Essential tremor research...

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Transpharmation is pleased to share another round of results with you!

Harmaline-induced tremor using the piezoelectric plate Validation with propranolol at 10 and 20mg/kg

Essential tremor (ET) is a progressive chronic neurological disorder characterised by involuntary and rhythmic tremor of a body part (hands and arms). ET can occur at any age but is most common in people aged 40 and older. ET are most visible when patients maintain a fixed position (postural tremor) or when patients execute a voluntary movement (action/intention tremor). Although it was classically called a “benign tremor”, ET can have a detrimental impact on the execution of daily tasks, such as eating, drinking, writing, talking, etc.

The first-line medication for ET are beta blockers such as propranolol (Jefferson et al., 1979; Thanvi et al., 2006). However, side effects occur in up to 66% of ET patients (bradycardia, light-headedness, fatigue, headache) and 50% of ET patients are not responsive to propranolol (Hedera et al., 2013), making it essential to develop novel treatments for ET.

Harmaline administration can be used as a model of ET in rodents to induce 9-12Hz tremors in rats and 10-15Hz tremors in mice (Martin et al., 2005; Paterson et al., 2009). Our goal was to implement the harmaline-induced model of ET in the Sprague Dawley rat using the SDI tremor monitor to record tremors.

The tremor monitor (San Diego Instruments, San Diego, CA) consists of a clear Plexiglas chamber with a piezoelectric unit placed underneath which transcribes animal movements into an electric signal. The recorded signal can be analysed to retrieve relevant data regarding animal vibrations recorded at certain frequencies, notably those occurring between 9 and 12Hz in the rat. In our preliminary study, Sprague-Dawley rats were dosed with propranolol at 10 and 20mg/kg 30 minutes prior to harmaline at 10mg/kg. 20, 40 and 60 minutes after the dosing of harmaline (or its vehicle solution), animals were placed in the tremor plate chamber, recorded for 5 minutes, and returned to their home cage between each recording.

The power spectrum is a quick tool used to oversee the global effect of compounds on the intensity, duration, and number of harmaline-induced tremor events without distinction between these three parameters. Our preliminary experiment showed a significant peak at the 9 to 12 Hz frequencies in harmaline-treated animals, as expected. Other vibrations are recorded, notably in the vehicle-treated animals, which correspond to normal biological cyclic activity (e.g., heart rate at 5-8Hz, respiratory rate at 1.5Hz).

The difference between the average power between 9-12Hz and the average power between 0-8Hz (or tremor index, Ossowska et al., 2015) was used to confirm the effect of propranolol on the harmaline-induced tremor. However, use of the power spectrum alone cannot determine whether the peak at 9-12Hz is due to tremors being recorded in a higher number, or at a higher intensity or both.

A B

Fig.1 Effect of propranolol at 10 and 20mg/kg on harmaline-induced tremors using the San Diego Instruments tremor monitor. A. Power spectrum at 60 minutes post-harmaline. Sprague-Dawley rats were dosed with propranolol at 10 and 20mg/kg 30 minutes prior to harmaline at 10mg/kg. 20, 40 and 60 minutes after dosing of harmaline, animals were placed in the tremor plate chamber, recorded for 5 minutes, and returned to their home cage between each recording. The harmaline-treated animals showed a significant power peak at the 9 to 12 Hz frequencies, which is consistent to what was expected. B. Tremor index at 20, 40, 60minutes post-harmaline. Tremor index is the difference between the average power between 9-12Hz and the average power between 0-8Hz. Tremor index was calculated to confirm the significant reduction in power induced by propranolol at 10 and 20mg/kg on the harmaline-induced tremor. Data were analysed with a Two-Way repeated measures ANOVA followed by a Dunnett’s multiple comparison test. Data are shown as mean ± S.E.M. *p<0.05 and **p<0.01 ± S.E.M. when compared to the vehicle/vehicle group at the same timepoint. ##p<0.01 and ####p<0.0001 when compared to the vehicle/harmaline group at the same timepoint.

