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DMSO Improves Motor Function and Survival in the Transgenic SOD1-G93AMouse Model of Amyotrophic Lateral Sclerosis

DMSO 투여된 근위축성 측삭경화증 SOD1-G93A 형질 변환 마우스 모델에서의 근육 기능과 생존 기간 증가 효과

  • Received : 2022.07.21
  • Accepted : 2022.08.25
  • Published : 2022.08.30
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Abstract

Dimethyl sulfoxide (DMSO) is commonly used as control or vehicle solvent in preclinical research of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) due to its ability to dissolve lipophilic compounds and cross the blood brain barrier. However, the biochemical effects of DMSO on the outcomes of preclinical research are often overlooked. In the present study, we investigated whether the long-term oral administration of 5% DMSO affects the neurological, functional, and histological disease phenotype of the copper/zinc superoxide dismutase glycine 93 to alanine mutation (SOD1-G93A) mouse model of amyotrophic lateral sclerosis. SOD1-G93A transgenic mice showed shortened survival time and reduced motor function. We found that administration with DMSO led to increased mean survival time, reduced neurological scores, and improved motor performance tested using the rotarod and grip strength tests. On the other hand, DMSO treatment did not attenuate motor neuron loss in the spinal cord and denervation of neuromuscular junctions in the skeletal muscle. These results suggest that DMSO administration could improve the quality of life of the SOD1-G93A mouse model of ALS without affecting motor neuron denervation. In conclusion, the use of DMSO as control or vehicle solvent in preclinical research may affect the behavioral outcomes in the SOD1-G93A mouse model. The effect of the vehicle should be thoroughly considered when interpreting therapeutic efficacy of candidate drugs in preclinical research.

DMSO (dimethyl sulfoxide)는 친유성 화합물을 용해하는 성질과 뇌혈관장벽(Blood-brain barrier)을 투과하는 화학적 특성으로 인해 근위축성 측삭경화증(amyotrophic lateral sclerosis) 등의 퇴행성 뇌신경질환을 타겟으로 하는 전임상 연구에서 용매로 널리 활용되고 있다. 그러나 DMSO를 활용한 연구 결과에 대하여 본 물질에 대한 생화학적 효과는 간과되고 있다. 본 연구에서는 근위축성 측삭경화증의 질환동물 모델인 SOD1-G93A형질 전환 마우스에 5% DMSO를 장기간 경구 투여하여 질병 표현형에 미치는 영향을 생존기간을 포함하여 신경학적, 기능학적, 조직학적으로 분석하였다. DMSO를 투여한 SOD1-G93A 동물군에서 DMSO 비투여군 보다 생존 기간과, 로타로드와 악력 측정으로 평가한 근육 기능이 유의미하게 증가했고, neurological score 가 감소했다. 반면, DMSO 투여군에서 DMSO 비투여군 대비하여 척수 운동 신경 세포와 신경근접합부가 보존되지 않았다. DMSO 투여는 SOD1-G93A형질 전환 마우스의 운동 신경 세포의 조직학적 영향을 미치지 않았지만, 신경 증상 완화와 생존 기간 등 개선된 마우스의 quality of life을 확인하였다. 본 연구 결과, DMSO를 이용한 퇴행성 뇌 질환 전임상 연구 및 후보 약물 효능 평가 시 DMSO의 생화학적 특성에 대한 종합적인 고려가 필요한 것으로 보인다.

Keywords

Introduction

Dimethyl sulfoxide (DMSO) is widely used as control or vehicle for efficacy testing of candidate drugs in preclinical research [6, 64]. DMSO is a small, amphiphilic molecule that is useful for dissolving lipophilic compounds, and its lipophilicity also facilitates crossing the blood brain barrier [10, 11, 60]. This makes DMSO a popular solvent in biomedical research [64]. Multiple studies have reported that treatment with DMSO can affect a wide range of clinical symptoms, which includes providing neuroprotective effects on central nervous system (CNS) pathology [40]. Therapeutic effects of DMSO have been reported in endotoxemia and septic shock [15], cerebral ischemia [5, 24], traumatic cerebral oedema [39], and Alzheimer’s disease [28], while others have reported negative or undesired effects of DMSO on the disease phenotype [12, 54]. While the mechanisms of neuroprotection by DMSO is often unclear, DMSO is a direct hydroxyl radical scavenger [56, 60], and it has been suggested that DMSO can provide protection against oxidative stress [5, 15, 26, 28, 39, 40]. Anti-inflammatory function [5, 40] and reduction of platelet aggregation in the case of cerebral ischemia models have also been suggested [5, 26]. Despite uncertainties in the exact mechanism by which DMSO affects these symp- toms, it is evident that DMSO cannot be considered as an inert vehicle, and its biological implications should be accounted for when interpreting results of preclinical studies using DMSO as a vehicle.

