A novel restrainer device for acquistion of brain images in awake rats

Abstract

Functional neuroimaging methods like fMRI and PET are vital in neuroscience research, but require that subjects remain still throughout the scan. In animal research, anesthetic agents are typically applied to facilitate the acquisition of high-quality data with minimal motion artifact. However, anesthesia can have profound effects on brain metabolism, selectively altering dynamic neural networks and confounding the acquired data. To overcome the challenge, we have developed a novel head fixation device designed to support awake rat brain imaging. A validation experiment demonstrated that the device effectively minimizes animal motion throughout the scan, with mean absolute displacement and mean relative displacement of 0.0256 (SD: 0.001) and 0.009 (SD: 0.002), across eight evaluated subjects throughout fMRI image acquisition (total scanning time per subject: 31 min, 12 s). Furthermore, the awake scans did not induce discernable stress to the animals, with stable physiological parameters throughout the scan (Mean HR: 344, Mean RR: 56, Mean SpO₂: 94 %) and unaltered serum corticosterone levels (p = 0.159). In conclusion, the device presented in this paper offers an effective and safe method of acquiring functional brain images in rats, allowing researchers to minimize the confounding effects of anesthetic use.

1. Introduction

The value of neuroimaging methods in neuroscience research has grown significantly in recent decades. Techniques such as functional MRI (fMRI), magnetic resonance spectroscopy (MRS), diffusion tensor imaging (DTI), and positron emission tomography (PET) provide a non-invasive method of exploring the structure and function of the nervous tissue. These advanced imaging techniques have played a crucial role in advancing many fundamental areas of neuroscience, including the study of multiple sclerosis (Wood, 2021), Parkinson’s disease (Lin et al., 2022), Alzheimer’s disease (Sheng et al., 2022), traumatic brain injury (Simchick et al., 2021) and other neurological conditions (Yen et al., 2023).

A major barrier in applying some of these advanced techniques in preclinical animal models is the necessity to minimize head motion during image acquisition. Typically, animal data is acquired under general anesthesia to ensure the animal’s head remains still throughout the imaging sequence. Nevertheless, anesthesia can have profound effects on brain metabolism and selectively alter dynamic neural networks by reducing functional connectivity within the frontotemporal association cortex and higher-order thalamocortical networks (Hudetz, 2012). Additionally, anesthetics can introduce confounding factors in fMRI by interfering with neuronal activity, cerebral vasculature, and neurovascular coupling (Masamoto and Kanno, 2012; Pan et al., 2015). Similarly, the use of anesthetics can affect cerebral metabolic rate and brain metabolite concentration, directly influencing the results of MR spectroscopy (Du et al., 2009). Therefore, to optimize data quality and ensure an accurate representation of neurological processes within a living animal, it is ideal to acquire functional brain images in fully awake and alert animals.

Over the years, numerous research groups have made efforts to obtain brain images in awake rodents, albeit with varying degrees of success. Some groups have explored the implantation of a head fixation clamp within the animal’s skull, which can be secured to a fixed plate (Chang et al., 2016; Hori et al., 2016). While this approach yielded promising results, it carries the risk of head injury and proves highly impractical for experiments requiring further manipulation of the skull. Other groups have evaluated the use of muscle relaxants to prevent movement during image acquisition (Xi et al., 2004). However, this method necessitates animal intubation and ventilation, which have been associated with significant morbidity and mortality (Zheng et al., 2020). Arguably, the most effective approach for obtaining awake images in rats involves utilizing a well-designed restrainer system that restricts any motion of the body or head without causing significant discomfort or distress to the animal. However, the number of such devices presented in the literature is highly limited, with each of them having their own intrinsic limitations (Mandino et al., 2024). For example, a restrainer device developed by Stenroos et al. (2018) is only compatible with a standard Bruker Biospin MRI rat bed, while another well-established device by Ferris et al. (2011) does not support the use of a surface coil.

