Brain atrophy patterns in multiple sclerosis patients treated with natalizumab and its clinical correlates

Abstract Background Multiple sclerosis (MS) is defined as a demyelinating disorder of the central nervous system, witnessing over the past years a remarkable progress in the therapeutic approaches of the inflammatory process. Yet, the ongoing neurodegenerative process is still ambiguous, under‐assessed, and probably under‐treated. Atrophy and cognitive dysfunction represent the radiological and clinical correlates of such process. In this study, we evaluated the effect of one specific MS treatment, which is natalizumab (NTZ), on brain atrophy evolution in different anatomical regions and its correlation with the cognitive profile and the physical disability. Methods We recruited 20 patients diagnosed with relapsing‐remitting MS (RR‐MS) and treated with NTZ. We tracked brain atrophy in different anatomical structures using MRI scans processed with an automated image segmentation technique. We also assessed the progression of physical disability and the cognitive function and its link with the progression of atrophy. Results During the first 2 years of treatment, a significant volume loss was noted within the corpus callosum and the cerebellum gray matter (GM). The annual atrophy rate of the cortical GM, the cerebellum GM, the thalamus, the amygdala, the globus pallidus, and the hippocampus correlated with greater memory impairment. As for the third and fourth years of treatment, a significant atrophy revolved around the gray matter, mainly the cortical one. We also noted an increase of the thalamus volume. Conclusion Atrophy in RR‐MS patients treated with NTZ is regional and targeting highly cognitive regions mainly of the subcortical gray matter and the cerebellum. The cerebellum atrophy was a marker of physical disability progression. NTZ did not accelerate the atrophy process in MS and may play a neuroprotective role by increasing the thalamus volume.


INTRODUCTION
Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) involving a complex combination of both demyelination and neurodegeneration (Kotelnikova et al., 2017). The most common course of the disease is relapsing-remitting MS (RR-MS), which represents 85% of MS forms (Lublin et al., 2014).
As for the clinical expression, cognitive dysfunction is a prominent feature of MS, occurring even in early stages of the disease (Oset et al., 2020) and has been reported during the pre-symptomatic phases as a potential revealing sign of the radiologically isolated syndrome (RIS) (Menascu et al., 2019). The radiological correlate of cognitive dysfunction is generally perceived through the atrophy pattern and its severity. Recent studies focused on gray matter (GM) analysis in MS, subcortical deep GM in particular, and demonstrated that it was correlated with cognitive dysfunction in MS patients, noticeable even in the earliest stages of the disease yet without consideration of the potential effect of the ongoing treatment (Eshaghi et al., 2018;Gilmore et al., 2009;Prins et al., 2015). The potential contributing role of treatment adds another layer of complexity to interpret factors leading to cognitive impairment and atrophy progression in MS (Sotirchos et al., 2020).
Even though such therapies are known to be efficient to control the disease's activity and disability's progression, its impact on the neurodegenerative process and its clinical expression in terms of cognition are mostly lacking (Compston & Coles, 2002).
Natalizumab (NTZ) is one among the therapies of RR-MS indicated mainly in forms presenting with high disease activity or aggressive evolution. Its efficacy is established in terms of reducing inflammation by preventing leucocytes from reaching the CNS (Polman et al., 2006). However, the effects of such an aggressive treatment on the neurodegenerative process in MS and its consequences upon the cognitive functions remain controversial (Alvarez et al., 2021;Preziosa, Rocca, Riccitelli, et al., 2020;Talmage et al., 2017). Studies examining atrophy evolution in MS patients treated with NTZ attributed it to the pseudoatrophy phenomenon due to the regression of inflammation in the white matter during the first year of treatment and was as a consequence considered as a further sign of treatment efficacy.
Nevertheless, these previous studies focused mainly on the white matter and specific structures such as the corpus callosum by measuring its index as a representative marker of atrophy in MS patients (Arpín et al., 2016). Lesion burden progression was also a subject of interest and showed no significant correlations with atrophy evolution and cognitive impairment in MS patients (Preziosa, Rocca, Riccitelli, et al., 2020). As for GM, findings about the effect of NTZ remain dispersed (Ciampi et al., 2017;Preziosa, Rocca, Pagani, et al., 2020;Preziosa, Rocca, Riccitelli, et al., 2020).
Thus, we chose to shed light on the effect of NTZ on the brain atrophy progression in RR-MS patients while focusing on both WM and GM structures and its correlates in terms of disability progression and cognitive impairment. We also aimed to investigate whether atrophy is a regional phenomenon that may potentially be considered as a biomarker of the disease progression. hospital. An MS-standardized protocol was used which included 5-mm slices obtained in axial T2, axial FLAIR, sagittal T1, sagittal FLAIR, and axial T1 before and after administration of gadolinium. T1-weighted sequence was used as an input for the image-processing pipeline.
Baseline-MRI (B-MRI) scan was available for all patients at the initiation of treatment with NTZ. Being part of the routine control, each patient underwent one MRI control per year in order to rule out a progressive multifocal leukoencephalopathy and the occurrence of new active lesions. We compared the baseline scan with the MRI scans at two different timepoints (at 2 years and 4 years of treatment) in order to ascertain the development of atrophy.

