In vivo monitoring of rat brain endocannabinoids using solid-phase microextraction

Abstract

Aim:

Solid-phase microextraction is proposed to measure concentrations of anandamide and 2-arachidonoyl glycerol in live rat brains in response to stress.

Materials & methods:

Solid-phase microextraction fibers were prepared from steel with 1.5 mm extraction coating. 24 male rats were divided into groups based on brain region, stria terminalis or posterior hypothalamus and loud noise or control groups. The fibers were desorbed in acetonitrile-water (75:25) and analyzed by ultraperformance LC–MS/MS. The linear range of the method was 0.05–50 ng/ml and the in vivo concentrations were found to be between 0.3 and 40 ng/ml.

Conclusion:

The new approach was successfully used to determine the concentrations of anandamide and 2-arachidonoyl glycerol in vivo and could be used in the future to measure other endogenous compounds.

The endocannabinoid (EC) system consists of a complex system of lipid ligands, their receptors and various enzymes involved in synthesis and deactivation of the ligands. The system is involved in regulating a wide variety of physiological processes, most prominent being its role in the modulation of neurotransmitter release [1]. Two different cannabinoid (CB) receptors have been identified. CB1 receptors are expressed primarily on central and peripheral neurons [2]. CB2 receptors are predominantly present on immune cells, though they are also present to a limited degree in the central nervous system [2].

The physiological ligands for the CB receptors are called ECs. The most studied ECs are anandamide (N-arachidonoylethanolamide, AEA) and 2-arachidonoyl glycerol (2-AG), shown in Figure 1. ECs have numerous cellular regulatory functions, including reducing programmed cellular death, helping with the upkeep of mitochondrial functions, and supporting cellular functions such as the influx of calcium and production of reactive oxygen species [3,4].

Figure 1. . Chemical structures for investigated endocannabinoids.

(A) Anandamide (N-arachidonoylethanolamide). (B) 2-arachidonoyl glycerol.

CB receptor agonists have been used as therapeutic agents for a broad spectrum of diseases such as multiple sclerosis (symptomatic treatment), spinal cord damage, aching, inflammation and similar syndromes, glaucoma, asthmatic problems, vasodilation related to progressive cirrhosis and malignacies; it is likely that CB agonists function as both neuromodulators and immunomodulators [5]. Various agonists were found to be involved in physiological and pathological functions including appetite, pain, sensation, memory and addiction [6]. Alteration of the EC system has been associated with a variety of disorders. For example, clinical EC deficiency is underlying the pathophysiology of migraine, fibromyalgia, irritable bowel syndrome and other dysfunctional conditions and is alleviated by medical cannabis [7]. Reductions in EC levels in different regions of the brain could lead to specific diseases. For example, reduction in the striatum is accompanied by cognitive impairments and Alzheimer's disease [6,8]. Alteration of EC concentrations in the cerebrospinal fluid plays a role in epileptic seizure disorders [9,10]. Also, ECs are involved in a cascade leading to long-term depression of hippocampal synaptic functions [11]. Thus, measuring the ECs – in brain tissue in particular – is crucial for diagnosis and prognosis of disease.

Measuring ECs accurately can be extremely challenging for several reasons; the highly lipophilic nature of the compounds makes them adsorb to the surfaces of sample preparation equipment and they are found at low concentrations in fluids and tissue. Importantly, ECs released in the interstitial fluid are quickly degraded enzymatically in order to terminate signaling [6]. At last, there is a considerable practical difficulty to accessing the brain tissue in vivo.

