast on voxels of the finger-tapping network (EPI > EPIK) did not reveal any significant supra-threshold voxels.
Labelling of the anatomical location was based on the Anatomy toolbox implemented in SPM 23 . To further demonstrate the different sensitivities of EPIK and EPI in these regions, signal time courses were extracted from the voxel in the primary motor area (Brodmann area (BA) 4a) exhibiting the most marked statistical difference (MNI: −42, 26, 64; T = 7.56). On average, "visually-guided finger tapping" induced a signal increase of 10.3%

Brain Region
Cluster size Side MNI-Coordinates T-score  Table 1. Brain regions whose activity has consistently been detected by EPIK and EPI. www.nature.com/scientificreports www.nature.com/scientificreports/ relative to the "baseline" when measured with EPI. In contrast, when measured with EPIK, the same analysis revealed a 36% increase in BOLD signal, corresponding to a signal increase by a factor of 3.5 (Fig. 5a).

DCM analyses: Sequence-specific differences in brain functional connectivity. Random-effects
Bayesian model selection revealed model 1 as being superior to all other models, as indicated by a protected exceedance probability 24 of 0.9471 compared to those from other models: 0.0072 (model 2), 0.0407 (model 3) and 0.0049 (model 4) in EPIK; the probabilities from EPI were 0.9957 (model 1), 1.5217e-04 (model 2), 0.0041 (model 3) and 5.2191e-05 (model 4). Hence, a bidirectional model, involving task-related modulations of connection between visual and motor areas, can be considered the best model. This finding has been confirmed by both the EPI, as well as the EPIK data.
In the next step, the accuracy with which the parameters were estimated was compared between the EPI and EPIK sessions. This was performed separately for every fixed connection (A-matrix) as well as for task-related changes in effective connectivity (B-matrix). In particular, using an F-test, it was tested whether the variance of DCM parameters across different scanning sessions was significantly smaller when data were acquired with EPIK as compared to EPI. The coupling parameters of the intrinsic connection between the hand area and the right visual area had a significantly smaller variance for EPIK compared to EPI (F(13,13) = 4.8796 p > 0.05, Bonferroni corrected for multiple comparisons). For all other connections, no difference in variance was observed (Table 3, all F-scores larger than F(13,13) = 0.45834 and all p-scores approximated a value of 1). Similarly, for task-related changes in connectivity as reflected by B-matrix parameters, no significant differences in variance were observed (Table 3, all F-scores larger than F(13,13) = 2.7952 and all p-values larger than p = 0.22).

Discussion
The work presented here demonstrates the utility of the EPIK sequence in a visually-guided finger-tapping task at 3 T. The reconstructed images from a representative fMRI session verify that EPIK outperforms the standard functional sequence, EPI, in terms of imaging resolution and robustness against geometric distortions. The functional data also show that EPIK identified stronger BOLD amplitudes than EPI. Moreover, effective connectivity analyses using DCM indicate a similar performance of EPIK and EPI with regard to parameters that reflect task-induced connectivity changes between brain regions. However, coupling parameters describing intrinsic connections from the left motor to the right visual cortex were more reliably estimated with EPIK.
The imaging parameters of the two sequences (e.g. EPI and EPIK) were adjusted in a way that the maximum possible matrix size and the number of slices were used under the condition that the same FOV, TR and TE were applied without any acceleration techniques; a matrix size of 96 × 96 with 32 slices and a matrix size of 64 × 64 with 36 slices were achieved by EPIK and EPI, respectively. We also noted that if parameters such as TR are changed to facilitate a better spatial resolution of EPI, the same change will lead to an even better resolution of EPIK; that is, EPIK stays a step ahead. It is further noted that the smaller number of slices acquired in EPIK was a direct consequence of the improved spatial resolution. Nevertheless, the acquired 32 slices (with a slice thickness of 3 mm -as for EPI) were clearly sufficient to assess all the relevant brain regions involved in the visually-guided finger tapping task, since the EPIK sequence covered almost the whole brain from the parietal cortex to the cerebellum (see Results). In adjusting the sequence protocols, acceleration techniques such as parallel imaging or partial Fourier were not employed in this study. This was done to avoid the deleterious effects thereof on the evaluation of the sequence performance, as the use of acceleration techniques would have made it more difficult to unambiguously attribute advantages and disadvantages to each sequence. Despite the more complicated reconstruction scheme, EPIK images were reconstructed without any degradation of image quality and exhibited enhanced spatial resolution compared to those from EPI.