In this paper, we present a novel head fixation device that has been developed in our laboratory. The device prioritizes the safety and well-being of animals while offering a high degree of stability and customizability. Furthermore, we present the findings of a comprehensive validation study, which supports an excellent level of motion control with minimal physiological stress. We anticipate that this advancement in motion control will serve as a valuable resource for fellow researchers, enabling them to acquire more consistent scans in awake rats.

2. Methods

2.1. Ethical statement

All experiments were conducted under a protocol approved by the Cleveland Clinic Institutional Animal Care and Use Committee (IACUC) with the protocol number 2019–2133. The use of animal images and videos was approved by the institutional veterinarian, adhering to Cleveland Clinic’s “Policy for Photography of Laboratory Animals in Animal Facilities and Laboratories”. The animal care was undertaken in line with the National Institues for Health guide for the care and use of laboratory animals, and directly supervised by the institutional Biological Resource Unit.

2.2. Head fixation device

We aimed to construct an effective and easy to use head fixation device, which can accommodate a range of animal sizes and MR imaging environments. The device, along with its key components, is shown in Fig. 1.

Fig. 1.

The images of the device highlighting its relevant components and features (Top– side view; bottom left– front view; bottom right – back view).

The primary function of this device is to immobilize the head of the awake rat while ensuring minimal discomfort. Fixation features are constructed around a central chamber shaped to accommodate the animal’s snout (A) situated at the anterior end (i.e. toward head) of the base (B). The chamber includes a passthrough port (C) to allow for volatile anesthetic administration as needed. The anterior end of the device features an adjustable-height controller (D) to allow the Delrin^® toothbar (E) to be raised or lowered in order to accommodate animals of different sizes. Once the animal is mounted, the toothbar can be raised to secure the animal’s snout between the toothbar and the roof of the chamber. To minimize discomfort, we recommend placing a layer of soft material on the chamber’s roof. Secondary support is provided by fixations arms (F) that extend posteriorly off the lateral margin of the central chamber along the side of the animal’s head. Four threaded slots in each arm provide flexibility for the placement of fixation screws that can be used to limit lateral head movement. The tips of the screws should be covered with a soft material to minimize discomfort. Depending on the size of the animal, a reusable neck positioning piece can be placed to optimize the horizontal alignment of the head. Finally, the base portion of the unit incorporates multiple slits (G) that allow for the attachment of gauze (H) or other restraint material to further limit movement of the animal’s neck and body. Once in place, a piece of light fabric can be used to wrap and secure the fore- and hindlimbs.

In order to minimize distress and ensure reliable placement, we recommend anesthetizing the animal just prior to its placement in the device. To facilitate this process as well as to allow for experimental designs that include prolonged or intermittent anesthesia use, the design includes an anesthesia port (C) that allows for continuous delivery of volatile anesthetic to the animal once it is placed in the device. For detailed instructions on how to use the device and appropriately place the animal, please refer to the supplementary materials, which include a video demonstration (Appendix A).

2.3. Animal acclimatization

To optimize the data collection process, it is important to provide adequate acclimatization for each animal. In this experiment, we utilized an acclimatation protocol developed by our laboratory based on the group’s previous experience and best available literature (King et al., 2005; Russo et al., 2021) This involves three repeated acclimation session taking place in the weeks prior to the initial scan acquisition. These sessions should be conducted in a designated area for animal handling and closely simulate the scanning process. It is advisable to place the animal onto the head fixation device and secure it to the MRI bed that will be used in the actual experiment.

During the first acclimation session, we allow the animal to become familiar with the device and the sensation of being restrained. This session should last approximately twenty minutes and it is recommended to cover the animal’s eyes during the process. Providing positive reinforcement (e.g., offering treats) when the desired behavior is exhibited can help reinforce positive associations. For the second session, we follow a similar procedure but extend the duration to forty minutes. This longer session allows the animal to further adapt to the device and the handling process. During the third session, also lasting forty minutes, we play the sound of the MRI machine at a loud volume near the animal. This step helps the animal become accustomed to the noise it will experience during the actual scanning procedure. It is recommended to provide at least one full day of rest between each of the acclimation sessions to minimize stress on the animal.