Brain segmentation
Segmentation of brain structures based on each subject T1-weighted MRI was performed automatically using auto-mated recon-all FreeSurfer processing pipeline (version 5.3.0; http://surfer.nmr.mgh.harvard.edu) to obtain the cortical surface reconstruction and tissue-class segmentation boundaries. No manual editing was performed to keep methods as automated as possible, and scans with segmentation errors/failures were excluded (Yaakub et al., 2020). The quality of brain segmentation was assessed by a neuroimage data processing expert and then by two neurologists  (2002,2004). FreeSurfer first affinely registers each T1-weighted MRI to a shared common space using MNI305 (Collins et al., 1994) atlas. Next, the variation in the white matter intensity is quantified to remove the B1 bias field estimation. A skull stripped algorithm is then applied using a deformable template model .
Following this nonlinear volumetric the MNI305 atlas, a simple label propagation algorithm is used to propagate the labels of the image template in the common atlas to the target-registered T1-weighted image (Fischl et al., 2002. The FreeSurfer-generated volumes are then measured following this step. Assessment of the progression of brain atrophy was based on the comparison of volumes of different structures at different timepoints of the treatment period with NTZ (at 2 years and 4 years of treatment) ( Figure 1).
For each anatomical structure, we computed the annual atrophy rate (AAR) using two consecutive timepoints during NTZ treatment period to investigate the progression of atrophy over time ( Figure 1). A p-value less than .05 was considered statistically significant in all of the different adopted statistical tests.

Ethics
Since the treatment and follow-up were managed according to usual clinical practice, ethics committee approval was not required. Nevertheless, informed oral consent was obtained from all participants to undergo the cognitive evaluation.

Population of study
Twenty patients were finally included in our study among the 25 eligible MS-patients undergoing NTZ treatment. We excluded one patient who was epileptic with history of two status epilepticus and four patients with distorted segmentation results in order to avoid errors in volume estimation.

Characteristics of the population of study at baseline
Baseline findings are summarized in Table 1.
One patient was treatment-naïve when NTZ was initiated. The choice of induction therapy was based on his age at disease onset (<18 years old) and the aggressive form of the disease (>2 relapses during the first year).