Brain Region
Cluster size Side MNI-Coordinates T-score    Table 3. F-scores and p-values testing for lower variance in EPIK compared to EPI. Tests were performed separately for the connectivity parameters of the fixed connections in the DCM-model (as given by the DCM A-matrix). Connection directionality is indicated from rows to columns, i.e. row labels indicate a connections source region, whereas column labels indicate the target brain region. The connection with a significant lower variance for EPIK is highlighted in bold. All p-values are corrected for multiple comparisons using a Bonferroni correction. (2019) 9:10978 | https://doi.org/10.1038/s41598-019-47341-y www.nature.com/scientificreports www.nature.com/scientificreports/ Essentially, EPIK shortens the effective readout time per k-space traversal by only applying the multi-shot scheme for the periphery of k-space. For the reconstruction of EPIK images, the peripheral k-space regions are completed by sharing the data from neighbouring scans. This repeated use of the k-space periphery may introduce an autocorrelation. However, in EPIK, only small parts of the k-space periphery are correlated in a limited number of scans. Importantly, this correlation is only given for the high spatial frequencies containing edge information. In contrast, keyhole data are unique to each shot. The feasibility of using EPIK for fMRI at 3 T has been previously demonstrated in our earlier work 6 showing that the temporal stability of EPIK, represented in temporal signal-to-noise ratio (tSNR), was comparable to that of EPI, and thus detection of BOLD responses was not significantly reduced for EPIK. Furthermore, the capability of EPIK in tracking the dynamic BOLD signal changes was investigated with actual functional data (16-subject data) as well as the BOLD simulations using the established MRI simulator, JEMRIS (Jülich Extensible MRI Simulator) 25 . Indeed, the simulation results demonstrated that the signal behaviour of the 'BOLD' response, as reproduced by EPIK, showed a higher degree of agreement with the input vector than did EPI. In addition, the autocorrelation issue has also been thoroughly investigated in our recent study 7 . In that study, it was shown that the autocorrelation effect induced from the EPIK reconstruction scheme does not have a significant impact on the detection of BOLD signals. Moreover, it has also been shown that the comparable multi-shot EPI method (i.e. three-shot EPI) had a substantially reduced performance in detecting the BOLD signals when compared to single-shot EPI and EPIK 6,7 . This was mostly due to the fact that the central k-space is also sparsely sampled in multi-shot EPI, leading to a considerable magnification of physiological noise resulting from the shot-to-shot instabilities. As a result, multi-shot EPI has been shown to have a significantly reduced tSNR. Therefore, in order to be feasible for use in dynamic MR applications, multi-shot EPI methods require additional correction for physiological noise.
In this work, a functional study with a visually-guided finger-tapping task has been performed using the EPIK sequence in direct comparison with a single-shot EPI sequence that is commonly used as the standard protocol for fMRI. The functional results obtained show that stronger sensitivity in the detection of BOLD activation was observed in EPIK. This was observed in the area involving the primary motor, as well as the supplementary motor cortex. The stronger activation was mostly due to the reduction of the partial volume effects in EPIK arising from the increased image resolution 26 . More specifically, a higher image resolution allows a more specific localisation of BOLD signals from activated voxels by minimising artefacts from neighbouring, non-activated regions. The actual echo spacing of each phase encoding line in EPIK was 0.890 ms. However, due to smaller number of readout lines in each shot of the EPIK scheme, the effective echo spacing required for each phase encoding line is reduced to half, which is 0.445 ms (i.e. 0.890/2); that is, the matrix size was 96 and each shot acquired 48 lines. This is shorter than that of EPI (0.510 ms) and as a consequence, it was demonstrated that EPIK was less prone to the susceptibility artefacts existing in the prefrontal area and fourth ventricle as shown in Fig. 3. As a consequence, the recovered signals (i.e. reduced susceptibility artefacts) in such an area can have a contribution to the stronger BOLD activations in a related fMRI study probing those regions.
The benefits of increased sensitivity in detecting BOLD activation and the higher spatial resolution not only affects the standard BOLD amplitude analyses but may also be beneficial for modelling approaches based on BOLD signals. DCM infers neural states and interactions between brain regions on the basis of measured BOLD signals. Increased image resolution and a less distorted, and hence more robust signal, due to reduced partial volume effects may allow a more reliable and efficient estimation of model parameters. This view is supported by the results of the present study. The overall connectivity pattern revealed by DCM was the same for EPI and EPIK. DCM analyses for both imaging sequences identified a bidirectional model, involving task-related modulations of connections between visual and motor areas as the best model. Yet, the hypothesis that the higher spatial resolution provided by EPIK might allow a more consistent and more reliable estimation of coupling parameters was confirmed for one of the connections investigated. Overall, parameter estimates based on EPIK were at least as robust as those observed with EPI. Moreover, significantly more robust parameter estimates were found from the EPIK data for intrinsic couplings from the left motor cortex to the visual cortex in the right hemisphere. This pattern is consistent with the findings from our analysis of BOLD amplitudes where EPIK, as compared to EPI, revealed stronger BOLD signals in the right occipital lobe, suggesting that brain regions that benefit from a higher spatial resolution due to EPIK with regard to BOLD amplitudes will also allow a more reliable modelling of coupling parameters.
The effects of local susceptibility gradients, which could effectively push the gradient echoes in the EPI train out of the sampling window 27 , need to be considered in EPIK as it shortens the total duration of the sampling window. Employing the formula given by Deichmann et al., the echo time modified by the local susceptibility gradients, TE' is given as follows: where , t and G SP are the gyromagnetic ratio, echo spacing time and local susceptibility gradient, respectively. The formula suggests that in certain brain regions where the G SP has a positive value, the effective echo time, TE' , is presented as an increased value of the nominal TE and hence, it leads to signal drop out. A typical case of this can be seen in the frontal region. As can be seen from the formula, for a given positive G SP , this TE change becomes bigger when echo spacing time ( t) is bigger. As calculated in this work, since the echo spacing of EPIK is shorter than that of EPI, it is expected that the TE' of EPIK is always smaller than that of EPI in such brain regions and hence, has a reduced signal drop out. This has been experimentally verified by the in vivo data (see Fig. 3).
Moreover, although the effective echo spacing is shorter in EPIK, as its matrix size (96) in this work is larger than that of EPI (64), the total sampling window of EPIK is not shorter. Considering the TE (30 ms) (2019) 9:10978 | https://doi.org/10.1038/s41598-019-47341-y www.nature.com/scientificreports www.nature.com/scientificreports/ and the effective echo spacing of each method, the sampling window of EPI is given as 13.68 ms (i.e. 30 ms − 0.510 ms × 32) < TE < 46.32 ms (i.e. 30 ms + 0.510 ms × 32) and for EPIK is given as 8.64 ms (i.e. 30 ms -0.445 ms × 48) < TE < 51.36 ms (i.e. 30 ms + 0.445 ms × 48), respectively. This calculation indicates that the employed EPIK sequence has a wider sampling window size (10.08 ms wider) and therefore, was able to cover more brain regions affected by various degrees of susceptibility gradients (G SP ) than the employed EPI.
The present work employed non-accelerated EPI and EPIK to avoid the complicating effects of acceleration techniques. The use of acceleration techniques (e.g. parallel imaging, partial Fourier or multi-band techniques) in EPIK is as straightforward as in EPI 6,7,28 . As the acquisition efficiency increases by combining the acceleration approaches introduced above, the use of EPIK method could potentially be extended to fMRI studies aiming at columnar resolution and/or sub-second image acquisition. Importantly, the advantages arising from these acceleration techniques are expected to accrue equally for both EPI and EPIK, as indeed are the disadvantages. However, the application of EPIK for the task-based BOLD and the resulting connectivity measurement have not been performed yet. Therefore, this work is focused on the performance evaluation of both EPI and EPIK methods without any acceleration conditions. Both EPI and EPIK would benefit equally from the use of acceleration techniques such as multiband excitation.
Furthermore, in high-resolution EPI, TE will be necessarily increased for a given set of parameters and therefore EPI will underperform compared to EPIK. That is, under these circumstances, a longer acquisition window accumulates more errors caused by field inhomogeneities, which may lead to unacceptable image quality for high-resolution applications. The claim of higher spatial resolution for EPIK may, at first sight, seem contradictory for a method where less time is spent acquiring the peripheral k-space lines. This is, however, manifestly not the case. First, the PSF of EPIK was shown to be sharper than that of EPI, which suggests that EPIK will have less blurring artefacts and thus a better spatial resolution than EPI 4 . This has been verified by the PSF simulation for the cases when time-series data are stationary as well as when dynamically varying. This, in turn, implies that EPIK is superior to EPI in localising the functional signals. Next, considering the fact that the haemodynamic response changes quite slowly, it is not always necessary to update the peripheral k-space at a high rate for high-resolution mapping. As long as the peripheral k-space is updated more quickly than the haemodynamic response changes, the spatial extent of the functional signals will be faithfully captured. In EPIK, the peripheral k-space update rate is given by 1/(TR · number of shots). In our study, it is 1/6.6 s (i.e. 0.15 Hz), which is much quicker than the period of haemodynamic response changes (1/32 s ≃ 0.03 Hz). The peripheral k-space update rate of EPIK in the present study also suggests the use of EPIK for resting-state fMRI as its target functional signals fluctuate very smoothly (<0.1 Hz) 29 . In event-related fMRI, the haemodynamic response changes are not, in general, as smooth as in blocked paradigms. However, if a shorter TR is used to further increase the peripheral update rate, then a utility of EPIK for event-related fMRI can be also expected. Nevertheless, the application of EPIK for resting-state and event-related fMRI needs to be carefully explored in future studies.

