The influence of ketamine’s repeated treatment on brain topology in 2020


Experimental design

Four parallel groups of the depressive-like NC and resilient PC male rats (N = 47; Sprague–Dawley; 83rd genetical generations; 8 weeks old; 246–344 g) underwent an escape test (for description see Supplement) and starting from this day received seven daily injections of either S-ketamine at 10 mg/kg (Ketanest, Pfizer Pharma GmBH, Berlin, Germany) (14 NC, 11 PC rats) or saline (12 NC, 10 PC rats) (subcutaneously (s.c.), 1 ml/kg) (Fig. 1). We chose the dose of 10 mg/kg as it corresponds to 0.5 mg/kg dose used in humans 23,24, resulting in a similar range of peak concentrations25, and is an optimum antidepressant dose in the animal models 13,15. In humans a regimen for Ketamine for sale repeated treatment mainly comprises six to eight intermittent injections; however, in rats, a schedule is often consecutive due to faster metabolic rates in rodents26. Similar to an intermittent regimen, in a rat model of depression, it demonstrates robust antidepressant-like effects13. We used a consecutive regimen in order to fit the injection schedule into a week instead of prolonging it to 2 weeks by intermittent treatments, thus keeping the ages of rats in alignment with our previous study and avoiding brain aging effects.

On the day following the last injection, the animals underwent fMRI scanning. Group (PC/NC), treatment (ketamine/saline), and time of day were randomized in the fMRI measurements. During the experiments, the investigator was blinded to the assignment of groups.

For the single injection, we used a separate previously acquired fMRI dataset consisting of NC and PC rats belonging to the same genetic generation as rats in the current dataset and treated with 10 mg/kg S-ketamine (12 NC, 13 PC rats) or saline (12 NC, 12 PC rats)16.

The experimental procedures (behavioral test, fMRI scanning, anesthesia protocol) and preprocessing pipeline for both datasets were identical.

Due to the exploratory nature of this study, no formal power or sample size estimation was carried out; however, the group sizes (N = 10–14 per group) were toward the high end of the range typically used in animal fMRI experiments.

The rats were housed in plastic cages (two rats per cage) at a constant temperature of 22 °C and 12-h light–dark cycle (lights on at 07:00 a.m.) and with food and water available ad libitum. At the end of the experiments the rats were sacrificed. The experiments were performed according to the regulations covering animal experimentation within the European Union (European Communities Council Directive 86/609/EEC) and within the German Animal Welfare Act, and were approved by the German animal welfare authorities (Regierungspräsidium Karlsruhe).

MRI acquisition and pre-processing

The rs-fMRI experiments were carried out at 9.4 T MRI scanner (Bruker BioSpec, Ettlingen, Germany) with Avance III hardware, BGA12S gradient system (maximum strength 705 mT/m), and Paravision 6 software. We used a linear whole-body volume transmitter coil combined with an anatomically shaped four-channel receive-only coil array. The rats were initially anesthetized with 4% isoflurane (Baxter Deutschland GmbH, Unterschleißheim, Germany) in a mixture of 70% N2 and 30% O2; then, after positioning in the scanner, isoflurane level was reduced to 2.5% and medetomidine (Domitor, Janssen-Cilag, Neuss) was injected as a bolus (0.5 ml, 0.07 mg/kg, s.c.). Isoflurane administration was slowly discontinued during 10 min (reduction by 0.5% every 2 min), and after switching it off, the animals continuously received medetomidine at 0.28 mg/kg/h. We monitored sedation depth via recording the physiological (respiratory and cardiac) parameters throughout the experiment at 10-ms resolution using the signal breakout module (Small Animal Instruments Inc., NY, USA) and a 4-channel recorder (Velleman® N.V., Gavere, Belgium). The physiological parameters stabilized at 15 min after the start of continuous medetomidine and remained stable during the whole experiment.




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