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52 (); 12-16
doi:
10.1016/j.jor.2024.02.012

Evaluation of brain activation related to resting pain using functional magnetic resonance imaging in cynomolgus macaques undergoing knee surgery

, , , , , ,
a Department of Orthopaedic Surgery, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan
b Pharmacology Group, Hamamatsu Pharma Research, Inc., Hamamatsu, Shizuoka, Japan

∗Corresponding author: Mitsuru Hanada. mitsuruhanada@gmail.com

Received: , Accepted: ,
Professor P K Surendran Memorial Education Foundation
Disclaimer:
This article was originally published by Reed Elsevier India Pvt. Ltd. and was migrated to Scientific Scholar after the change of Publisher.

Abstract

Abstract

Functional magnetic resonance imaging (fMRI) visualizes hemodynamic responses associated with brain and spinal cord activation. Various types of pain have been objectively assessed using fMRI as considerable brain activations. This study aimed to develop a pain model in cynomolgus macaques undergoing knee surgery and confirm brain activation due to resting pain after knee surgery.

An osteochondral graft surgery on the femoral condyle in the unilateral knee was performed on four cynomolgus macaques (Macaca fascicularis). Resting pain was evaluated as changes in brain fMRI findings with a 3.0-T MRI scanner preoperatively, postoperatively, and after postoperative administration of morphine. In the fMRI analysis, Z-values >1.96 were considered statistically significant.

Brain activation without stimulation after surgery in the cingulate cortex (3.09) and insular cortex (3.06) on the opposite side of the surgery was significantly greater than that before surgery (1.05 and 1.03, respectively) according to fMRI. After the administration of morphine, activation due to resting pain decreased in the cingulate cortex (1.38) and insular cortex (1.21).

Osteochondral graft surgery on the femoral condyle can lead to postoperative resting pain. fMRI can reveal activation in pain-related brain areas and evaluate resting pain due to knee surgery.

Keywords

Cingulate cortex
fMRI
Insular cortex
Knee surgery
Macaques
Resting pain
1

1 Introduction

Pain is a common disorder that leads to adverse effects on physical and social activities. Therefore, effective tools to understand the degree of pain and to standardize the assessment of treatment efficacy are needed. Because objective assessment of pain is difficult, it is assessed subjectively in clinical settings through patient self-reports using visual analog, numerical rating, verbal rating, and face rating scales. In animals, pain can be assessed by gait posture and limping during movement or reactivity to thermal stimulation.1–3 However, rhesus macaques might not exhibit responses against thermal stimulation because non-human primates (NHPs) hide awareness of pain and exhibit few behaviors against pain.4 Therefore, in clinical examinations of humans and animals, an objective evaluation of pain is important.

Brain hemodynamic responses associated with brain activation can be visualized by the procedure of functional magnetic resonance imaging (fMRI). fMRI can reveal brain reactions to pain stimulation and changes in the brain function related to chronic pain. Several studies have shown brain activation in animal models using fMRI.5–7 Amirmohseni et al.5 reported brain activation to noxious stimulations using a rat of postoperative pain model. Nagasaka et al.6 demonstrated that a cold stimulation to a macaque of chemotherapy-induced peripheral neuropathy model increased brain activation in the secondary somatosensory cortex and anterior insular cortex (IC). Hama et al.7 showed that in a macaque model with an abdominal incision, the IC and cingulate cortex (CC) were activated while applying pressure to the incision. Thus, brain activity may be a useful objective marker of the incidence and magnitude of pain. Furthermore, brain activation that reflects the incidence and magnitude of pain using fMRI in humans was reported,8 and the effect of analgesic treatment on pain was revealed by fMRI as a decrease in pain-related brain activation,9 and treatment strategies for pain have been suggested.10 Therefore, fMRI can assess the reaction of the brain to pain stimulation and can be used for the objective evaluation of pain.11,12

In previous studies investigating pain using fMRI, mechanical, chemical, or thermal stimulation was applied to patients.13–16 Furthermore, electrical, mechanical, chemical, and thermal stimulations have been used in animal models.17–20 In these studies, the stimuli were provided intermittently and turned on and off during fMRI. However, there are no previous studies to assess resting pain without any stimulation.