conclusions
In order to evaluate the performance of EPIK in terms of BOLD detection and functional connectivity, the application of EPIK to a visually-guided finger-tapping paradigm has been demonstrated here at 3 T. The performance of the EPIK method was evaluated by inspecting the quality of the reconstructed images, as well as the functional quantities obtained, and comparisons were made with those from a standard, single-shot EPI method. The reconstructed images from a representative fMRI session verified that EPIK yielded higher resolution images than EPI, with a slight decrease in the number of slices acquired. Furthermore, a substantial reduction of geometric distortions, arising from susceptibility differences, was observed in EPIK compared to EPI. The analysis of functional data revealed that when directly compared to EPI, EPIK yielded stronger BOLD responses in the primary and supplementary motor areas as well as in the cerebellar regions and in the visual cortex. This indicates that EPIK was especially sensitive to BOLD signal changes in the core regions of the network. The analysis of effective functional connectivity between the visual and motor areas suggests that EPIK can be also used in functional connectivity studies and may allow for a more robust estimation of coupling parameters. Overall, it has been shown that EPIK performs better than EPI and can be one of the methods of choice for fMRI.


s2fieldsofstudy: Biology Medicine

text:
Melatonin MT2 receptor agonist IIK-7 produces antinociception by modulation of ROS and suppression of spinal microglial activation in neuropathic pain rats