By emulating the actual scanning environment, these acclimation sessions help the animals to adjust the head fixation device and reduce potential stress during the scan acquisition.

2.4. Validation experiment

A total of eight naïve male rats (Long-Evans, 225 +/− 25 g, purchased from Envigo, LLC) were used to validate the head fixation device. The rats were individually housed on a 12-h light/dark cycle and kept in a vivarium maintained at a temperature of 15–21 °C and humidity of 40–60 %. All subjects underwent the acclimatization protocol described above prior to imaging.

The rats were anesthetized using 2 % isoflurane and carefully placed onto the device following the steps demonstrated in the Supplementary video. At this stage, 150 μL of venous blood was drawn from the tail vein to obtain a baseline, pre-scan corticosterone measurement. Subsequently, the device was positioned onto the MRI bed and inserted into the 7T BRUKER BioSpec 70/20 USR MRI System (Bruker, 2023).To monitor the rat’s vital signs, a respiratory probe (Small Animal Instruments, Inc) was securely taped to its body and a fiber optic pulse oxygen meter (Small Animal Instruments, Inc.) was attached to the hind paw. The respiratory probe facilitated continuous monitoring of respiratory rate and pattern, while the pulse oximeter facilitated monitoring of animal’s heart rate and oxygen saturation. Finally, a thirty-millimeter surface coil (manufacturer) was placed over the rat’s scalp to enhance image quality. Once the subject was stable and exhibited appropriate physiological parameters, an MRI localizer scan was acquired, and the position within the scanner readjusted as necessary. This was followed by B0 map to quantify and correct subject-induced geometric distortions. Lastly, a high-resolution anatomic data were acquired using a 3D TurboRARE with 60 0.2 mm thick slices (TE=36 ms; TR=1400 ms; RARE factor=8; FOV = 30×35 mm2; matrix=150×175; in-plane resolution = 0.2 × 0.2 mm2).

Upon the of the aforementioned steps (requiring between seven to ten minutes), the delivery of isoflurane was stopped, and a five-minute count began. At the conclusion of the five-minute countdown, three consecutive whole-brain fMRI scans were acquired, each scan lasting 10 min and 24 s. The scans were acquired using segmented spin-echo, echo planar imaging with 35 contiguous 0.8 mm thick axial slices (echo time (TE)=15 ms; repetition time (TR)=1040 ms; flip angle (FA)=90°; field of view (FOV)=35×18 mm2; matrix=125×64; in-plane resolution = 0.28×0.28 mm2; 200 vol). Following a total period of 31 min and 12 s of awake fMRI scan acquisition, isoflurane (1.5 %) administration was resumed and 150μL of venous blood was again drawn from the tail vein to obtain a post-scan corticosterone measurement. Thereafter, anesthesia was discontinued and the animal was carefully removed from the device.

2.5. Data analysis

The fMRI scans obtained in this validation experiment were used to quantify the level of motion during the awake stage. Across the three fMRI scans, a total of 600 volumes were acquired, each providing a ~3.12 second snapshot of the rat’s spatial position in relation to the center of the MRI scanner. The volumes that were significantly affected by the motion artifact on visual inspection, or displayed a relative displacement of > 0.28 mm (i.e., the size of the smallest voxel dimension), were identified and replaced with a ‘dummy volume’ (i.e. the exact copy of the preceding volume) as per established research protocols (Power et al., 2012, 2014) (Appendix B). The MCFLIRT algorithm was employed to evaluate the extent of motion between each acquired volume (Jenkinson et al., 2002). The analysis focused on determining the mean absolute and relative displacement, with the former reflecting the overall displacement of the subject during the entire imaging session, and the latter representing the average displacement between individual fMRI volumes. Additionally, we examined the extent of rotation and translation across all three planes throughout the scan acquisition.

