Potentiation of pentylenetetrazole-induced neuronal damage by dimethyl sulfoxide in chemical kindling model in rats.

OBJECTIVES: Dimethyl sulfoxide (DMSO) is commonly used as a vehicle for many hydrophobic drugs. This study aimed at evaluating the effect of low dose of DMSO (0.1%) on Pentylenetetrazole(PTZ) induced neuronal damage in rats.
MATERIALS AND METHODS: Young male Wistar rats (n = 32) were divided into four groups as follows: saline control group, DMSO control group, PTZ group (35 mg/kg), and combination group (DMSO + PTZ). Animals were observed for seizure score, latency to develop kindling, percentage of animals kindled, and histopathological score of hippocampus.
RESULTS: There was a significant increase in the seizure scores and histopathological scores in the combination group as compared to PTZ-treated group. The latency to develop kindling was, however, decreased in the combination group (4th week) as compared to PTZ (6th week) group.
CONCLUSIONS: The present study has concluded that 0.1% DMSO in PTZ-induced rat model of epileptogenesis needs further optimization and should be used cautiously.

Introduction

Dimethyl sulfoxide (DMSO) is a widely used polar organic solvent in neuroscience research. Its amphipathic nature makes it soluble in both aqueous and nonaqueous media.[1] Studies in the literature have demonstrated both neuroprotective and neurotoxic effects of DMSO at different concentrations in different experimental models.[12] Some studies have illustrated that DMSO is used as a therapeutic agent because of its antioxidative, anti-inflammatory, neuroprotective, and antinociceptive properties,[13] while other studies have demonstrated several side effects of DMSO, including effects on the cell membranes and receptor affinity.[12] DMSO is a commonly used solvent for many antiepileptic drugs in various in vitro and in vivo experimental models of epilepsy. Animal models of epilepsy are an important prerequisite to study the process of epileptogenesis. Pentylenetetrazole (PTZ)-kindling model is one of the most commonly used animal models to study the antiepileptic potential of a compound.[4]

Studies have demonstrated the apoptotic and seizure-inducing potential of DMSO in the central nervous system (CNS).[56] Hence, it is of utmost concern to evaluate the effect of DMSO on neuronal damage before using it as a vehicle in neuroscience research. This study was designed to assess the effect of low dose of DMSO (0.1%) in a PTZ-kindling model of epileptogenesis.

Materials and Methods

Chemicals

PTZ was procured from Sigma Pharmaceutical Industrial Co, and DMSO was purchased from Merck (Merck Millipore, USA).

Animals

A total of 32 (n = 8 in each group) male Wistar rats (150–250 g) were divided into four groups: (1) saline (0.9% NaCl w/v, i.p); (2) DMSO (1 ml/kg of 0.1% v/v, i.p); (3) PTZ (35 mg/kg, i.p), and (4) DMSO + PTZ (35 mg/kg). Animals were kept at a standard temperature of 23°C ± 2°C and relative humidity of 65% ±5%. They were provided with the standard rat diet and tap water. All animal procedures and the experimental protocols were executed after getting approval from the Institutional Animal Ethics Committee (IAEC number: 68/IAEC/407).

Pentylenetetrazole-kindling model and behavioral studies

PTZ was injected every alternate day at a submaximal dose of 35 mg/kg (i.p). After PTZ injection, rats were placed separately in transparent plexiglass cages and scored according to the Racine 5-point scale.[7] An animal was said to be fully kindled when it showed a seizure score of 4 or 5 on three consecutive dosing. DMSO was given 30 min before PTZ administration every alternate day until the animal develops kindling or up to 10 weeks. For behavioral studies, seizure score, latency to develop kindling in each group, and percentage (%) of animals being kindled in each group were recorded.

Histopathological scoring for neuronal damage

Fully kindled rats were sacrificed by decapitation under the overdose of pentobarbital sodium. The brain was dissected out, and coronal sections containing hippocampus were kept in 10% formalin. Paraffin-embedded blocks were prepared, trimmed, and then stained with hematoxylin and eosin (H and E). The sections were observed for degenerative changes in the neurons in CA1, CA2, and CA3 regions of the hippocampus. The relative percentage of damaged neurons were scored using a semiquantitative scoring system,[8] which ranges from 0 (no injury) to 4 (diffuse injury).

Statistical analysis

Statistical analysis was done using SPSS statistics V22.0 (International Business Machine Corp., India). One-way ANOVA followed by Scheffe post hoc analysis was applied for seizure score, and percentage of animals kindled was analyzed using the Chi-square test. Kruskal–Wallis test was applied to histopathological score data and Mann–Whitney U-test for comparison in between two groups. P < 0.05 was considered statistically significant.