The current study was a preliminary experiment to develop a resting pain model in cynomolgus macaques after knee surgery. Brain activation was examined using fMRI to objectively evaluate resting pain.

2

2 Materials and methods

2.1

2.1 Animals

This study was approved by the ethics committee in our institution. It was complied with all relevant guidelines and laws in Japan related to the care and use of laboratory animals. We used four male cynomolgus macaques (Macaca fascicularis; EBS Co., Hashimoto, Japan), with weight range: 5.6–6.3 kg. The macaques were individually accommodated in cages with stainless steel that the AAALAC International accredited for primate. The care conditions were as follows, light-dark cycle per 12 h, feeding a diet (Oriental Yeast Co., Chiba, Japan), and having water freely. Furthermore, fresh fruits and vegetables were given, and animal care and research staff performed treatment.

2.2

2.2 Autologous osteochondral transplantation as a surgical procedure for the knee pain model

Macaques were anesthetized using ketamine (Daiichi Sankyo, Tokyo, Japan) with an intramuscular injection with 10 mg/kg. Body temperature was kept during the surgery using a heating pad. Deep anesthesia was performed based on no reaction to digit pinch and absence of corneal reflex. The animal was placed in the supine position, and under ketamine sedation, the right knee was shaved, and was disinfected using povidone-iodine. Under aseptic conditions, the articular surface was exposed with anterior skin incision of the right knee, and a medial parapatellar approach. An osteochondral graft (4 mm in diameter and 4 mm deep) was harvested from the proximal end of the femoral groove using a disposable biopsy device. In the same knee, the donee site was made at the weight bearing area of the femoral condyle as a cylindrical osteochondral defect (4 mm diameter), and the prepared osteochondral graft was transplanted. The capsule and skin wounds were sutured after all procedures.

2.3

2.3 fMRI examination

Brain activation without stimulations was visualized with magnetic resonance imaging (MRI) at 3 T (GE Healthcare, Milwaukee, WI) before surgery, approximately 48–49 h after surgery, and following morphine administration. Propofol was used to prevent the macaques from moving during fMRI examination. Persistence intravenous administration of propofol was done with 0.2 mg/kg/min to sedate the macaques., and the dose administered had little analgesic effect.21 Morphine (6 mg/kg) was intramuscularly administered to macaques, and fMRI was examined for 30–42 min after morphine administration.

The anatomical MRI examination was applied with a T1-weighted fast spoiled gradient-recalled sequence (repetition time [TR]/echo time [TE], 15.8/7.0 ms; number of averages, 1; flip angle, 12°; field of view, 150 mm × 150 mm; matrix, 256 × 224; slice thickness/interval, 1.0/0.5 mm; number of slices, 168). Sequences of functional scan included field-echo, echo-planar imaging (TR/TE, 3000/35 ms; flip angle, 90°; field of view, 140 × 140 mm; matrix, 64 × 64; slice thickness, 2.4 mm; number of slices, 30). The time interval of each scan was 10 min. The functional images were registered in the standard space through anatomical images. After MRI examinations was completed, the macaques were observed for recovery from propofol and were returned to their home cages.

2.4

2.4 Data analysis of fMRI

The slice time correction, motion correction using SPM12 (Wellcome Centre for Human Neuroimaging, London, UK), motion correction using the Functional Magnetic Resonance Imaging of the Brain Expert Analysis Tool (FMRIB Software Library, Oxford, UK), spatial smoothing due to a Gaussian kernel of full width at half maximum (4 mm × 4 mm × 4 mm), nonlinear and high-pass temporally filtering, and subtraction of the mean of each voxel time course from particular time course were done as preprocessing of the time series of fMRI volumes.

Two elements were derived from the spontaneous fluctuations in the magnitude of postoperative pain to examine brain activation. First, brain activation was identified for phases when the ongoing pain was high versus low, and the pain ratings were binarized relative to the mean of rating. Second, brain activation was identified for phases when subjective pain increased, according to a previous report by Baliki et al.22 The time epochs were binarized for pain whose positive change rate was sustained over the minimum period (9 s, 3 TRs) compared to all other periods. The visual vector was derived similarly.