Although several reliable approaches for identification and measurement of ECs in the brain have been developed and validated for various animal species, as shown in Table 1, the main limitation of these methods is the requirement to analyze the samples after sacrificing animals [12,13]. Typically, the levels of ECs are analyzed directly by subjecting brain tissue to extraction followed by isolating or purifying the compounds of interest [14]. LC–MS or GC–MS can then be utilized to quantify the levels, but this method does not account for any postmortem changes that occur in the tissue. LC–MS has been used to quantify EC levels from blood plasma and serum using many different approaches [15]. It has been utilized to quantify a variety of biologically important lipids including ECs in combination with other metabolites and steroids as well as other important bioactive lipids [16,17]. Microflow LC–MS has also been utilized to increase sensitivity while measuring ECs in serum [18]. To prevent isomerization and increase stability, ultraperformance LC–MS was used in conjunction with toluene extraction for quantification of ECs [19]. Similarly, GC–MS and GC–MS/MS have been used to quantify ECs in bulk tissue and blood plasma. Isotope dilution-based GC–MS was used to determine AEA amount in rat plasma [20] and GC–MS/MS was used to measure AEA with a single solvent extraction as sample preparation [21]. The method has also been used in clinical studies to determine how EC levels change in the cerebrospinal fluid for patients who have multiple sclerosis [22]. Solid-phase extraction was used with LC–MS/MS to measure 14 different ECs from serum using an online approach which reduces analysis time [23].

Table 1. . Overview of current methods for measuring anandamide and 2-arachidonoyl glycerol.

Species	Biological sample	Purpose of study	Sample preparation and analysis	Analytical figures for AEA and 2-AG respectively	Ref.
Human	Plasma	Quantitation of ECs, prostanoids and steroids	Solid-phase extraction; LC–MS/MS	LOQ 0.1 ng/ml; 3.2 ng/ml	[16]
Rat	Tissues	Quantitation of ECs, ceramides and oxysterols	Solid-phase extraction; LC–MS	LOQ 1.7 pmol; 2.5 pmol (on column)	[17]
Black bear	Serum	Quantitation of 5 ECs in serum	Liquid–liquid extraction; microflow LC–MS/MS	LOQ 1.7 pg/ml; 13.4 pg/l	[18]
Human	Plasma	Quantitation of ECs	Liquid–liquid extraction; LC–MS/MS	LOQ 0.5 nM (AEA only)	[19]
Human	Plasma	Quantitation of AEA and ethanol amides	Liquid–liquid extraction; GC–MS/MS	LOQ 0.25 nM (AEA only)	[21]
Human	Serum	Quantitation of 14 ECs in serum	Solid-phase extraction; LC–MS/MS	LOQ at pg/ml level for all 14 ECs	[23]
Rat	Hypothalamus	Regulation of EC release by CB1 receptors	MD, microSPE; LC–MS/MS	LOQ 30 amol; 300 amol (on column)	[24]

2-AG: 2-arachidonoyl glycerol; AEA: Anandamide; CB: Cannabinoid; EC: Endocannabinoid; LOQ: Limit of quantitation; MD: Microdialysis; SPE: Solid phase extraction.

In addition, in vivo methods have also been developed to determine the concentration of ECs. Several microdialysis (MD)-based methods have been proposed, but they suffer from high variability due to the inherent difficulty of sampling lipids using cellulose membranes and aqueous buffers (artificial cerebrospinal fluid) which can cause low recovery rate [14,25,26]. The difficulty in sampling hydrophobic molecules can be alleviated by using low dialysate flow rate or using binding agents on the probes, but these modifications affect the temporal resolution and the surrounding tissue [26]. With MD there can also be variability in quantity of ECs measured caused by trauma due to MD probes which are usually a few millimeters in diameter [26]. MD has been used to sample ECs in peripheral blood on several occasions, but the technique was deemed to be unsuitable for accurate quantification of EC concentrations in tissues due to poor temporal resolution and any local physiological changes that might occur due to hydroxypropyl-β-cyclodextrine (added to facilitate lipid transfer) crossing the MD membrane into the tissue [27]. In another study, MD was combined with solid-phase extraction and chromatography with tandem mass spectrometry to detect AEA and 2-AG in hypothalamus of live rat brains to determine role of CB1 receptors in regulation of ECs [24]. These findings indicate that a new more accurate approach is needed to measure ECs in vivo accurately.

Solid-phase microextraction (SPME) is a new minimally invasive in vivo technique for collecting samples that is gaining popularity as an alternative to MD and aliquot removal; the experimental approach consists of exposing a small amount of immobilized extraction phase to the sample for a precise period of time. Major advantages of the technique include small size of the probe, tubing- and attachment-free operation, and excellent sensitivity for lipophilic compounds [28–31]. Microextraction has been used for in vivo sampling for more than a decade, and has evolved to the point where applications for brain sampling and even monitoring in human tissues are being reported [32–34].