Results

Behavioral studies

A seizure score of zero (0) was observed in rats belonging to both saline and DMSO control groups. An increase in the seizure score from 1.71 ± 0.23 at 1st week to 3.37 ± 0.88 at the end of 10 weeks was observed in the rats of PTZ group. In DMSO + PTZ group, there was a significant increase in the seizure score from 2.27 ± 0.44 (P = 0.002) at 1st week to 4.12 ± 0.35 (P = 0.034) at the end of 10 weeks [Table 1]. At the end of 10th week, the number of animals became kindled in the PTZ control group was 50.0% (4/8), whereas it was 75.00% (6/8) in the combination group [Table 2].

Table 1

Effect of various treatments on seizure score in Pentylenetetrazole-kindling model in rats

Table 2

Effect of various treatments on induction of kindling by Pentylenetetrazole and total percentage of animals kindled at various points of time

Neuronal damage

The neurons of the H and E-stained sections of rat hippocampus in the saline control group showed a normal morphology with intact shape and vesicular nucleus (mean histopathological score = 0.00 ± 0.00). However, in DMSO control group, some of the neurons exhibited hypereosinophilia, nuclear chromatin clumping, and condensation of cytoplasm in the H and E-stained sections of rat hippocampus, which is significantly greater (P = 0.002) than the saline control group (mean HS = 0.87 ± 0.37). On the other hand, most of the neurons of the H and E-stained sections of rat hippocampus in the PTZ group (mean HS = 2.75 ± 0.70) as well as in the combination group (mean HS = 3.75 ± 0.46) have demonstrated the condensation of cytoplasm, hypereosinophilia, nuclear chromatin clumping, and fragmentation of cell [Figure 1]. However, the damage of the neurons in the combination group was significantly more (P = 0.015) severe.

Figure 1

Histopathological analysis of rat hippocampus in Pentylenetetrazole-kindling model of rats: (a) Effect of various treatments on histopathological scores of rat hippocampus – statistical analysis was done by Kruskal–Wallis test (P = 0.000) and comparison between two groups with Mann–Whitney U-test. Values are expressed as mean ± standard deviation and n = 8 for all groups. *P < 0.05 compared to saline control group, †P < 0.05 compared to dimethyl sulfoxide control group, ‡P < 0.05 compared to saline + Pentylenetetrazole group. (b) Microphotograph (×40) of rat hippocampus showing neuronal morphology: (i) saline control group showing normal neuronal cells; (ii) dimethyl sulfoxide control group showing some damage in neuronal cells; (iii) Pentylenetetrazole group showing diffuse neuronal injury of the cells, and (iv) dimethyl sulfoxide + Pentylenetetrazole group showing diffuse neuronal injury of the cells. Arrow – densely stained nuclei of neurons indicating chromatin clumping

Discussion

DMSO is very useful and popular solvent for hydrophobic drugs by virtue of its physicochemical properties.[1] Its use in various experimental researches including neuroscience research is very common. Despite being commonly used as a vehicle for various compounds in many in vivo and in vitro experiments, only few studies have demonstrated its neurotoxic side effects. Apoptosis caused by DMSO (0.5% and 1%) has been reported in the CNS of young mice,[5] and behavioral neurotoxicity of DMSO has been reported in rats.[9] Some animal studies have also demonstrated the incidence of seizures with the use of DMSO.[510] A study by Kovács et al. has mentioned that absence-like epileptic activity of WAG/Rij rats decreased with low concentration of DMSO and increased with high concentration of DMSO.[11]

In the present study, we have reported that when 0.1% DMSO was used along with PTZ, the seizure score and the number of kindled rats were significantly increased in the DMSO + PTZ group as compared to PTZ group. Furthermore, 0.1% DMSO exacerbates neuronal damage in PTZ-kindled rats and fastens the latency of kindling in this model. Even 0.1% DMSO alone had shown potential to significantly increase the histopathological score (0.87) as compared to saline control group (0), indicating that there might have been some neuronal damage in the hippocampus of rats treated with DMSO. The present study is the first in vivo study to demonstrate the adverse effects of very low dose of DMSO (1 ml/kg of 0.1% DMSO v/v) as there were neurological damage and decreased seizure threshold in PTZ-kindled rats.

The mechanism behind this is yet to be explored. A study by Yuan et al. has demonstrated the neurotoxic mechanism of DMSO and reported that it is because of the disruption of mitochondrial membrane potential and its integrity, which ultimately lead to the development of oxidative stress, apoptosis, and decreased glial glutamate transporter expression.[12] Another study by Schroeder et al. has illustrated that PTZ kindling is also responsible for alteration in the activity of glutamatergic system of the rat hippocampus.[13] Hence, DMSO and PTZ kindling collectively enhance the glutamatergic system, and this might be a reason for the enhancement of hippocampal damage when used together in rat model of epileptogenesis. Additional studies are required to explore the mechanism behind the interaction of DMSO with PTZ.

Conclusion

It can be concluded that even a low dose of DMSO can enhance the hippocampal damage when used in PTZ-induced rat model of epileptogenesis and may generate false-negative results. It may also enhance the neurotoxicity of drugs dissolved in it. Hence, the use of DMSO in PTZ-induced rat model of epileptogenesis needs further optimization and should be used cautiously as it may lead to misinterpretation of the results.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.