Background In recent years, several melatonin (MLT) receptor agonists have been approved by FDA for the treatment of sleep disorders and depression. Very few studies have shed light on their efficacy against neuropathic pain (NP). IIK-7 is an MT-2 agonist known to promote sleep. Whether IIK-7 suppresses NP has not been reported, and the signaling profile is unknown. Objective To investigate the effect of melatonin type 2 receptor agonist IIK-7 on partial sciatic nerve transection-induced NP in rats and elucidate the underlying molecular mechanisms. Methods NP was induced by the PSNT in the left leg of adult male Wistar rats. On post-transection day 7, rats were implanted with intrathecal (i.t) catheter connected to an infusion pump and divided in to four groups: sham-operated/vehicle, PSNT/vehicle, PSNT/0.5 μg/hr IIK-7 and PSNT/0.5 μg IIK-7/1 μg 4-p/hr. To test the MT-2 dependence on IIK-7 activity, the animals were implanted with a single i.t catheter and injected MT-2 antagonist 4-Phenyl-2-propionamidotetralin (4-p) 20 mins prior to IIK-7 injection on day 7 after PSNT. The antinociceptive response was measured using a mechanical paw withdrawal threshold. Activation of microglial cells and the expression of NP-associated proteins in the spinal cord dorsal horn was assessed by immunofluorescence assay (IFA) and Western blotting (WB). Reactive oxygen species (ROS) scavenging ability of IIK-7 was evaluated by using bone marrow-derived macrophages (BMDM). Results Treatment with the MT-2 agonist IIK-7 significantly alleviated PSNT-induced mechanical allodynia and glial activation along with the inhibition of P44/42 MAPK, HMGB-1, STAT3, iNOS and casp-3 proteins. Conclusion IIK-7 attenuates NP through the suppression of glial activation and suppression of proteins involved in inflammation and apoptosis. MT-2 receptor agonists may establish a promising and unique therapeutic approach for the treatment of NP.

Introduction
Neuropathic pain (NP) is a chronic pain condition resulting from damage or disease to the somatosensory system and is difficult to diagnose, with limited treatment options. In addition, modern lifestyles and chronic diseases have significantly increased the number of new cases. Some of the common conditions that are known to damage the somatosensory nervous system include HIV infection, chemotherapy, leprosy, diabetes, syphilis, myocardial ischemia, amputation, alcoholism and surgery. 1 Some of the USFDA-approved firstand second-line treatment options for NP include tricyclic antidepressants and calcium channel α2-delta ligands, with opioids as a last resort for the pain management. 2 Nevertheless, insufficient pain relief, addiction, tolerance and severe side effects are some of the limitations that necessitate novel therapeutic interventions. Collective evidence has proven antinociceptive effects of MLT and its receptor agonists against nociceptive and NP in preclinical models, and some of the approved MLT agonists for the treatment of sleep disorders are known to have excellent safety and tolerability. 3 In recent years, several MLT receptor agonists like Ramelteon and Tasimelteon were approved for the treatment of sleep and depressive disorders and several others have entered clinical trials including TIK-301 and Piromelatine. 4 Although MLT receptor agonists are approved for the treatment of sleep and depressive disorders, none of them have been yet approved for the treatment of NP. However, many promising pre-clinical findings suggest that MLT and its receptor agonists have a strong potential in managing chronic pain conditions. [5][6][7][8][9] Recent evidence shows that there is a relation between sleep and NP. One recent study concluded that about 80% of patients suffering from NP have sleep disorders. 10 It has also been reported that sleep deprivation can greatly reduce the pain thresholds along with signs of depression and anxiety which can aggravate pain. 11 It is also known that sleep disturbances can greatly increase the risk of developing chronic pain. 12 Recently, Gobbi et al demonstrated that analgesic properties of MLT are mediated specifically by MT-2 receptors by using MT-2 antagonists. 13,14 However, the receptorindependent mechanisms underlying the antinociceptive actions of the MT-2 agonists are not explored so far. IIK-7 is a tetracyclic analog of MLT that specifically binds to MT receptor. 15 This agonist is known to have 90folds more selectivity toward MT2 receptor (pKi =10.30) subtype over MT1 (pKi =8.34) with a 6-fold higher affinity toward MT2 receptor subtype than MLT. 16 IIK-7 is known to promote sleep and protect the cells from electromagnetic stress like MLT. 17,18 However, to date, there are no reports on the anti-nociceptive properties of this agonist. In the present study, we for the first time assessed the anti-nociceptive effects of the selective MT-2 agonist IIK-7 in partial sciatic nerve transection (PSNT) rats, and, we studied the underlying molecular mechanisms, involving glial cells activation and signaling pathways.

Methods
The protocols used in our study are reviewed and approved by the Animal Care and Use Committee of the National Defense Medical Center and comply with the guidelines given by National Institute of Health Guide for the Care and Use of Laboratory Animals. Male Wistar at 7 weeks of age (BioLASCO Taiwan Co., Ltd., Taiwan) were housed (2 per cage) with soft bedding material on a 12 hrs night/day cycle with unrestricted access to food and water. All efforts were taken to minimize the animal number and suffering.