To the authors’ best knowledge, there are no previous reports dealing with measurement of EC levels using in vivo microextraction or biosensors. The present research details the application of biocompatible solid-phase microextraction probes for in vivo monitoring of AEA and 2-AG in two brain regions in rats exposed to various stress-inducing noise conditions.

Materials & methods

Chemicals & materials

AEA (5 mg/ml), AEA-D4 (internal standard, 1 mg/ml, deuterated at the hydroxyethyl 1,1′,2, and 2′ locations), 2-AG (5 mg/ml) and 2-AG-D5 (internal standard, 0.5 mg/ml, deuterated at the 1, 1, 2, 3 and 3 locations on the glycerol group) were acquired from Cayman Chemical (MI, USA). Polyacrylonitrile (PAN; precursor contamination below 4.85 ppm), methanoic acid, N,N-dimethylformamide, ammonium acetate (MS grade), gradient grade acetonitrile, gradient grade methyl alcohol and phosphate buffer saline (pH = 7.4) were purchased from Krackeler Scientific (NY, USA, vendor for Sigma-Aldrich). Stainless steel wires (125 μm radius) were acquired from Small Parts Inc (FL, USA). The RP-amide-C16-silica particles, which contain palmitamido-propyl as the chemically linked phase, were purchased from Supelco (PA, USA). This extraction phase is composed of granules with 0.2 m²/mg surface area, 18 nm pore size and are spherical in shape with a radius of 2.5 μm.

Preparation of SPME fibers

The microextraction fibers with bonded extraction phase were manufactured as shown in previous publications [35,36]; furthermore, these probes were previously shown to be biocompatible and suitable for in vivo experiments. 10% PAN in dimethyl-formamide (by mass) was mixed with RP-amide-C16 and heated to 40°C to make the coating suspension. Medical-grade steel threads (with a radius of 125 μm) were trimmed to 20 mm length, the last 1.5 mm from the terminal end was covered with the PAN/RP-amide suspension and then allowed to dry.

In vivo microextraction approach

The newly proposed approach for in vivo monitoring of EC concentrations was developed and validated according guidelines established in previous publications [31,37,38].

Analysis of SPME fibers

The fibers employed in the current research were analyzed by desorption in acetonitrile (with 25% water by volume). The fibers were placed in total-recovery poly-propylene conic inserts with 35 μl fluid for desorption and shaken for a previously determined interval (starting with a few minutes and increasing in increments up to 45 min) at 12.5 rotations per second on a shaking platform. Subsequently, the fibers were withdrawn from the total-recovery inserts and 5 μl solution with deuterium-labeled analogs as internal standards were added in order to control for variation in injection volume and mass spectrometry ionization conditions. The resulting solution was thoroughly mixed to homogeneously distribute the deuterium-labeled analogs within the liquid to be analyzed. The fluid with internal standard was submitted for analysis by LC–MS/MS.

Sampling matrix for laboratory tests

To establish novel microextraction approaches, matrices with standard constitution that resemble the actual samples are typically utilized. As formerly demonstrated, agarose gel mimics the viscosity of the interstitial fluid of live organisms to a remarkable degree [39–42]. Accordingly, to determine the optimal sample extraction time and quantifiable concentration range, all samples were prepared in 1% agarose gel to which relevant amounts of the investigated ECs were added.

Optimal extraction time

21 SPME fibers were inserted in 21 tubes with agarose gel samples containing ECs at a level of 10 ng/ml. The probes were then divided into groups of three to be collected and analyzed at the following time points: 5, 10, 15, 20, 30, 45 and 60 min.

Quantifiable concentration range

To determine the linear dynamic range, 21 coated fibers were used in groups of three for seven predetermined EC levels. The fibers were then inserted in the test tubes with agarose gel making sure that all the extraction coating at the end of the fiber was introduced in the sample solution. Extraction occurred for the optimal time interval formerly established from the extraction time profile experiments (30 min for both compounds).