Clinical progression of the disability
After initiation of treatment, the ARR decreased from 1.6 ± 1 relapse/year before the initiation of NTZ to 0.2 ±1 relapse/year after switching to NTZ (p = 0.325).
The mean F-EDSS score was of 3.8 ± 2.26 points [1, 6.5] which was higher than the mean B-EDSS. Yet, the difference remains statis-

Cognitive and psychiatric assessment
The main findings of the different cognitive tests and questionnaires are summarized in Table 2.
The SDMT score correlated with the cerebellar function at baseline (p = 0.028, r = − 0.506) and during the final assessment (p = 0.002, r = −0.789). As for the SPART score, it was correlated with the baseline thalamic volume (p = 0.008, r = −0.849).  Table 3, the volume of corpus callosum decreased significantly (p = 0.032) along with the cerebellum GM volume (p = 0.047). The AAR of the thalamus, total GM, cortical GM, GP, and the hippocampus correlated with SPART test score ( Figure 4). As for the ARP of the cerebellar function, it was negatively correlated with AAR of the cerebellum GM (p = 0.033, r = −0, 909).

-During the third and fourth years of treatment (T2-T1)
Regarding the GM, significant atrophy was noted (p = 0.038) mainly due to cortical atrophy (p = 0.005). As for the subcortical GM, it was globally stable. It is noteworthy that the thalamus volume increased significantly (p = 0.016).
The AAR of the cortical GM and the cerebellum GM during the third and fourth years of treatment were negatively correlated with the QPC score. Categorical fluency score was positively correlated with the AAR (T2-T1) of the putamen and the thalamus (Figure 4).
While comparing the AAR of the two first years of treatment and the third and fourth years, we noted that the CN and the amygdala were subject to significant progression of annual atrophy rate (Table 3).

DISCUSSION
In our study, RR-MS patients treated with NTZ showed a regional atrophy pattern that progressed dynamically during the 4-year treatment period. The two first years of treatment mainly reflected a reduction in the CC volume and the cerebellum GM. The third and the fourth years of treatment were characterized with the reduction in GM volume mainly the cortical one. As for the cognitive profile, memory impair- In bold are the p values which are statistically significant, meaning a p value < 0.05.

F I G U R E 4
Correlations (Pearson's coefficient "r") between the AAR of different brain regions and cognitive assessment findings. The "*" indicates a p value < .05. Abbreviations: AAR1, AAR during the first two years of NTZ therapy; AAR2, AAR during the third and fourth years of treatment; SPART-D, SPART delayed recall; SPART-I, SPART immediate recall cortical GM, subcortical GM including the thalamus and the GP during the first two years of treatment. It was also associated to the AAR of the hippocampus and the amygdala.

The atrophy pattern: A dynamic feature
In accordance with previous studies ( However, the pseudo-atrophy seems to be an overlapping phenomenon unrestricted to white matter. In fact, demyelination is seen in the cerebellar GM five times more than in the white matter (Gilmore et al., 2009). Meningeal inflammation in the deep folia accommodates a persistent inflammation in direct contact with the cortical GM of the cerebellum (Howell et al., 2011), which explains the decrease in its GM volume due to the anti-inflammatory effect of the treatment as demonstrated in our study.
Regarding supra-tentorial GM, our study demonstrated a significant loss in the cortical GM volume (p = 0.005) and, as a consequence, the total GM (p = 0.038) during the third and fourth years of treatment. At first glance, such finding might be considered atypical since NTZ is known to slowdown the atrophy of GM beyond the two first As for subcortical GM, it remained globally stable with a significant increase in the thalamus volume which may indicate a neuroprotective effect of NTZ by acting on specific regions of brain that are highly implicated in the cognitive aspect in MS such as the thalamus (Bisecco et al., 2021;DeLuca et al., 2015;Rojas et al., 2018;Schoonheim et al., 2015).
Regarding the dynamic aspect of atrophy, the CN showed a progression of the AAR (p = 0.008). Such fact may not be specific to MS patients treated with NTZ since it has already been established as an early characteristic marker of atrophy in the relapsing remitted form of the disease (Eshaghi et al., 2018). Such finding might be comprehensible considering that, on a histopathological level, the CN is one of the most affected deep GM regions by the demyelination process (Haider et al., 2014).