During the acquisition of the awake scan, a close monitoring of vital signs (i.e., heart rate, respiratory rate, and oxygen saturation) was carried out. The mean, maximum, and minimum values for each of these parameters were recorded and are presented in the result section.

Finally, the blood samples obtained during the validation experiment were centrifuged at 3500RPM for 15 min to separate out the serum, which was stored at −80C°. The enzyme-linked immunosorbent assay (ELISA) of corticosterone was performed with Abcam AB108821 Assay Kit as per the manufacturer’s instructions.

3. Results

3.1. Motion assessment

The number of fMRI volumes that have to be replaced with “dummy volumes” due to motion artifact can serve as index of the overall motion contamination present throughout the study. For the experiments described, the number of motion-contaminated volumes ranged from 4 to 11 across the eight animals tested, representing 0.6 % to 1.8 % of the total volumes acquired. Individual values for each animal are presented in Table 1.

Table 1.

The number of acquired volumes that had to be removed due to motion.

Subject name	Number of Volumes Affected by Motion Artifact (across all three fMRI scans [600 volumes total])
Rat 1	10
Rat 2	5
Rat 3	8
Rat 4	4
Rat 5	8
Rat 6	5
Rat 7	9
Rat 8	11

After the exclusion of the motion affected volumes, the MCFLIRT algorithm was utilized to assess the overall level of motion between each individual volume across the entire awake acquisition. The mean absolute and relative displacement across the entire cohort were 0.0256 (SD: 0.011) and 0.009 (SD: 0.002) millimeters, respectively. The motion data for each individual rat is further summarized in Table 2. Importantly, there was no statistically significant difference in the total displacement across the three fMRI scanning sessions (ANOVA, p = 0.27) For a more detailed summary regarding the overall displacement, rotation, and translation along the x, y, and z axes throughout the acquisition of individual scans, please refer to the supplementary material (Appendix C).

Table 2.

The mean absolute and relative displacement for each subject, across all three fMRI scans.

Subject name	Mean absolute displacement (in mm)	Mean relative displacement (in mm)
Rat 1	0.0200	0.0075
Rat 2	0.0125	0.0075
Rat 3	0.0125	0.0075
Rat 4	0.0363	0.0113
Rat 5	0.0325	0.0113
Rat 6	0.0213	0.0088
Rat 7	0.0275	0.0100
Rat 8	0.0425	0.0088
Cohort Mean & Standard deviation	0.0256 (SD: 0.011)	0.009 (SD: 0.002)

3.2. Physiological measurements

The mean, minimum, and maximum recorded values of heart rate, respiratory rate, and oxygen saturation for each of the eight rats are further summarized in Fig. 2.

Fig. 2.

The recorded mean, maximum, and minimum values of heart rate, respiratory rate, and oxygen saturation across the eight rats.

Moreover, we assessed serum corticosterone levels both before and after the awake scan acquisition to gauge the extent of stress induced by the scanning process. Fig. 3 provides a visual representation of cortisol measurements at the beginning and end of the scanning session for all eight rats. Among the subjects, the average baseline serum corticosterone level stood at 210.74 ng/ml, increasing slightly to 216.52 ng/ml by the conclusion of the scan. Notably, there was no statistically significant disparity in cortisol values between the scan’s onset and conclusion, as indicated by a paired t-test (p = 0.159).

Fig. 3.

Serum corticosterone concentration at baseline and at the conclusion of the scanning session.

Finally, it is crucial to note that none of the rats suffered mortality or displayed any signs of disability throughout the course of the experiment.