Quantitative analysis by chromatography with tandem mass spectrometry

The polypropylene tubes obtained after desorbing the fibers were placed in the proper sample phials and processed with a Waters ACQUITY UPLC instrument (Waters, MA, USA) furnished with a triple quadrupole detector with electrospray ionization (AP-ESI) source. The temperature of the chromatography and autosampler modules were kept at 40 and 4°C. Partial loop with needle overfill sample introduction was used to inject 10 μl desorption fluid onto the column. After each injection, the autosampler was washed with 1 ml solution with high elution power (50% methyl alcohol in water by volume) and 0.6 ml solution with low elution power (5% methyl alcohol in water by volume). The AP-ESI source was set to positive ionization with an ion spray voltage of 3500 V. N₂ was utilized as ion source gas at flow rates of 50 l/h for nebulization and 500 l/h for desolvating the mobile phase. The mass spectrometer inlet and the nebulization gas were kept at 150 and 400°C.

Analytical separation was done on a bridged ethylene hybrid octadecyl column, 0.21 × 5 cm, containing 1.7-micron particles (Waters). The mobile phases A and B consisted of 0.01 M CH₃COONH₄ and acetonitrile, flowing at 300 μl/min. The gradient program started from 90% A which was decreased to 10% A until 3 min and was returned to the opening percentage of 90% A from 3 to 3.5 min. Argon flowing at 300 μl/min was utilized for collisionally activated dissociation in the second quadrupole. The target compounds and the internal standards were quantified in multiple reaction monitoring (MRM) mode with the specific transitions 348.4→62.0 (quantitation) and 348.4→287.3 (confirmation) for AEA, 352.4→66.0 for AEA-D4, 379.4→287.3 (quantitation) and 379.4→91.0 (confirmation) for 2-AG, and 384.4→287.3 for 2-AG-D5.

The Waters Acquity system was operated with Empower 2 program (Waters) and chromatography data were processed automatically.

In vivo analysis protocol

Subjects

24 male Sprogue–Dawley rats (IN, USA) with a weight of around 285 g at onset time were used. The rats were kept in plastic containers with timber chips, with lined covers allowing the rats access to food and drink at will. The environment in the animal facility was kept at the same moisture and warmth, with a half day light and half day dark cycle. The experiments were done in the interval from 08:00 to 12:00 during the daily low point of the hypothalamic–pituitary–adrenal axis. The experimental methods were examined and authorized by the Institutional Animal Care and Use Committee of the University of Colorado and conformed to the United States of America National Institute of Health Guide for the Care and Use of Laboratory Animals (IACUC protocol number 2573, approved on 11/08/2017). The experimental approach was designed to reduce as much as possible the discomfort and the number of animals utilized for the study.

Surgery

Following a week of acclimation to the colony and daily handling, all rats were surgically implanted with a unilateral indwelling guide cannula (21 G – #C312G, Plastics One, Inc., VA, USA) above the anterior bed nucleus of the stria terminalis (BST – coordinates, 0 mm anterior, 0.8 mm lateral and 7.2 mm ventral from bregma; n = 11) or the posterior hypothalamus (PH – coordinates, 3.3 mm posterior, 0.2 mm lateral and 7.4 mm ventral to bregma; n = 12), according to the rat brain atlas of Paxinos and Watson, 1998. The surgical procedures were performed as in Day et al. [43], and following guide implantation, a stylet extending 1 mm below the guides were inserted and dust caps screwed on to keep them clean and patent. Rats were then housed individually in polycarbonate tubs and allowed to recover for a period of 5–8 days prior to any additional experimental procedures.