Amyotrophic lateral sclerosis (ALS) is one of the most prevalent neurodegenerative diseases caused by loss of motor neurons in the cerebral motor cortex, brainstem, and spinal cord [29, 37]. ALS usually affects patients between 50-60 years of age, and progresses rapidly leading to paralysis and death by respiratory failure within 3-5 years after onset [2, 38]. While the exact cause and the mechanism of neuro degeneration of ALS are not fully understood, it is known that approximately 10% of ALS cases are inherited (familial ALS), and approximately 20% of inherited cases are caused by autosomal dominant mutations of the copper/zinc superoxide dismutase (Cu/Zn SOD, SOD1) [4, 23, 52, 58]. The SOD1 plays a role in defense against oxidative stress, which is a key factor of ALS pathogenesis [1, 29], by removing superoxide radicals in a dismutase reaction [4, 9, 27]. Out of the several transgenic mouse models that have been developed based on this mutation, the most common and widely used variant is the SOD1 glycine 93 to alanine mutation transgenic mouse model (SOD1-G93A) [32, 51]. In the SOD1- G93A mutant, the SOD1 gains an additional toxic function that elevates the generation of reactive oxygen species (ROS) [4, 67]. This model displays rapid degeneration of motor neurons and corresponding behavioral deficits including asymmetric hind limb paralysis and reduction in motor performance [18, 46, 51]. The SOD1-G93A model is widely used for preclinical research of candidate drugs for ALS [7, 30, 36, 61, 65].

DMSO has often been used as vehicle for small molecule drugs with hydrophobic properties in preclinical research on the SOD1-G93A mouse model targeting ALS [19, 33, 49, 68, 69]. While DMSO has been used as a vehicle in preclinical research on the SOD1-G93A mouse model targeting ALS (Table 1), the effect of DMSO on the phenotype of the SOD1-G93A mouse model has not yet been properly tested. In this study, we investigated the therapeutic effect of DMSO in motor function loss and neurodegeneration in SOD1-G93A mice.

Table 1. Examples of the use of DMSO as vehicle in preclinical studies of using the G93A-SOD1 mouse model

Materials and Methods

Animals

All animal experiments were carried out ethically and sci-entifically according to the KBIO (Osong, Korea) Institutional Animal Care and Use Committee (IACUC) guidelines for the care and use of laboratory animals (KBIO-IACUC-2021- 209). In accordance with IACUC standards, mice were housed in cages with clean bedding materials, which were regularly changed to maintain a clean environment. Tunnels and igloos for enrichment of the cage environment and materials for nest building were provided in every cage. Food and water were available ad libitum to all mice in their home cages.

For the main behavioral study, 28 transgenic male mice expressing the mutated human SOD1-G93A (hemizygous TgN-SOD1-G93A-1Gur) [32] and 10 wildtype littermate (B6SJL strain background) mice were used for this experi- ment. Male SOD1-G93A (high-copy SOD1-G93A, G1H with 25 transgenic copies; The Jackson Laboratory, stock# 002726) and female B6SJL strain background (non-carrier, wildtype) mice obtained from The Jackson Laboratory were mated to generate F1 mice for the experiment.

Formulation and dosing

For the study, a solution of 5% DMSO and 95% sterile water was treated via oral gavage once a day, starting from 10 weeks of age to wildtype (“WT + DMSO” group; n=10,) and transgenic (“SOD1-G93A + DMSO” group; n=8,) mice, until the end of the study. Injection volume was 5 ml/kg (5 µl/g). The DMSO solution was administered in the morning, and behavioral tests were performed in the afternoon. No treatment was given to the wildtype (“WT” group; n=10) and transgenic (“SOD1-G93A” group; n=10) control groups.

Body weight and survival

Body weight was measured once a week on the same day of the week starting at the age of 9 weeks up to the age of 21 weeks. The mice were monitored for survival on a daily basis. The time of death of the test mice were recorded, and the survival time and probability of survival were presented as a Kaplan Meier survival plot.