Cognitive mapping of MS patients treated with NTZ
Exploring the relationship between the volumes and AAR of different Regarding memory evaluation, our study highlighted that the AAR of GM, cortical GM, and specific regions of the subcortical GM along with the hippocampus were associated to SPART score (Figure 4). In fact, cortical GM includes the orbitofrontal cortex and the medial prefrontal cortex, which are functionally linked to the hippocampus via a network for memory formation, maintenance, and retrieval (Benarroch, 2013). Furthermore, the SPART score was also correlated with the baseline thalamic volume and with the AAR of the thalamus. Such finding lines with the currently sparse literature pinpointing the cognitive function of the thalamus in MS including specific areas such as the ventral anterior nucleus and ventral lateral nuclei (Bisecco et al., 2015(Bisecco et al., , 2021. Visuospatial memory seems to be altered in thalamus pathology, and it is explained partly by the disruption of the thalamo-frontal circuits and the connection between the medio-dorsal thalamus and the hippocampus (Parnaudeau et al., 2018). However, even though the AAR of the thalamus correlated with the SPART score, the fact that the thalamus volume increased significantly during the third and fourth years of treatment and that baseline volume was already associated to a greater SPART score could reflect that NTZ may have decelerated neurodegeneration within the thalamus. Thus, memory impairment might be just a sequela of the damage prior to the initiation of NTZ therapy or might be due to the neurodegenerative process of the disease going at its normal pace.
Our results also showed that SPART delayed recall test was correlated with the AAR of the amygdala (Figure 4), replicating the finding that the amygdala is linked to the visuospatial memory and psychosocial functioning in pediatric onset MS patients (Green et al., 2018). The increasing AAR of the amygdala, as shown in our study (p = 0.003), calls for a greater attention to this structure as it may particularly be vulnerable to the neurodegenerative process with great cognitive, emotional, and social impact.
As for the GP, its AAR correlated, as well, with the SPART score ( Figure 4) which is plausible given the intricate involvement of deep GM in memory. In fact, the GP is implicated in memory networks involving subsets of cortico-basal loops (Middleton & Strick, 2000).

Physical disability and the neurodegenerative process
The progression of the cerebellar signs was the key clinical feature that correlated with volumetric measures. It was correlated with the AAR of the GM of the cerebellum during the first year of treatment. Such finding emphasizes further the crucial role of the cerebellum in causing both physical and cognitive handicap in MS (Damasceno et al., 2014;Weier et al., 2014).
However, our study is not without limitations. The seemingly small sample size might be explained by the fact that Tunisia is characterized with a moderate prevalence of MS ranging from 9 to 20 per 100,000 habitants and by the adopted exclusion criteria (Yamout et al., 2020). Even though the sample size may not allow to draw definite conclusion about the impact of NTZ on atrophy progression, it provides preliminary data that calls for further investigations. The single timepoint assessment of the cognitive profile did not allow to better study the cognitive aspect and its progression since the initiation of NTZ.

CONCLUSION
Through this study, we analyzed a large number of clinical, neurocognitive, and MRI variables acquired at baseline and during the first 4 years of NTZ treatment. A robust and consistent finding is that atrophy affects white matter during the first 2 years of treatment and the GM later. A key finding to highlight is the increase in the thalamus volume, which may indicate a neuroprotective effect of NTZ. This study also allowed to map vulnerable highly cognitive regions involved in memory and executive function such as the cortical GM, the cerebellar GM, the deep GM mainly the thalamus, the GT, and the putamen. It emphasized the particular vulnerability of the caudate nucleus to neurodegeneration with an increasing atrophy rate during the third and fourth years of treatment.
This study also opens the door for future research that could conduct long-term follow-up of patients before and after the initiation of NTZ with larger samples and appropriate controls in order to better comprehend the supplemental impact of NTZ on atrophy progression.
By highlighting brain regions highly involved in cognition, our study provides insight into the potential role of these structures and the possibility to use them as biomarkers of the disease progression.

CONFLICT OF INTEREST
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

PEER REVIEW
The peer review history for this article is available at https://publons.