Experimental design

Experimental animals were arbitrarily allocated to a type treatment (one group of 5–6 rats for four types of treatment). The groups were based on brain region as the first factor (anterior BST or PH) and loud noise stress or no noise control treatment as the second factor (2x2 factorial design) to assess the concentrations of the compounds AEA and 2-AG as a function of the following conditions: no noise control, acute loud noise and repeated loud noise exposures. On the first experimental day, the stylet from each rat was gently replaced with a solid-phase microextraction probe (Figure 2) that allowed the capture of AEA and 2-AG and the rats were placed in their home cages in acoustically attenuating chambers (described in detail in [43]) for 30 min in the presence of background noise (mostly fan noise of approximately 57 dBA) or loud noise (100 dBA). Treatment initiation was staggered by 5-min intervals to ensure precise standardization of procedure timing. Immediately following the 30-min control or noise procedure, rats were removed from the chambers and the microextraction probes were removed and frozen to −80°C. The same control or loud noise exposures were repeated for ten additional, consecutive days and on the last day (day 11), the same procedures were carried out as described for day 1, providing a second assessment of ECs, in the same subjects following control or repeated loud noise exposures.

Figure 2. . Schematic representation of the placement of microextraction probes in the brain for extracting endocannabinoids (not to scale).

Stainless steel wires (0.25 mm in diameter) coated with RP-amide-C16 with 10% polyacrylonitrile in dimethylformamide solution were placed in the surgically implanted unilateral indwelling guide cannula above either bed nucleus of the stria terminalis or posterior hypothalamus.

Probe & sample stability

To test the stability of the analytes during transportation and storage, 15 probes were loaded with analytes from agarose gel samples containing 10 ng/ml AEA and 2-AG. Three probes were analyzed on day 0, and the rest were frozen at -80°C. Batches of three probes were removed every 7 days and tested as described for probe desorption and LC–MS/MS analysis.

Results & discussion

SPME offers numerous advantages for in vivo analysis in the brain, such as rapidity, minimal invasiveness as there is no need to draw blood or excise tissue, unchanged sample volume, accuracy and ruggedness [28,44]. Compared with other in vivo techniques such as MD which allows for continuous extraction of various compounds, SPME can offer a more practical and useful approach to EC quantitation due to its smaller size; also, SPME probes do not need tubing and pumps to work which makes it easier to collect samples from freely moving organisms [26,28,44]. SPME can be used with both hydrophobic and hydrophilic substances as well as substances that disintegrate quickly and have a short lifespan [26]. It also reduces ionization suppression which could occur when using MD [28,44]. Although MD has been used to measure ECs in the past, this is the first time to our knowledge that SPME has been used for EC quantitation directly from tissue.

SPME method validation

Assay validation and standardization were performed as recommended by the previous studies for creating microextraction approaches for investigation of live organisms [28,30,32,37,44–47]. Intraday relative standard deviations (RSD) and interday RSDs were calculated at three different concentration levels (0.1, 1.0 and 10 ng/ml; n = 6) and were found to be less than 15 and 20%, respectively. It should be noted here that RSDs and matrix effects were kept significantly low by using isotopically-labeled internal standards (which elute at the same retention time as the analytes and are influenced the same way by the matrix or injection volume). The lower limit of the assay, determined based on a ten-times signal to noise ratio, was below 0.05 ng/ml, which was sufficient for the current in vivo application. Assay accuracy was calculated based on the ratio of the level determined by SPME to the level added to agarose gel and was shown to be in the range 121% for lower levels (0.1 ng/ml) and 97.8% for upper levels (40 ng/ml).

Analysis of SPME fibers

To determine the amount of residual compound on the probe after the first desorption, carryover tests were performed. These trials were performed by extracting from samples with known concentrations, followed by repeated desorption procedures. Usually, a carryover below 5% is satisfactory for a microextraction method. Using this set of experiments, it was found that the best desorption solution for back-extracting the ECs from the probes is acetonitrile with 25% water by volume. With this procedure, the carryover was less than 3% for AEA and less than 4% for 2-AG after 20 min desorption (Figure 3). Although longer desorption times provided somewhat lower carryover values, a duration of 20 min desorption was chosen for convenience.

Figure 3. . Desorption time profile for probes loaded with anandamide and 2-arachidonoyl glycerol.

After extraction, microextraction probes were placed in acetonitrile-water (75:25 by volume) for 10, 20, 30 and 45 min to determine the optimal desorption time. As desorption time increased, % carryover decreased (experiments were performed in triplicate).