Neurological Score

Mice were evaluated for disease symptoms using a modified neurological scoring system originally developed by the ALS Therapy Development Institute (ALS TDI) [35, 61]. The following scoring criterion was used: 0 points for full extension of the hind limbs away from the lateral midline when suspending the mouse by its tail, 1 point for trembling of the hind limbs during tail suspension, 2 points for clasping of hind limbs during tail suspension, 3 points for dragging one hind limb when walking, 4 points for dragging both hind limbs when walking, and 5 points when the mouse cannot walk and cannot right itself within 30 seconds when placed on it back. Scoring was performed starting at 10 weeks of age up to 21 weeks of age, once a week on the same day of the week.

Rotarod test

The rotarod test (BS technolab, Korea) was performed at 12, 14, 16 and 18 weeks of age. Before testing, each mouse was given a training trial for 5 minutes at 4 rpm on the rota rod apparatus. One hour later, the mouse was tested for two consecutive trials with the rotation velocity increasing from 0 to 40 rpm over 360 seconds. A 30-minute resting period was given between the trials. The latency to fall from the rotarod was recorded. Mice that remained on the rotarod for more than 360 seconds were removed from the rotarod, and their score was recorded as 360 seconds.

Grip strength test

Grip strength measurement was performed at 12, 14, 16 and 18 weeks of age. Each mouse was placed on the grip strength apparatus (Duratool, China) so that the mouse could grab the small mesh grip with its forepaws. The mouse was then slowly pulled away from the mesh grip by the tail until the animal released the mesh grip. The apparatus automatically measured the strength of the animal’s grip in kilogram strength (Kgf)×10-3. In each session, each mouse was tested five times in a consecutive sequence. The mean grip strength of the five tests was recorded.

Tissue sample collection

A separate cohort of mice in the satellite group was euthanized at the age of 109 days for tissue sample collection (n=6 per group) for histological sectioning, staining and analysis. The mice were euthanized with terminal dose of pentobarbital. Immediately after cardiac puncture and blood removal, the mice were perfused transcardially with heparinized saline fol-lowed by perfusion with 4% paraformaldehyde until the mice were fixed. The spinal cord of lumbar 1 (L1) vertebra and gastrocnemius muscle samples were dissected and post-fixed by immersion in 4% paraformaldehyde for 24 hr at 4℃. The spinal cord and gastrocnemius muscle samples were then stored in 30% sucrose at 4℃ for 3 days. The spinal cord and gastrocnemius samples were embedded in optical cutting temperature compounds (Sakura Finetek, USA) and sectioned to slides of 20 µm thickness, mounted on a microscope slide, and stored at -80℃ until staining.

Nissl staining for motor neuron counting

Spinal cord (L1) samples were sectioned with a thickness of 20 µm and immunofluorescence of motor neurons were performed. Two slices of spinal cord samples per mouse were mounted on a microscope slide. Each slide was washed in phosphate buffered saline (PBS) solution and permeabilized with 0.1% Triton X-100 in PBS for 10 minutes at room temperature. The fluorescent Nissl dye NeuroTrace 500/525 (Invitrogen, USA) was subjected to 1:300 dilution and allowed to stand at room temperature for 20 minutes. After washing the sample with PBS, it was completely dried and mounted with a mounting solution containing 4',6-diamidino- 2-phenylindole, dihydrochloride (DAPI) (Vector, USA). The samples were imaged using the Immunofluorescence microscope (Olympus, BX51). The mean number of motor neurons in the ventral horn of the left and right side of the spinal cord was counted. Stained cells of sizes of 900 pixels or larger were counted using the Image J software.

Neuromuscular junction staining and analysis

Immunofluorescence evaluation of neuromuscular junctions (NMJ) and muscular innervation were evaluated. Immunohistochemical analyses using anti-synaptotagmin-2 (SV2) as pre-synaptic marker (DSHB, USA) and a-bungarotoxin (Invitrogen, USA) as post-synaptic marker were performed on gastrocnemius muscle samples. Gastrocnemius muscle samples were sectioned with a thickness of 20 µm. One slice per gastrocnemius muscle was mounted on a microscope slide. The slides were washed in tris-buffered saline (TBS) solution. The sections were then permeabilized for 15 minutes in TBS-Tween (TBST), rinsed twice in TBS and blocked in a solution containing TBS, 5% normal goat serum and 0.2% Triton X-100 for 1 hr at room temperature. The sections were incubated overnight at 4℃ with the appropriate primary anti- bodies. The sections were washed in TBS solution and incubated in secondary antibody for 1 hr at room temperature. Finally, the sections were washed in TBS solution and completely dried and mounted with a mounting solution containing DAPI.