Establishment of the neuropathic pain animal model & intrathecal catheterization
PSNT is accomplished according to previously reported procedures. 19 In the PSNT rats, the left sciatic nerve was carefully exposed till mid-thigh level, and a prolene 7-0 ligature was inserted through the midpoint of the nerve just cranially to the branch running to the musculus biceps femoris, resulting in half of the nerve being transected in the ventrocranial direction up to the ligature. In the sham group, the left sciatic nerve was exposed and then the wound was closed with sutures. From operation day to day 7, the paw withdrawal thresholds are measured in sham-operated rats and PSNT rats to mechanical stimulus. Rats presenting any sign of motor deficit were omitted from the study. On day 7 after PSNT, intrathecal catheter/osmotic pump is implanted under 2-2.5% isoflurane anesthesia. 20,21 The intrathecal catheter was constructed as described previously. 22 The rat's cisternal membrane is carefully punctured to insert a polyethylene catheter (PE 10 tubing, 8.0 cm) and threaded caudally to reach the lumbar enlargement of the spinal cord. The rostral end of the catheter was exteriorized at the top of the head, and the wound closed with sutures. IIK-7 (Santa Cruz Biotech, Dallas, Texas) is first dissolved in 4% DMSO, then diluted with 45% w/v (2-hydroxypropyl)beta-cyclodextrin to improve solubility. To test the dose response, a single catheter was implanted in animals (n=6) and various doses of drug are intrathecally administered through a syringe (10, 100, 200, 400, 500 and 5000 ng) 7 days post PSNT. After drug administration, the catheters were flushed with 15 μL saline and 30 mins post-injection the animals are randomly assigned to undergo paw withdrawal threshold test. For intrathecal infusion, an osmotic pump (flow rate 1 μL/hr; ALZET, Cupertino, California) filled with vehicle (drug diluent), MT-2 agonist IIK-7 (0.5 μg/hr), MT-2 antagonist 4-p(Tocris Biosciences Bristol UK) (1 μg/hr)/IIK-7(0.5 μg/hr) (all at 1/μL/hr, n=6 in each group) was connected to the intrathecal catheter. The pumps were installed subcutaneously, and the wound was closed with silk sutures. Rats that showed neurological deficits after intrathecal catheterization were eliminated from the study. To evaluate the efficacy of the drug or drug in combination with antagonist, the paw withdrawal threshold is measured from day 8 to day 13 after osmotic pump installation.
To test the effect of pretreating antagonist prior to the agonist, a single intrathecal catheter is implanted in rats as described above (n=6 in each group) and injected with 10 μL saline followed by the injection of vehicle, IIK-7 (5 μg/ 10 μL) or 4-p (10 μg/10 μL) followed by a single injection of IIK-7 (5 μg/10 μL) 20 mins post-injection of antagonist. After drug administration, the catheters were flushed with 15 μL saline and 30 mins post-injection the animals are randomly assigned to undergo paw withdrawal threshold test from day 8 to day 11.

Behavior test for tactile allodynia
In this observer-blinded experiment, the sensitivity of the plantar region was measured using an automatic von Frey Dynamic Plantar Aesthesiometer (Ugo Basile, Comerio, Italy). Rats were individually placed in a plastic cage (25 cm long ×10 cm wides ×14 cm high) with a wire-meshed floor and acclimatized for 20 mins before each session of testing and then a paw withdrawal response was induced by gradually increasing the force using a blunt-end metal filament (0.5 mm) fixated on the mid-plantar surface of the hind paw. The force was primarily below the detection threshold, and then was increased from 1 to 50 g in 1 g steps over 25 seconds, then maintained at 50 g for another 10 seconds. The mechanical withdrawal threshold was taken as the force required initiating hind paw reflex and was recorded as the mean of 3 measurements at 2 mins time intervals.

Spinal cord preparation and Western blotting analysis
After the behavioral testing, all the rats were euthanized by exsanguination under anesthesia with isoflurane (Abbott Laboratories Ltd., Queenborough, Kent, United Kingdom), and the left dorsal quadrant portion of the lumbar spinal cord enlargement was detached and stored at −80°C till usage. The spinal cord samples were homogenized in icecold 1X RIPA lysis buffer (Temecula, CA) using an ultrasonic cell disruptor (Misonix, Inc. USA) and then the homogenate was centrifuged at 13,000 RPM for 30 mins at 4°C. The supernatant was collected and its protein concentration was determined using the Bradford protein assay. The proteins were denatured by heating at 90°C for 10 mins in an equal volume of reducing sample buffer and subjected to separation using 12% SDS-PAGE followed by protein transfer on to a PVDF membrane (Pall, Ann Arbor, MI, USA) and blocked using 5% skim milk in TBST

Bone-marrow-derived cell culture and ROS evaluation
Murine bone-marrow-derived macrophages (BMDMs) were generated by flushing bone marrow cells from femurs and tibias of 6-10-week-old BALB/c mice. These cells were cultured for 5-6 days in RPMI (Gibco) containing 10% FBS and 10 ng/mL recombinant mouse M-CSF (Peprotech, Rocky Hill, NJ, USA). To check the ROS scavenging ability of IIK-7, the cultured BMDM cells 4×10 5 cells are seeded in 6-well plates and divided into four groups. The first group is left without any treatment (Mock), the second group is treated with IIK-7 (25 ng) and the third group is treated with PAM3CSK4 (5 μg/mL). The fourth group is pre-incubated for 30 mins with IIK-7 followed by the addition of PAM3CSK4 with additional incubation for 12 hrs. Finally, DCFHDA is added to all four groups and readings are obtained at FL-1 channel using a flow cytometer.

Immunofluorescence assay
For the spinal cord IF, rats were deeply anesthetized with isoflurane and perfused with 0.15 M phosphate buffer (PB, pH 7.2) followed by 4% paraformaldehyde in PB. The dorsal quadrant of the lumbar spinal cord enlargement was detached and embedded in optimal cutting temperature compound until usage. The samples were sectioned (10 μM) and air-dried for 30 mins and first washed with PBS (3x) and then treated with acetone for 2 mins followed by a wash with PBS (3x). The slides were incubated overnight with primary antibody, Alexa Fluor® 488 antirat CD11b/c (Biolegend, San Diego, CA, USA, CAT# 201812) (1:200) and washed with PBS (3x) followed by the addition of secondary antibody (1:100 dilution) and DAPI. The slides were imaged using a fluorescence microscope.