AEA: Anandamide; 2-AG: 2-arachidonoyl glycerol.

Extraction time profile

The extraction time is the interval of time it takes for the analytes to migrate from the sample into the extraction phase while the extraction phase is in contact with the sample. To determine and select an optimal equilibrium or pre-equilibrium extraction time for in vivo experiments, in vitro extraction time course experiments were performed. The best extraction time should give an optimal range for method reproducibility, sensitivity and convenience. The SPME fibers were created and designed specifically for in vivo experiments by changing the size and material of the coating to work well with brief extraction times and at an adequate limit of quantitation. EC levels in the laboratory-prepared samples for the extraction time profiles were chosen to be around the predicted in vivo interval (10 ng/ml). The time needed to achieve extraction equilibrium was found from the data for AEA and 2-AG as presented in Figure 4. When conducting quantitative determinations, the time chosen for contact with the sample should at least the same as the time required to get to equilibrium in order to obtain suitable reproducibility and optimum sensitivity. For the compounds used in this study, the time to get to at least 95% of equilibrium was 30 min for both compounds, with AEA reaching equilibrium a little faster.

Figure 4. . Extraction time profile for anandamide and 2-arachidonoyl glycerol.

The experiments were performed in vitro using solutions of 10 ng/ml of each compound in 1% agarose gel; groups of three probes were collected and analyzed at the following time points: 5, 10, 15, 20, 30, 45 and 60 min. ‘n’ represents the amount extracted and ‘n0’ the maximum amount extracted (at equilibrium); error bars represent one standard deviation (experiments were performed in triplicate).

2-AG: 2-arachidonoyl glycerol; AEA: Anandamide.

Quantifiable concentration range

The levels for assay validation were chosen to cover the predicted endogenous window for both analytes. The quantity of compound detected was graphed versus the prepared level and the window within which the signal was proportional to concentration (r² >0.99) was obtained. The lower LOQ was determined as the level calculated from a signal ten-times higher than the noise of the chromatogram. The largest level when a proportional signal was obtained with a coefficient of variation less than 15% was considered the higher LOQ. The quantifiable range evaluated in this study was 0.05–50 ng/ml in agarose gel for both compounds. A positive result was reported if the concentration detected was above the lower LOQ. The concentrations measured in vivo were found to be in the range of 0.3–40 ng/ml.

Probe & sample stability

No significant changes in the amount of analyte found on the probes was observed over a duration of 4 weeks of storage at −80°C. Differences from values at day 0 were similar to interprobe variability.

EC concentrations brain

The EC concentration was measured in BST or PH by using SPME probes on day 1 and day 11 to determine the levels acutely and after the rat had become habituated to noise. The SPME probes were placed in a desorption fluid composed of acetonitrile with 25% water by volume and analyzed using LC–MS/MS (see ‘Materials & Methods’ section). To show how the levels of ECs change in individual animals, Figure 5 presents the EC concentration for four different rats who were either in the no noise or noise condition and had SPME probes in either BST or PH. The levels of 2-AG were greater than AEA for three rats shown except the rat in ‘BST no noise’ condition, where the acute concentration of AEA was about 40 ng/ml. For the rats in ‘BST no noise’ and ‘BST noise’ conditions, the acute levels of 2-AG are greater than habituated levels, but the habituated AEA levels are slightly greater than acute in the ‘BST noise’ condition and the acute levels are greater in ‘BST no noise’ condition. The AEA and 2-AG levels are slightly greater in acute ‘PH no noise’ and ‘PH noise’ condition compared with the habituated concentration.

Figure 5. . Endocannabinoid concentrations in the brain in acute versus habituated conditions in four individual rats.

Each rat was assigned to treatment groups based on different brain region (BST or PH) and a no noise control or loud noise condition. AEA and 2-AG concentrations were measured on day 1 (acute) and repeated on day 11 (last day) using solid-phase microextraction probes after the rats had become habituated to their treatment condition. The concentrations represent measurements in individual rats, so no error bars were included.