A surrogate control was generated by inverting the recorded pain rating besides visual control. This protocol preserved all statistical properties of the original examinations, however the relationship between the ratings and actual pain fluctuations were scrambled, and non-specific activations were controlled. Each element (pain and vision) could constitute to generate a hemodynamic response by the convolution of the corresponding vector with a generalized hemodynamic function (gamma function lag, 6 s; standard deviation, 3 s). The head motion vector which derived from the motion correction was used as a covariate of no interest to exclude residual variance caused by head motion. The significance in the model fit for time series in each voxel was calculated, yielding statistical parametric maps for each participant and condition. Average group activity maps were generated by subtracting the visual and surrogate activity maps from the pain activity map in a second-level random and fixed effects group analysis, after co-registration of individual scans to a macaque brain template.23 A Z-value of statistically significant voxels revealing pain-related activation was obtained. Z-value >1.96 (p < 0.05) as peak voxels was considered significant using one-tailed t-test with an uncorrected for multiple comparisons.24

3

3 Results

All macaques recovered from anesthesia without complications after right knee surgery. Brain fMRI before surgery did not reveal significant activation (i.e., mean peak voxel Z values < 1.96) (Table 1, Fig. 1a). Forty-eight hours after right knee surgery, significant mean peak voxel Z-values were observed in the contralateral IC and CC (Table 1, Fig. 1b). After morphine administration, the activation in these areas was reduced (Table 1, Fig. 1c). This brain alteration was observed in all four macaques.

Table 1 Resting state peak brain voxel Z values before and after autologous osteochondral transplantation of the right knee, and after administration of morphine. Values in parentheses are stereotaxic coordinates (X/Y/Z in mm) according to the Horsley-Clarke stereotaxic coordinates.
Area Hemisphere Z values
Pre-surgery Post-surgery Post-administration
Insula Right 0.74 (−14/18/2) 0.96 (−14/18/4) 0.51 (−12/18/4)
Left 1.03 (20/20/4) 3.06a (18/20/4) 1.21 (16/20/6)
Cingulate cortex 1.05 (−2/-18/-6) 3.09a (−2/-18/-10) 1.38 (0/-20/-10)
a Peak voxels were considered significant (p < 0.05) at Z values > 1.96.
Coronal brain sections arranged from rostral (upper left) to caudal (lower right) according to fMRI. The images show the mean peak voxels of the four macaques. a: Prior to knee surgery, there is little or no brain activation. b: Forty-eight hours after surgery, significant activation of the CC and left IC was observed. c: Following a single dose of morphine, activation in the CC and IC is significantly decreased. R, right; L, left; fMRI, functional magnetic resonance imaging; CC, cingulate cortex; IC, insular cortex.
Fig. 1 Coronal brain sections arranged from rostral (upper left) to caudal (lower right) according to fMRI. The images show the mean peak voxels of the four macaques. a: Prior to knee surgery, there is little or no brain activation. b: Forty-eight hours after surgery, significant activation of the CC and left IC was observed. c: Following a single dose of morphine, activation in the CC and IC is significantly decreased. R, right; L, left; fMRI, functional magnetic resonance imaging; CC, cingulate cortex; IC, insular cortex.
4

4 Discussion

The current study demonstrated brain activation in the contralateral IC and CC without stimulation after unilateral knee surgery. As the IC and CC are key brain areas related to nociceptive processing, we consider that activation of the IC and CC without somatosensory stimulation suggests significant resting or spontaneous pain following knee surgery. Morphine reduced the post-surgery activation of the IC and CC, supporting the notion that these regions are involved in pain perception. The current findings reveal that it is possible to measure spontaneous brain activation as measured using fMRI in macaques, which could serve as an objective indicator of postsurgical pain. Furthermore, these findings suggest that these brain regions can be targeted for the evaluation and development of painkiller regimens for traumatic and postoperative pain.