Statistical analysis
Two-way mixed ANOVA analysis was used to study the effect of time on the different drug-treated groups, as well as interaction between time and groups. Two-way repeated measures ANOVA was used for the analyses of main effect of time, main effect of paws and interactions between time and paws. Withdrawal threshold data were converted to maximal possible effect (%MPE), according to the formula: %MPE=[(postdrug threshold-post-injured baseline threshold)/(cutoff threshold-post-injured baseline threshold)] ×100. One-way ANOVA was used to detect the group differences where no time course was involved (eg, in vitro test in BMDM for ROS scavenging). Bonferroni post hoc (for all comparisons) and Bonferroni's multiple comparison tests (for making a restricted set of comparisons) were performed for all pairwise comparisons. GraphPad Prism version 5.01 (GraphPad Software, San Diego, CA) was used to analyze the data. P<0.001 was considered statistically significant. Figures were generated using GraphPad Prismversion 5.01 (GraphPad Software, San Diego, CA). Data are expressed as mean ± S.E.M.

IIK-7 attenuates sciatic nerve-transectioninduced pain
The anti-nociceptive effect of intrathecal MT-2 agonist IIK-7 was examined in the PSNT rats. After baseline measurement for seven days, IIK-7 was intrathecally administered (10, 100, 200, 400, 500 and 5000 ng), and then the PWT was measured after 20 mins. IIK-7 dosedependently reversed the mechanical allodynia induced by PSNT. The ED50 of intrathecal IIK-7 is 342 ng ( Figure S1). Figure 1 shows the temporal changes of left hind paw withdrawal to the plantar metal filament during infusion of drugs/saline (say 7 to day 13). There was no significant difference in the baseline of paw withdrawal threshold (PWT) in rats prior to nerve transection (depicted as day −1 in Figure 1). In the nerve-transected groups, mechanical allodynia developed on the first day and the withdrawal threshold remained stable from 3rd day after nerve transection till day 7 ( Figure 1). The mean withdrawal threshold pre-operation was (mean ± SD) 40 ±2.0 g and decreased progressively to 20±2.0 g, on day 7 after PSNT. In contrast, PWT of transection/IIK-7,  IIK-7-4-p group were significantly higher than those of the transection/vehicle group on all day's post-surgery (day 8 to day13 in Figure 1). On 6th day post-surgery (Day 13), the PWT of ligation/IIK-7, IIK-7-4-p group were 33.1 g and 31.7 g, significantly higher than those of the transected/vehicle group (21.2 g) ( Figure 1). Additionally, we examined if 4-p continuous infusion had any effect on the nociceptive response; we found no significant difference from that of control PSNT groups (data not shown). Next, we further explored, if pre-treatment of rats with a single injection of MT-2 antagonist 4-p can suppress the anti-nociception of IIK-7 ( Figure 2). The analgesic effects of IIK-7 were not antagonized reversed when co-infused or pre-treated with MT-2 antagonist 4-p for four days prior to IIK-7 single injection, respectively (Figures 1 and 2). At a tested dose of 0.1-5 μg, melatonin alone did not display any anti-allodynic effects (data not shown).

IIK-7 inhibited the expression of p 44/42 MAPK, HMGB-1 and iNOS expression induced by partial sciatic nerve transection
To determine the expression of p 44/42 MAPK, HMGB1 and iNOS in PSNT group and IIK-7-infused group, the spinal cord protein lysates were analyzed by Western blotting (Figure 3). The results showed that the levels of 44/42 MAPK, HMGB-1 and iNOS proteins were significantly higher in PSNT group in comparison to the Sham group. The expression of p 44/42 MAPK, HMGB-1 and iNOS significantly decreased in the IIK-7 infused group IIK-7 scavenge ROS generated in bonemarrow-derived macrophages in response to TL2 receptor ligand PAM3CSK4 The anti-oxidant properties of IIK-7 are examined in bonemarrow-derived macrophages (BMDM). The ROS generated in BMDM is quantified following the treatment with Toll-like receptor-2 (TLR2) ligand PAM3CSK4 and finally treated with redox-sensitive dye DCFDA. Significant elevation of cellular ROS (P<0.05) is observed in BMDM treated with PAM3CSK4 as compared to untreated controls Mock and IIK-7 ( Figure 4). Pre-treating BMDM 30 mins with IIK-7 significantly inhibited the elevation of ROS levels on treatment with PAM3CSK4, indicating the ROS scavenging capabilities in PAM3CSK4-treated cells thereby maintaining a redox-environment comparable to that of mock and IIK-7-alone treated macrophages ( Figure 4).

IIK-7 attenuates nerve-transectionevoked microglia activation and iNOS expression
In the dorsal horn of the spinal cord of vehicle-infused PSNT rats, ameboid shape (active, OX-42 cells) microglia with a strong green fluorescence was found ( Figure 5A). In contrast, in the animals infused with IIK-7, the morphology of the microglia remains in a ramified shape (inactive form) with a very weak fluorescence. Likewise, the expression of iNOS was significantly high in PSNT rats infused with the vehicle in comparison with IIK-7 infused groups ( Figure 6A). The iNOS immunoreaction intensity was significantly reduced in IIK-7-treated rats, showing very rapid effect on iNOS activation ( Figure 6B).  Treatment with IIK-7-attenuated PSNTinduced expression of STAT3 and cleaved casp-3 We further investigated the effect of PSNT on STAT3 and cleaved casp-3 expression. It is found that PSNT persistently increased the expression of STAT3 along with cleaved casp-3, an indicator for the initiation of apoptotic signaling. The PSNT rats infused with IIK-7 significantly decreased the expression of STAT3 and cleaved casp-3 suggesting that IIK-7 could suppress the immune responses in PSNT rats (Figure 3).

