Development and validation of brain target controlled infusion of propofol in mice

Abstract

Mechanisms through which anesthetics disrupt neuronal activity are incompletely understood. In order to study anesthetic mechanisms in the intact brain, tight control over anesthetic pharmacology in a genetically and neurophysiologically accessible animal model is essential. Here, we developed a pharmacokinetic model that quantitatively describes propofol distribution into and elimination out of the brain. To develop the model, we used jugular venous catheters to infuse propofol in mice and measured propofol concentration in serial timed brain and blood samples using high performance liquid chromatography (HPLC). We then used adaptive fitting procedures to find parameters of a three compartment pharmacokinetic model such that all measurements collected in the blood and in the brain across different infusion schemes are fit by a single model. The purpose of the model was to develop target controlled infusion (TCI) capable of maintaining constant brain propofol concentration at the desired level. We validated the model for two different targeted concentrations in independent cohorts of experiments not used for model fitting. The predictions made by the model were unbiased, and the measured brain concentration was indistinguishable from the targeted concentration. We also verified that at the targeted concentration, state of anesthesia evidenced by slowing of the electroencephalogram and behavioral unresponsiveness was attained. Thus, we developed a useful tool for performing experiments necessitating use of anesthetics and for the investigation of mechanisms of action of propofol in mice.

Fig 1. Schematic of sampling procedure.

In order to minimize the effect of repeated sampling from the same animal, samples were collected in a consistent order alternating between biopsies shown in (A) along with a drawing of the tool used to obtain brain biopsies. To further establish that this method did not cause significant harm to the surrounding brain tissue, a single large biopsy of brain tissue was removed and sectioned following a 1-hour infusion at 2 mg⋅kg^-1 ⋅min^-1. The propofol concentration in each section was analysed. (B) shows the median and interquartile range for the propofol concentration measured in the separate sections of the large biopsy (n = 8, median = 10.05, IQR = 1.5). (C) Drug only enters and exits the model through compartment 1. Compartment 1 models blood, compartment 2 models brain, and compartment 3 models all other tissues. k₁₂, k₁₃, k₂₁, and k₃₁ are the inter-compartmental rate constants, k₁₀ is the rate of elimination, and s_inf is a constant that was necessary to convert the body weight of the mouse to the theoretical volume of compartment 1.

Fig 2. Schematic of model creation methods.

The first image represents the initial infusion used, which was delivered at a fixed rate. The second image shows that data collected from these experiments were fit and used to produce estimates of the pharmacokinetic parameters, as described in the methods. Third, these parameter estimates were used to calculate the infusion rate necessary to maintain a target brain concentration for the brain TCI experiments. After each experimental set, accuracy of TCI model was determined and fit was updated to incorporate all experimental findings. The methodology represented in images 2 and 3 was repeated until time-invariant and unbiased target brain concentration of propofol was maintained for at least 1 hour.

Fig 3. Brain propofol concentration data used for model fitting.

Brain propofol concentration measured and fitted for all experiments used in model creation. In each graph, the shaded grey area shows the infusion rate in mg⋅kg^-1⋅min^-1 used in each experiment. These rate data are plotted on a log scale displayed on the right y-axis. Heavy black lines show the propofol concentration predicted in the brain tissue in response to the infusion used in that experimental set according to the final set of pharmacokinetic rate constants obtained after fitting all four experimental paradigms. Connected points indicate the propofol concentration measured in the brain tissue samples from a single subject. (A) and (B) show the simple infusions used. (A) 150 mg⋅kg^-1⋅min^-1 for 6 seconds. (B) 2 mg⋅kg^-1⋅min^-1 for 1 hour. (C) and (D) show the infusions resulting from the first and second attempts at achieving brain TCI, both targeting 10 μg/g in the brain tissue.

Fig 4. Blood propofol concentration data used for model fitting.

Blood propofol concentrations measured from the same experiments represented in Fig 3. As in Fig 3, the shaded grey area shows the infusion rate in mg⋅kg^-1⋅min^-1 used in each set of experiments. Heavy red lines show the propofol concentration predicted in the blood. Connected points indicate the propofol concentration measured in the blood of a single subject. (A) and (B) show the simple infusions used. (A) 150 mg⋅kg^-1⋅min^-1 for 6 seconds. (B) 2 mg⋅kg^-1⋅min^-1 for 1 hour. (C) and (D) show the infusions resulting from the first and second attempts at achieving brain TCI, both targeting 10 μg/g in the brain.

Fig 5. Model validation data.

The infusion (grey shaded area) was computed to target brain concentration of 10 μg⋅g^-1 (A and B) or 15 μg⋅g^-1 (C). Predicted propofol concentration in the brain (A and C) and blood (B) are shown by thick black and red lines, respectively. Measured propofol concentration in the brain (A and C) and blood (B) are shown as points. Points are colour coded by subject. The data from panels A and C were replotted in (D) as the moving average and standard deviation of the normalized propofol concentration in brain tissue, relative to the target concentration.

Fig 6. Electrocorticographic verification of anesthetic depth.

Each trace represents a 10 second segment of spontaneous ECoG recorded over the mouse somatosensory cortex while the mouse is receiving TCI propofol. This data was collected beginning 20 minutes into a 30 minute recording. The target brain concentration of propofol is 10 μg⋅g^-1 in the top trace (A). This trace shows that the alpha frequency is prominent from 4–7 seconds. In contrast, the target brain concentration in the bottom is 15 μg⋅g^-1 (B), during which a deeper anesthetic state is illustrated by burst suppression.