4. Discussion

In this manuscript, we presented a head fixation device developed in our laboratory to facilitate the acquisition of awake scans in rats. In general, employing a restrainer device has several distinct advantages when compared to alternative methods of immobilization. Unlike the “head fixation clamp” method, it allows for scan acquisition in naïve rodents whilst permitting additional surgical manipulation of the skull. In contrast to the “muscle relaxant” approach, a restrainer device is generally considered safer, with a lower associated mortality and morbidity rate. To the best of our knowledge, only two such devices have been frequently cited in the literature (Ferris et al., 2011; Stenroos et al., 2018)

Directly comparing the efficacy of our device with other devices in the literature presents a unique challenge due to varying methods of quantifying motion. The majority of studies utilize framewise displacement and translation to quantify motion (Mandino et al., 2014), while our group opted to use a related, yet distinctly different metric of mean / absolute relative displacement. However, it should be emphasized that our device provided a satisfactory level of motion control with the mean absolute displacement representing less than 10 % of the size of the shortest voxel – a value widely considered appropriate for fMRI acquisition (Stenroos et al., 2018). Similar to the vast majority of other devices, our restrainer necessitated the use of robust acclimation protocol to achieve optimal results (Reed et al., 2013). Additionally, our device required the use of anesthesia for animal mounting, which represents a common practice across the awake MRI restraining devices. Nonetheless, it is critical to acknowledge that even brief exposure to anesthesia may induce lasting effects on cerebral networks, thus confounding the fMRI results (Stenroos et al., 2021). Importantly, the main distinguishing feature of our device is its versatility, as it can be used with nearly any MRI bed, as well as support the use of both surface and volume coils.

It is also important to note that we did not observe any substantial impact on the animals’ stress levels and physiological parameters during the awake scans. The average change in serum corticosterone levels before and after the awake scan was negligible, implying that the animals experienced little to no physiological stress during the procedure. This observation is not common in the field of awake animal acquisition, with the majority of research groups reporting that the use of their respective restrainer devices induced a significant stress response in experimental animals. (Suzuki et al., 2021; Sung et al., 2009). The absence of such a response in our cohort may be attributed to a robust acclimatation protocol, as well as the use of isoflurane for animal placement, which could have attenuated the overall stress response (Flezzani et al., 1986).

While most physiological measurements fell within acceptable limits, respiratory rate tended to hover towards the lower end of the physiological range. This could be attributed to the residual effect of isoflurane used for animal placement, which is known to decrease respiratory rate. It is also plausible that the low respiration could have been due to the compression of rat’s body by the restraining fabric, thus in increasing the work of breathing. However, stable and high oxygen saturation metrics were reassuring, suggesting that despite the low respiratory rates, the experimental rats were not hypoxic.

Finally, it is essential to highlight that none of the animals exhibited any long-term morbidity or mortality as a result of this experiment. Nevertheless, it should be noted that during the development of the device, we did encounter a few isolated cases of restrainer-associated mortality. We suspect these incidents were primarily a result of an acute restraint stress response, leading to sudden cardiac arrest (Liu et al., 2016; Stalnikowicz and Tsafrir, 2002). In the event of such an incident, characterized by a sudden drop in oxygen saturation and heart rate, we advise the operator to promptly remove the animal from the restrainer. This immediate action can significantly mitigate the risk of mortality and enhance the chances of successful recovery.

In summary, our study has introduced and assessed the utility of a head fixation device developed in our laboratory for conducting awake scans in rats. This device demonstrates effective motion control, facilitating the acquisition of high-quality awake brain scans while minimizing the impact on animals’ stress levels.

Abbreviations:

MRI

Magnetic resonance imaging

fMRI

Functional magnetic resonance imaging

MRS

Magnetic resonance spectroscopy

DTI

Diffuse tensor imaging

CT

Compute tomography

SPECT

Single photon emission computed tomorgraphy

PET

Positron emission tomography

ELISA

Enzyme-linked immunosorbent assay

Data availability

Some data has been made available through a public repository, while other data points will be made available on request. For further information, please view Data Availability Statement