Discussion
In the present study, for the first time, we show that intrathecal infusion of the MT-2 receptor agonist IIK-7 relieved mechanical allodynia in hind paws of PSNT male Wistar rats at an ultra-low dose of 0.5 μg/hr (Figure 1). PSNT resulted in extreme sensitivity to mechanical stimulus as demonstrated by plantar filament tests (Figures 1 and 2). The co-infusion or pre-treatment of PSNT rats with selective MT-2 receptor antagonist 4-p did not significantly inhibit the analgesic potency of IIK-7. Previous studies have shown that pre-treatment with 10 μg of MT-2 selective antagonist 4-p, 30 mins prior to MLT administration suppressed MLT-induced increase of paw withdrawal threshold in SNL (sciatic nerve ligation)-induced NP. 23 In our study 10 μg 4-p administration did not show any significant impact on IIK-7's anti-nociception; therefore, it seemed that the anti-nociceptive effect of IIK-7 was irrelevant to MT-2 receptors. However, the receptor-independent free-radical scavenging mechanisms might play a key role in antinociception, and those have been demonstrated previously by several studies on MLT and its agonists. 19 The receptor-independent mechanisms through which MLT and its agonists expunge the radical scavenging involves the following: 1) through glutathione synthesis, 2) stimulation of antioxidant and inhibition of pro-oxidant enzymes thereby reducing oxidative stress, 3) detoxification of free radicals and maintenance of mitochondria membrane potential. 24   Oxidative stress is considered as one of the most important mechanisms in the development and maintenance of NP. 25 It can result in the hyperexcitability of nerves in the afferent nociceptors and central neurons triggering spontaneous impulses inside the axons and the DRG-producing NP. The mechanisms through which oxidative stress induces nerve damage or dysfunction include the ROS (Reactive oxygen species) and RNS (Reactive nitrogen species). ROS is known to activate/inhibit several crucial pathways that mediate pain such as activation of P44/42 MAPK, reduction of GABA release, initiation of pro-inflammatory cytokine from the glial cells and inhibition of γ-aminobutyric acid, which are key pain transmitters (Scheme 1). 25,26 Both p44 MAPK (ERK1) and p42 MAPK (ERK2) are distributed ubiquitously in the brain and spinal cord at high levels. Various extracellular stress resulting from nerve damage is known to activate P44/42 MAPK. 27 Inhibition of ROS is known to downregulate MAPK pathway in various animal models. 28 MAPK pathway is known to be activated as early as 10 mins post nerve injury in the dorsal horn and 12 hrs post-injury in the microglia through transcriptional modulation of genes involved in central sensitization. 29 One recent clinical trial has shown that MAPK inhibitor dilmapimod is efficient in relieving NP in patients with good tolerance at higher doses. 30 To this end, our findings herein show that the IIK-7 blocked PSNT-induced p44/p42 MAPK activation in the dorsal horn of the spinal cord, with the net result being pain relief. p44/42 MAPK activation is known to regulate the expression of several cytokines such as COX-2 and iNOS through transcriptional and translational modifications. 31 NOS (Nitric oxide synthase) produced during nerve damage can cause nitrosative stress by the production of NO (nitric oxide) and is known to have a significant role in nociception, neuroinflammation and analgesic tolerance, and its inhibition/knockdown is known to alleviate NP. 32 NO produced in neurons serves as an important signaling molecule by two isoforms of NOS: neuronal isoform (nNOS) and an inducible isoform (iNOS). Among both, iNOS is known to play a key role in the pathogenesis of neuroinflammation, through the prolonged synthesis of a highly uncontrolled quantity of NO that can be toxic for cells. MLT and its analogs are known to inhibit NOS through receptor-independent mechanisms. 33 In one recent study, Lin and colleagues have revealed that MLT's effect on NOS is unaffected by MT-2 & ROR α-specific drugs/agonists and further confirmed that MLT can suppress NP receptor-independently by the suppression on NOS. 6 In the present study, the rats treated with IIK-7 displayed markedly lower levels of iNOS expression compared with sham and PSNT-vehicle-infused rats in both Western blot and immunofluorescence assays (IFAs) (Figures 3 and 6). These results suggest that IIK-7 may attenuate NP by inhibiting iNOS activity. Recent evidence suggests that iNOS expression can potentiate ROS generation in macrophages. 34 Though emerging evidence suggests the crucial role of ROS in pain, the primary source for ROS generation in NP is still elusive. ROS is produced through various sources in the central nervous system (CNS) including the mitochondria, NADPH oxidase, nitric oxide synthase (NOS), etc. 35 One recent study by Park et al demonstrated that ROS production is confined to mitochondria of dorsal horn neurons in a rat L5 spinal nerve ligation (SNL) model. 36 Similarly, intradermal capsaicin injection is shown to induce ROS accumulation primarily in the dorsal horn neurons, indicating the chief role of mitochondrial ROS in nociception. 37 In another study, Gwak et al demonstrated that T10 spinal cord injury can increase the production of ROS in neurons and glial cells in the L4-L5 dorsal horn. 38 In addition, it was demonstrated that ROS causes NP and locomotor deficit through the activation of neuronal calcium/calmodulin-dependent protein kinase II (CaMKII) by phosphorylation. 38 Kallenborn-Gerhardt et al demonstrated that knocking out of NOX4, a ROS protein, alleviated nerve-injury-induced NP through the suppression of ROS levels in mice. 39 Recently, several studies have shown the efficacy of anti-oxidants like phenyl N-tert-Butyl-α-phenylnitrone (PBN), 5,5-dimethyl-pyrroline-N-oxide(DMPPO),4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) and vitamin A, C and E against various NP conditions. 