(T1430-03-18) Effect of Intranasally Administered Midazolam, Diazepam or Lorazepam on Neuroinflammation in Acute Seizures Mice

Purpose: Epilepsy is a brain disorder that causes sudden seizures, subsequently leading to neuroinflammation. Recently, various non-intravenous administration routes or first-line seizure control drugs, including midazolam [1-6], diazepam [1-6], and lorazepam [7], have been developed, with intranasal delivery of these drugs as one of the most promising option. Although our previous research demonstrated that intranasal delivered diazepam outperformed intranasally administered midazolam or lorazepam in acute seizure control [8], their effect on neuroinflammation remains unknown. Therefore, the current study is proposed aiming to evaluate the effects of intranasally administered midazolam, diazepam or lorazepam on neuroinflammation via monitoring mRNA levels and/or protein expressions of GABARα1, TNF-α, and IL-1β in cortex and hippocampus of mice with acute seizures.

Methods: A pilocarpine-induced acute seizures model was established using C57 mice. To establish the model, scopolamine methyl bromide and pilocarpine hydrochloride prepared in saline at concentrations of 0.15 mg/ml and 52.5 mg/ml, respectively, were adopted. To prepare the tested drugs, midazolam, diazepam, and lorazepam solution were prepared by dissolving 3 mg of each drug into 200 μl polyethylene glycol 400 (PEG 400) followed by sonicating for 15 minutes, and subsequent addition of 200 μl of propylene glycol (PLG) per time for four times to reach a final concentration of 3 mg/ml for each drug. Animal grouping and drug treatments in the pilocarpine-induced acute seizures mice model were shown in Figure 1. The study involved 30 mice, divided into five groups (n=6/group), including Group I (negative control group): Vehicle (intranasal, i.n.) + saline (intraperitoneal, i.p.) + saline (i.p.), Group II (seizure model control group): Vehicle (i.n.) + 1 mg/kg scopolamine methyl bromide (i.p.) + 350 mg/kg pilocarpine (i.p.), Group III (midazolam treatment group): 1 mg/kg midazolam (i.n.) + scopolamine (i.p.) + pilocarpine (i.p.), Group IV (diazepam treatment group): 1 mg/kg diazepam (i.n.) + scopolamine (i.p.) + pilocarpine (i.p.), and Group V (lorazepam treatment group): 1 mg/kg lorazepam (i.n.) + scopolamine (i.p.) + pilocarpine (i.p.). As indicated by the dosing schedule shown in Figure 1, drugs/vehicle were administered intranasally to mice from all groups at 0 min, followed by i.p. administrations of scopolamine /saline at 5 min and pilocarpine /saline at 25 min. The seizure scores of mice all groups were evaluated at 35 minutes, followed by sacrificing the mice to collect their cortex and hippocampus to monitor the levels of GABARα1, TNF-α, and IL-1β mRNA by RT-PCR and the expression of TNF-α protein level by western blot.

Results: The seizure scores following intranasal administrations of vehicle, midazolam, diazepam, and lorazepam in the pilocarpine-induced acute seizures mice were shown in Figure 2. The mean seizure scores of Groups II, III, and v were 2.67, 2.50, and 2.67, respectively, which were all significantly higher (p < 0.001) than that of Group IV (mean seizure score of 1.33), consistently suggesting more potent seizure control by intranasally administered diazepam than that from midazolam and lorazepam. Comparing the inflammation marker mRNA levels in the cortex, it was noted that TNF-α was the only one significantly different (four times higher) from those in Group II (p < 0.05), indicating that TNF-α could serve as the inflammation marker in acute seizure model. Our further studies demonstrated that the significantly increased TNF-α mRNA (Figure 3A) and protein levels (Figure 3B) in both the cortex (Figure 3A i) & Figure 3B i)) and hippocampus (Figure 3A ii) & Figure 3B ii)) in seizure model control Group II could be significantly reduced by intranasally treated midazolam, diazepam, or lorazepam. Among these drug treatments, intranasally administered diazepam exhibited the greatest efficacy in terms of reducing TNF-α mRNA (p < 0.05 vs. Lorazepam, p < 0.05 vs. Midazolam) and protein levels (p < 0.05 vs. Lorazepam, p < 0.01 vs. Midazolam).

Conclusion: Intranasally administered midazolam, diazepam or lorazepam could reduce not only the seizure score but also the neuroinflammation in pilocarpine-induced acute seizures mice model. Among the tested drug treatment, intranasally administered diazepam was most effective in controlling acute seizures and reducing TNF-α mRNA and protein levels.

References: [1] T. Fişgin, Y. Gurer, T. Tezic, N. Senbil, P. Zorlu, C. Okuyaz, D. Akgün, Effects of intranasal midazolam and rectal diazepam on acute convulsions in children: prospective randomized study, J. Child Neurol., 17 (2002) 123-126.
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[8] A. Tong, J. Zhao, J. Wang, Y. Xie, Y. Zhang, Z. Zuo (2023). Comparing the Efficacy of Intranasal Midazolam, Diazepam and Lorazepam as First-Line Treatments of Acute Seizures: Pilot Investigation in Pilocarpine-Induced Mice Model [poster]. AAPS National Biotechnology Conference, Philadelphia, USA


Figure 1. Animal grouping and drug treatment in the pilocarpine-induced acute seizures mice model.


Figure 2. Seizure scores after intranasal administrations of vehicle, midazolam (1 mg/kg), diazepam (1 mg/kg) and lorazepam (1 mg/kg) in pilocarpine-induced acute seizures mice model.


Figure 3. Effect of midazolam, diazepam or lorazepam treatment on (A) TNF-α mRNA level and (B) TNF-α protein level from (i) cortex and (ii) hippocampus in the brain of pilocarpine-induced acute seizures mice model.