The samples were imaged using the ZEISS’s Confocal microscope. For analysis, the degree of overlap between the SV2 and α-bungarotoxin (post-synaptic) regions was classified into three different categories: ‘Fully-innervated’ for completely overlapping, ‘partially innervated’ for partial overlapping and ‘denervated’ for non-overlapping pre-synaptic and post-synaptic regions.

Statistics

For mean comparisons at multiple timepoints (body weight, neurological score, rotarod, grip and strength), the Kruskal- Wallis test was used followed by the pairwise Wilcoxon sign- ed-rank test at each timepoint. For the Nissl motor neuron count and NMJ innervation, the Kruskal-Wallis test was used followed by the pairwise Wilcoxon signed-rank test was used. For survival analysis, the Kaplan-Meir (log rank) test followed by with was used. For all statistical analyses, the holm adjusted p value was reported.

Results

Body weight

The SOD1-G93A and SOD1-G93A+DMSO groups showed significantly lower body weight compared to the WT and WT + DMSO groups throughout the study (Wilcoxon signed-rank test) (Fig. 1). There was no significant difference in body weight between SOD1-G93A and SOD1-G93A + DMSO groups.

Fig. 1. Body weight of the study groups. Mean body weight is presented with error bar showing standard deviation (SD). Significance levels: p<0.05*, p<0.01**, p< 0.001*** compared with WT. p<0.05&, p<0.01&& com- pared with WT + DMSO (pairwise Wilcoxon signed- rank test).

Survival

The survival of the experimental mice was observed up to 22 weeks of age (Fig. 2, Table 2). Survival time of the SOD1-G93A + DMSO group (mean survival time: 138 ± 7.15 days, median survival time: 138 days) was significantly longer compared with the SOD1-G93A group (mean survival time: 125±7.84 days, median survival time: 125 days) (Kaplan-Meier (log rank) test, p<0.01). None of the mice in the wild- type groups (WT, WT + DMSO) died within the observation period.

Table 2. Survival analysis results and pairwise statistical analysis of survival time (Kaplan-Meier (log rank) test)

Fig. 2. Kaplan-Meier survival plot for the effect of DMSO treatment on the survival duration of WT and SOD1- G93A mice. All mice in the WT groups survived until the end of the study. SOD1-G93A mice that were treated with DMSO (138±7.15 days) lived for signifi- cantly longer compared with the untreated SOD1-G93A mice (125±7.84 days) (log-rank test, p<0.01**).

Neurological score

The neurological score of the SOD1-G93A group started to increase relative to the WT and WT + DMSO groups starting at approximately 14 weeks of age and reached mean score of 3- 4 at approximately 16-17 weeks of age (Fig. 3). The neurological score of SOD1-G93A + DMSO increased more slowly compared with the SOD1-G93A group, reaching mean score of 3-4 at approximately 17-19 weeks of age. Despite the delayed trend in disease onset of SOD1-G93A + DMSO compared with SOD1-G93A, there was no significant difference in mean neurological score after between the two groups. The score for the WT and WT + DMSO groups was maintained constant, at below 2 throughout the study.

Fig. 3. Neurological score of the study groups presented with SD. Significance levels: p<0.05*, p<0.01** compared with WT. p<0.05&, p<0.01&& compared with WT + DMSO (pairwise Wilcoxon signed-rank test).

Rotarod test

Motor function was measured using the rotarod test, and the time spent on the rotarod before falling was compared between groups. At 12 weeks of age, time on rotarod of the SOD1-G93Aand SOD1-G93A+ DMSO groups was significantly lower compared to the WT group (pairwise Wilcoxon signed-rank test, p<0.05) (Fig. 4A). Time on rotarod of the SOD1-G93A and SOD1-G93A+DMSO decreased over time. The rate of decrease was greater in the SOD1-G93A group than the SOD1-G93A + DMSO group (Fig. 4B). At 16 weeks of age, most mice in the SOD1-G93A group struggled to remain on the rotarod for over 10 seconds (mean time on rotarod: 11.4 seconds; 7.3% relative to week 12). The time on rotarod of the SOD1-G93A + DMSO group was significantly higher (mean time rotarod: 93.8 seconds; 55.0% relative to week 12) at 16 weeks of age (pairwise Wilcoxon sign- ed-rank test, p<0.01) (Fig. 4A). At 18 weeks of age, all the surviving mice in the SOD1-G93A group (n=3) failed to cling to the rotarod (0 seconds on rotarod). There was no significant difference in rotarod performance between the WT and WT+DMSO groups.