40 To evaluate the ROS scavenging ability of IIK-7, bone-marrow-derived macrophages are used and ROS generation is triggered using Toll-like receptor-2 (TLR2) ligand PAM3CSK4. TLRs are key initiators of innate response and are triggered upon microbial attack or nerve injury. They are also known to play an important role in microglial activation, inflammation and the development of NP. 41 The ROS generated upon the treatment of BMDM with TLR2 ligand is detected by redox-sensitive dye DCFDA. Significantly elevated levels of cellular ROS (P<0.05) were observed in BMDM treated with PAM3CSK4 as compared to untreated control and IIK-7-treated cells. Pretreatment with 25 ng IIK-7 followed by treatment with PAM3CSK4 significantly reduced the ROS levels as evident from Figure 4. These results indicate that ROS rapidly accumulates in macrophages treated with PAM3CSK4, while IIK-7 pretreatment can scavenge ROS, and levels remain like those observed for untreated control.
ROS is known to serve as an important trigger for the activation of STAT3 by JAK2-dependent mechanism promoting the expression of inflammation-associated genes. 42 Studies have also shown that blockade of STAT3 can relieve NP. 43 In our present study, vehicle-infused PSNT rats showed significant over-expression of STAT-3 when compared to sham groups and intrathecal infusion of IIK-7 prevented the STAT3 activation induced by PSNT (Figure 3). STAT-3-induced apoptosis through caspase activation is one of the well-known mechanisms for neuronal inflammation. 44 In addition, caspase activation is also known to induce microglial activation and play a pivotal role in the pathogenesis of several neurodegenerative disorders including NP. 45 Casp-3 activation is one amongst the key indicators for apoptosis as is necessary for protein cleavage. Inhibition of casp-3 is known to suppress NP. 46 The casp-3 proteins are virtually inactive until they are cleaved by an initiator caspase upon apoptotic signaling and some recent reports suggest that NP increases the cleaved casp-3 levels. 47 Blocking casp-3 signaling pathway is also known to reduce the apoptotic neurons by the down-regulation of GAP-43 expression, another key regulator of NP. 46 Our results suggested that treatment with IIK-7 can significantly inhibit cleaved casp-3 ( Figure 3) displaying anti-apoptotic properties. Recently, it was demonstrated that Caspases signaling regulates microglial activation through a protein kinase C (PKC)-δ-dependent pathway. 48 Microglial cells are considered as the gatekeepers of NP and their activation can trigger the release of several neurotransmitters including glutamate and substance P, resulting in the release of inflammatory mediators, such as interleukin-11β (IL-1β), IL-12, ROS, iNOS, Tumor necrosis factor-α (TNF-α), prostaglandin E2, chemokines and adenosine triphosphate (ATP) thereby bringing other immune cells to the injured site and intensifying the inflammatory response. 49 In one recent study, Kim et al confirmed that NOX-2 ROS production is the crucial step in the induction of NP and demonstrated that ROS is required for pro-inflammatory gene expression in microglial cells. 50 Blocking microglial activity is an effective strategy in preventing NP. During the resting phase, microglial cells are small with long and thin fine structures; upon activation, they display an amoeboid morphology with large retracted structures. Microglial activation can be detected by OX-42 antibody immunocytochemical staining; vehicle-infused PSNT rat spinal cords in contrast to IIK-7-infused rats displayed enlarged areas of OX42 green immunofluorescence with a distinct morphology indicating their activated state ( Figure 5A). The OX42 immunoreaction intensity was significantly reduced in IIK-treated rats ( Figure 5B), thus showing a very rapid effect on microglial activation. HMGB1 is a nuclear protein, but upon encountering mild oxidative stress it translocates to the cytoplasm with subsequent activation of autophagy. 51 Additionally, several studies reported a spontaneous increase of ROS because of HMGB1 release through the expression of iNOS. 52 Some evidence indicated that effects of HMGB1 in NP are mediated via its activation of toll-like receptor-4. 53 The release of HMGB1 in the neuronal cells of CNS is believed to regulate the endogenous inflammatory components and influence the adjacent neurons and microglial cells. 54 It has been previously reported that the exogenous administration of HMGB-1 induces excitability of primary afferent neurons contributing to NP after nerve injury. 55 Inhibition of HMGB-1 is known to relieve hyperalgesia and allodynia after nerve injury. 55 Our present study has conclusively found that HMGB1 is actively released upon nerve injury induced by PSNT ( Figure 3) and infusion of IIK-7 significantly blocked the expression of HMGB-1.

Conclusion
In conclusion, we present several lines of evidence to show that MLT MT-2 agonist IIK-7 play an important role in modulating nerve transection-induced pain and neuronal inflammation. First, continuous intrathecal infusion of IIK-7 attenuates PSNT-induced mechanical allodynia. Second, the anti-nociceptive action is not antagonized either with co-infusion or pre-treatment with MT-2 antagonist 4-p, suggesting MT-2 receptor-independent mechanism of anti-nociception. The mechanisms underlying the influence of IIK-7 on NP are yet unclear. IIK-7 may inhibit spinal microglial activation and attenuate NP. Further investigation in determining the underlying mechanisms of IIK-7 anti-nociception may be beneficial.

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