To determine if the significant attenuation of the harmaline-induced tremor by propranolol was caused by a reduction in the number of recorded tremors or in the duration of one tremor or both, we developed a script that can decipher the number of tremors, the average duration of each tremor, and the cumulated time spent in tremor within each 5-minute recording.

Fig.2: Effect of propranolol 10-20mg/kg on the average duration of tremor in a harmaline-induced model of essential tremor in the SD rat. Propranolol at 10 and 20mg/kg were given 30 minutes before harmaline. Tremor event data were recorded for 5 minutes at 20min, 40min, and 60min post-harmaline. The average duration of one tremor was calculated at the 20-, 40- and 60- minute timepoints, and analysed with a One-Way ANOVA followed by a Dunnett’s multiple comparison test. ****p<0.0001 when compared to the vehicle/vehicle group. ####p<0.0001 when compared to the vehicle/harmaline group. Data were shown as mean+S.E.M.

Preliminary data showed that propranolol had a significant effect on the duration (Fig. 2) and intensity of harmaline-induced tremors. However, it was also found in later studies that propranolol can significantly reduce the number of harmaline-induced tremors (data not shown).

More potent and consistent results were observed when animals were recorded past the 60- minute timepoint or with a higher dose of harmaline (data not shown), which makes this assay a robust tool for the development of future treatment of ET.

Advantages of using the SDI piezoelectric plate

to record harmaline-induced tremor

Can be used with rats or mice (up to 4 animals recorded simultaneously)

Record undisturbed animals in sound-attenuated and light-controlled conditions

Retrieve various quantifiable data from a single recording (power spectrum, intensity, duration, time spent in tremor)

Avoid the use of visual scoring to count harmaline-induced tremors

Screen novel candidate drugs using a preclinical model of essential tremor with distinct features from Parkinson’s disease (Kosmowska et al., 2017; Milner et al., 1995; Thanvi et al., 2006)

Authors: Rina Ramarohetra, Senior Scientist M.Sc and Chris Cassidy, Research Assistant M.Sc at Transpharmation.

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References

Hedera P, Cibulčík F, Davis TL. (2013) Pharmacotherapy of essential tremor. J Cent Nerv Syst Dis. 2013 Dec 22;5:43-55.

Jefferson D, Jenner P, Marsden CD (1979) beta-Adrenoreceptor antagonists in essential tremor. J Neurol Neurosurg Psychiatry. 1979 Oct;42(10):904-9.

Kosmowska B, Ossowska K, Głowacka U, Wardas J. (2017) Tremorolytic effect of 5'-chloro-5'-deoxy-(±)-ENBA, a potent and selective adenosine A1 receptor agonist, evaluated in the harmaline-induced model in rats. CNS Neurosci Ther. 2017 May;23(5):438-446.

Martin FC, Thu Le A, Handforth A. (2005) Harmaline-induced tremor as a potential preclinical screening method for essential tremor medications. Mov Disord. 2005 Mar;20(3):298-305.

Milner TE, Cadoret G, Lessard L, Smith AM. (1995) EMG analysis of harmaline-induced tremor in normal and three strains of mutant mice with Purkinje cell degeneration and the role of the inferior olive. J Neurophysiol. 1995 Jun;73(6):2568-77.

Ossowska K, Głowacka U, Kosmowska B, Wardas J. (2015) Apomorphine enhances harmaline-induced tremor in rats. Pharmacol Rep. 2015 Jun;67(3):435-41.

Paterson NE, Malekiani SA, Foreman MM, Olivier B, Hanania T. (2009) Pharmacological characterization of harmaline-induced tremor activity in mice. Eur J Pharmacol. 2009 Aug 15;616(1-3):73-80.

Thanvi B, Lo N, Robinson T. (2006) Essential tremor-the most common movement disorder in older people. Age Ageing. 2006 Jul;35(4):344-9.

Copyright © Transpharmation Harmaline Tremor Newsletter August 2022

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