2-AG: 2-arachidonoyl glycerol; AEA: Anandamide; BST: Bed nucleus of the stria terminalis; PH: Posterior hypothalamus.

The fold change in the average acute and habituated concentrations of AEA and 2-AG was determined in all animals for the four treatment groups (Figure 6). There was almost no change (ratio acute/habituated of 1) in concentrations for ‘BST, noise’ condition (both ECs) and ‘PH noise’ condition (for AEA) on day 1 versus day 11. For all the other experimental conditions, there were large changes in the levels of ECs (ratio more than 1). ‘BST no noise’ condition showed the greatest change for both AEA and 2-AG levels with 16-fold higher acute AEA levels. A previous study reports that with stress the AEA levels in the brain decrease and 2-AG levels increase and this effect is amplified by repeated exposure to stress; however, this can vary based on brain region and type of stressor [48]. Figure 6 shows that for AEA, average acute levels of AEA were greater than habituated levels for BST and PH no noise conditions, where the only stress was placement in a new cage, but the levels did not change when the rats were exposed to noise. The average acute 2-AG levels were greater than habituated for BST and PH no noise, and PH noise, but did not change for BST noise conditions. The variations in this study can be explained due to several factors. The rats were moved to a different box for audio exposure and EC level monitoring, and the stress caused by new environment could have affected the EC levels measured. In addition, since each rat could only be assigned to one experimental condition, the variations in endogenous levels of ECs and response to stress include interindividual variability.

Figure 6. . Fold change in the average levels of endocannabinoids in the brain for all experimental animals in acute versus habituated conditions.

BST no noise condition showed the greatest fold change for both AEA and 2-AG. Error bars represent the standard error of the mean.

2-AG: 2-arachidonoyl glycerol; AEA: Anandamide; BST: Bed nucleus of the stria terminalis; PH: Posterior hypothalamus.

Conclusion

SPME in vivo in the brain was found to be between 0.3 and 40 ng/ml, within the linear range of the proposed method. The EC response to stress varied in the rats for different brain regions which could be due to stress induced by a new environment as well as variation in brain chemistry. It would be important in future studies to add an additional naive control group (in which ECs are measured in the home cage within the animal colony) to consider putative effects of environmental novelty.

Future perspective

The proposed technique can be used in the future to measure EC levels in brain tissue from several different regions with accuracy to establish a baseline and monitor any variations. The EC system affects a variety of physiological processes and changes in EC levels can lead to a variety of disorders and diseases. Accurately monitoring the EC levels for individuals can lead to proper diagnosis and treatment. In addition, this simple extraction technique can be used to measure other endogenous substances to diagnose and treat other diseases and disorders.

Executive summary.

Background

• Endocannabinoids (ECs) regulate many cellular functions and their deficiency can cause many diseases and disorders.

• Current in vivo techniques used to measure ECs have trouble measuring hydrophobic and fast disintegrating compounds.

• Solid-phase microextraction (SPME) is a minimally invasive technique that uses small sized probes and can be used to measure endogenous compounds directly from the brain.

Methodology

• SPME fibers were manufactured by covering 1.5 mm of the terminal end of medical-grade steel threads with polyacrylonitrile/RP-amide mixture.

• Subsequently, the probes were used for in vivo sampling of ECs in 24 male rats from bed nucleus of the stria terminalis (BST) and posterior hypothalamus (PH) on day 1 and day 11 after repeated exposure to loud noise or no noise control.

• After sampling, the fibers were analyzed by desorption in acetonitrile-water (75:25 by volume).

• The desorption solution was analyzed using chromatography coupled with tandem mass spectrometry.

Results

• The quantifiable window was from 0.05 to 50 ng/ml.

• The in-lot reproducibility of fiber manufacturing was less than 14%; between-lot reproducibility was less than 19%.

• In vivo EC concentration range in the BST and PH regions was found to be 0.3–40 ng/ml.

Conclusion

• The SPME probes provided a minimally invasive way to collect ECs from rat brain while they were moving without the hassle of large machinery, tubing or pumps.

• The proposed assay was effectively used to quantify anandamide and 2-arachidonoyl glycerol levels from BST and PH in rat brain.