Based on preclinical rodent studies, several potential therapeutics have been developed; however, only a few drugs that have been tested in rodents demonstrated efficacy in clinical trials.25–27 Since rodents are phylogenetically distant from humans as animal models, results in rodents may not always translate to humans. Conversely, NHPs are more phylogenetically and neuroanatomically similar to humans compared with rodents.28–31 Additionally, it may be possible to develop clinically relevant endpoints in large animal species, such as NHPs. Studies in humans have demonstrated significant activation of brain areas, including the IC, CC, thalamus, and brainstem, in reaction to brief harmful stimulation.17,20,32–34 Davis et al.32 showed that the IC and CC were enhanced because of pain from noxious cold stimulation as demonstrated by fMRI findings; this finding was similar to our observation. As in our study, Bingel et al.33 showed that the IC and CC were enhanced because of laser-evoked pain on fMRI. Lahti et al.17 reported that in reaction to electrical shock to the hind paw, the signal intensity increased in the contralateral somatosensory cortex under propofol anesthesia. Using fMRI as the imaging modality, several previous reports have shown signal enhancement of ICs in not only the contra-affected side but also the affected side and secondary somatosensory cortex following stimulation in only one extremity.20,34 However, pain stimulation in these studies might not be strictly performed in just one unilateral extremity, because pain due to pulsed or electrical stimulation might have been slightly transmitted to the other extremity. Although these previous reports showed contrasting results, the signal changes in the IC and secondary somatosensory cortex in the present study were considered indicative of resting pain after knee surgery. Additionally, significant signal enhancement on fMRI was not observed before knee surgery and disappeared after morphine administration.

This study has several limitations. First, animals for our examination were limited since experimental macaques were rare and very expensive. Statistical power analysis could not be performed due to the small sample size. However, Shirai et al.20 revealed that mean peak Z-values for brain activation following somatosensory stimulation could be calculated from eight macaques. Furthermore, several reports have included a similar number of subjects as in our study.35–38 Second, it is uncertain whether the spontaneous brain activation shown in the current study can be generalized to other trauma and surgeries of the knee. Experiments using macaques with varying magnitudes of knee damage may be required. Third, anesthetizing the macaques could have underestimated spontaneous pain and overestimated the effect of morphine on brain activation. As opioids are usually administered with propofol for anesthesia, the effect of the interaction between morphine and propofol on brain activation should be examined. However, the dose of propofol was reported no analgesic efficacy in either macaques or humans.21

Despite the concerns mentioned, the present study's results could still be useful in future experiments that focus on the association between the activation of brain regions and resting pain without somatosensory stimulation after trauma and surgery of the knee.

5

5 Conclusion

An osteochondral graft procedure on the femoral condyle can lead to postoperative resting pain. fMRI can reveal activation in pain-related brain areas and evaluate resting pain due to knee surgery.

Ethical statement

All study procedures involving human participants followed the ethical standards of the Hamamatsu University School of Medicine and the 1964 Declaration of Helsinki and the amendments or comparable ethical standards. The study was approved by the ethics committee of Hamamatsu University School of Medicine (No. 21-145). The need for informed consent was waived because of the retrospective nature of the study.

Funding

This research did not receive any specific grants from funding agencies in the public, commercial, or not-for-profit sectors.

Guardian/patient's consent

The study was performed retrospectively and approved by the ethics committee of our institution (No. 21-145). The need for informed consent was waived because of the retrospective nature of the study.

CRediT authorship contribution statement

Hatsumi Ichinose: Conceptualization, design, data collection, Formal analysis, and interpretation, manuscript writing, and final approval of the manuscript. Takahiro Natsume: Conceptualization, and design, data collection, Formal analysis. Mizuho Yano: Supervision, All authors have read and approved the final version of the manuscript. Yuji Awaga: Animal care, experiment support. Mitsuru Hanada: Conceptualization, design, data collection, Formal analysis, and interpretation, manuscript writing, and final approval of the manuscript. Hiroyuki Takamatsu: Supervision, All authors have read and approved the final version of the manuscript. Yukihiro Matsuyama: Experiment support.

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