Fig. 4. Rotarod test results of the study groups. A) Mean time on rotarod (seconds) before falling presented with SD. B) Time on rotarod relative to the mean result on 12 weeks of age in percentages presented with SD. Signi- ficance levels: p<0.05*, p<0.01** compared with WT. p<0.05#, p<0.01## compared with SOD1-G93A. p< 0.05&, p<0.01&& compared with WT + DMSO (pair- wise Wilcoxon signed-rank test).

Grip strength test

At 12 weeks of age, grip strength of the SOD1-G93A and SOD1-G93A + DMSO groups was significantly lower compared with the WT and WT + DMSO groups (pairwise Wilcoxon signed-rank test, p<0.05) (Fig. 5A). Grip strength of the SOD1-G93A and SOD1-G93A + DMSO groups decreased over time, and the rate of decrease was greater for SOD1-G93A than SOD1-G93A + DMSO (Fig. 5B). At 16 weeks of age, grip strength of 12 weeks of age of the SOD1- G93A with DMSO treatment (mean grip strength: 41.8 Kgf ×10-3; 74.7% relative to week 12) trended higher compared to the SOD1-G93A without treatment (mean grip strength: 26.6 Kgf×10-3; 42.1% relative to week 12), but this difference was not statistically significant. Statistical significance was detected at 18 weeks of age, but this is confounded by the low sample size of the SOD1-G93A group (n=3) in which most mice had died before 18 weeks of age (Fig. 5A, Fig. 5 B).

Fig. 5. Grip strength results of the study groups. A) Mean grip strength (Kgf×10-3) presented with SD. B) Grip strength relative to the mean result on 12 weeks of age in percentages presented with SD. Significance lev- els: p<0.05*, p<0.01**, p<0.001*** compared with WT. p<0.05# compared with SOD1-G93A. p<0.05&, p< 0.01&& compared with WT+DMSO (pairwise Wilcoxon signed-rank test).

Tissue sample analysis

In a cohort of wildtype and SOD1-G93A mice separate from the cohort used for the behavioral study, the spinal cord of the L1 vertebra and gastrocnemius muscles were collected at the age of 109 days for immunohistochemical analyses of motor neuron integrity.

The motor neuron count measured by Nissl staining in the spinal cord of the SOD1-G93A and SOD1-G93A + DMSO groups was significantly lower compared with the WT and WT + DMSO groups (pairwise Wilcoxon signed-rank test, p<0.05) (Fig. 6, Fig. 7). The relative connectivity of the NMJs in the gastrocnemius muscle was reduced in the SOD1-G93A and SOD1-G93A + DMSO groups, as evident in the significant reduction in the proportion of fully innervated NMJs and increase in denervated NMJs compared to the WT and WT + DMSO groups (Fig. 8, Fig. 9). There was no significant difference in motor neuron count or NMJ connectivity between the SOD1-G93A and SOD1-G93A + DMSO groups, or between the WT and WT + DMSO groups.

Fig. 6. Motor neuron counts by Nissl staining in the spinal cord (L1) of the study groups. The mean number of the counted motor neurons are presented with SD. Significance levels: p<0.01**, p<0.001*** compared with WT. p<0.05&, p<0.01&& compared with WT + DMSO (pairwise Wilcoxon signed-rank test).

Fig. 7. Representative images of motor neurons in the spinal cord (L1) stained by Nissl. Motor neuron count of the SOD1-G93A groups was significantly lower compared with that of the WT groups (pairwise Wilcoxon sign- ed-rank test).

Fig. 8. Percent of NMJ categorized as fully innervated, parti- ally innervated, or denervated of the study groups. The mean percentages in each category are presented with SD. Significance levels: p<0.05* compared with WT. p<0.05& compared with WT + DMSO (pairwise Wil- coxon signed-rank test).

Fig. 9. Representative images of NMJ staining with anti-syn- aptotagmin-2 (pre-synaptic marker) and a-bungarotox- in (post-synaptic marker) in the gastrocnemius muscle. In the SOD1-G93A groups, the relative frequency of fully innervated NMJs significantly decreased, and the relative frequency of denervated NMJs increased, com- pared with that of the WT groups (pairwise Wilcoxon signed-rank test).

Discussion

In the present study, we evaluated the therapeutic effect of DMSO on the SOD1-G93A mouse model for ALS. In accordance with previous reports, the SOD1-G93A mice displayed significant motor function deficits and shortened survival time, as well as loss of motor neurons in the spinal cord and reduced NMJ connectivity in the skeletal muscle, compared to the WT mice [20, 25]. We found that administration of DMSO by oral gavage to SOD1-G93A mice improved survival time and motor performance compared to the untreated SOD1-G93A mice. Overall, the clinical status of the WT mice administered with DMSO was in good con- dition, with no notable side-effects.

A potential explanation for this outcome is that the antioxidant property of DMSO provided neuroprotective function. The SOD1-G93A mutation leads to a toxic gain in function mutation that results in increased production of ROS [4, 67]. Oxidative stress is therefore a major target for preclinical research on ALS [4, 41, 53, 55, 59, 69, 71]. Consistent with our results, treatment of SOD1-G93A transgenic mice with antioxidants delayed disease onset [8, 42, 44, 72], increased survival duration [8, 41, 42, 44, 50, 66, 72], and improved rotarod performance [16, 44, 55] and grip strength [44, 50].

Potential neuroprotective effects of DMSO have been also reported on other pathologies related to the central nervous system. For example, multiple studies report that treatment with DMSO led to reduced neurodegeneration in animal models of cerebral ischemia [5, 22, 24]. One study reported that intraperitoneal injection of pure DMSO attenuated age-related loss in cognitive performance in Lurcher mice [47]. Potential effectiveness of DMSO have also been indicated in Alzheimer’s disease [28] and cold-induced traumatic brain oedema [39]. On the other hand, some studies have reported toxic effects of DMSO [3, 12, 13], such as acute reduction in rotarod performance after intraperitoneal injection with pure DMSO or 5% DMSO in PEG-400 in mice [48] and increased time required to complete beam walking test after injection with 10-15% DMSO in mice [12]. It is possible that the effect of DMSO on animal models of pathology may vary depending multiple factors such as the dose, schedule of dosing, type of pathology, animal strain and species.

The effects of DMSO on behavior and motor function were not correlated with the subcellular analyses of motor neurons in the spinal cord and motor neuron connectivity in the skeletal muscle. Motor neuron loss and denervation of NMJ that act under the influence of acetylcholine are molecular hallmarks of the early phase of ALS in SOD1-G93A mice [14, 63, 65, 70]. It is possible that other mechanisms may contribute to the improvement in behavior. Some studies report that treatment with DMSO provided anti-inflammatory function in zymosan-induced edema [21] and encapsulating peritoneal sclerosis [43]. As we did not test the effect of DMSO treatment on inflammatory response or gliosis in the spinal cord, the precise mode of action of DMSO in the SOD1-G93A mouse model cannot be determined, and further experiments to address a key mechanism by which DMSO improves the behavior and survival benefits in SOD1-G93A mice will be needed. Nevertheless, in clinical research, in neurodegenerative diseases such as ALS, improving the behavioral outcome of treatment – the “quality of life (QOL)” – is a major goal in efficacy evaluation and palliative therapy [17, 34, 45, 62]. In our preclinical study, it can be interpreted that treatment with DMSO increased the QOL of the test animals by attenuating the loss of motor function and increasing survival time [31]. Therefore, the capacity for DMSO to improve motor function of SOD1-G93A mice should not be overlooked when interpreting results of therapeutic efficacy of candidate drugs when using DMSO as low as 5% as the solvent/vehicle control.

In conclusion, to the author’s knowledge, this is the first study to report therapeutic effect of DMSO in the SOD1- G93A mouse model for ALS. We observed that long term oral treatment of DMSO improved survival time and motor function assessed in vivo with little or no improvement in motor neuronal damage. To further investigate the effect of DMSO on the SOD1-G93A model, a comparative study using commercially available antioxidants such as Edaravone is required [57]. Furthermore, molecular analyses of inflammatory markers to test the involvement of inflammatory pathways in also desired. The beneficial effect of DMSO on the behavioral parameters should be carefully considered when using DMSO as vehicle for drug candidates for ALS. Furthermore, it is important to thoroughly consider the effect of the vehicle of choice when conducting safety or efficacy tests of drug candidates targeting neurodegenerative disease.

The Conflict of Interest Statement

The authors declare that they have no conflicts of interest with the contents of this article.

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