The purpose of this study is to determine the possible effect of photoluminescence of bioceramic (PLB) on ischemic cerebral infarction (stroke), by using an animal model of transient middle cerebral artery occlusion (MCAO). Sprague-Dawley rats were used to induce MCAO to block the origin of the left MCAO; three months later, the positive chronic stroke rats were selected by running tunnel maze; the MCAO rats with significant chronic stroke and neurological defects were used for treadmill experiments with varying speed settings to test their capability for restoration after muscular fatigue under conditions of with and without PLB irradiation. As a result, PLB irradiation could improve exercise completion rate and average running speed during slow and fast treadmill settings. After PLB irradiation, the selected MCAO rats successfully completed all the second-round treadmill exercises at the maximum speed setting, and they had better restoration from muscular fatigue. An in vitro cell study on astrocytes of rats by bioceramic irradiation further demonstrated increased intracellular nitric oxide. To explain these results, we suggest that cortical brain stimulation of microcirculation and enhancement of peripheral muscular activity are the main causes of the improved exercise performance in MCAO rats by PLB.
Ischemic stroke is the result of reduction in blood flow to the affected brain regions. It causes sudden loss of neurological function after hypoxia of brain tissue and then activates pathogenic cascades after ischemia, disrupting cell metabolism, which eventually results in brain cell death. Ischemic stroke is the biggest cause of physical disability in both developed and developing countries [
Our previous studies showed that bioceramic materials treatment can promote microcirculation [
Bioceramic irradiation treatment has also been shown to activate the parasympathetic nervous system, which may improve the recovery of resting cardiac and respiratory rates following submaximal exercise [
Recently, we presented a series of physical-biological experiments on the material based on the characteristics of biological effects of electromagnetic nonionizing radiation [
Conceptual picture of PLB, a combination of bioceramic material and visible light spectrum.
The ceramic powder was obtained from the Laboratory of Radiology in Taipei Hospital (New Taipei City, Taiwan). The bioceramic material consisted of microsized particles produced primarily from different elemental components.
The concept and procedure of PLB irradiation involve the application of specially manufactured bioceramic materials that consists of microsized particles produced from various elemental components [
Sprague-Dawley rats (200 g;
Transient MCAO was induced by advancing a 4-0 surgical nylon suture into the internal carotid artery (ICA) to block the origin of the MCA. A length of 18.5 to 19.5 mm 4-0 surgical nylon suture, determined by animal weight, was advanced from the left external carotid artery (ECA) into the lumen of the ICA until it blocked the origin of the MCA. The suture was withdrawn to restore blood flow and reperfusion after two hours and then the wound was clipped. As mentioned, selected rats were irradiated using a light source of combining LED (wavelength of visible light, as determined by spectroscopy by the Taiwan Textile Research Institute) and a photonic crystal of bioceramic material. The light sources were kept 10 cm from the rats’ bodies (Figure
PLB irradiation on MCAO rats with surgical clips.
To assess the amount of cerebral damage three months after the MCAO procedure (to be defined as chronic stroke status), we used a simple tunnel maze (60 × 45 × 60 cm), made of nontransparent material. Before a real test was performed, each rat received five practice runs in the tunnel maze, so as to develop experience and prevent fearfulness.
We selected the MCAO rats that expressed poor maze performance, those finishing the tunnel maze in over 200 seconds, to continue our tests.
The selected MCAO rats ran the tunnel maze without PLB and then received PLB treatment on another day at the same time, 2:00 p.m., so as to prevent uncontrollable factors on the levels of adrenaline and metabolic rates. The MCAO rats were kept in the tunnel maze for 200 seconds and 30-minute PLB treatments were then provided for each rat, and their subsequent tunnel maze performance was used for comparison.
MCAO rats underwent the treadmill experiment at different speed settings of 10 cm/s, 20 cm/s, 30 cm/s, 40 cm/s, 50 cm/s, 60 cm/s, high speed (80 cm/s), and maximum speed (100 cm/s). From start to finish, the rats would complete three rounds of running at each speed setting. To be successful, the rat had to complete the entire distance; inability to complete the entire distance would be regarded as “failure” (Figure
Running rat without PLB irradiation (a) and with PLB irradiation (b).
In the beginning, the selected MCAO rats finished the treadmill running and rested for 30 minutes without PLB irradiation. They were then divided into control and experimental (PLB irradiation) groups. The second round of treadmill running was designed with different treadmill speed settings to assess whether PLB irradiation could restore muscle fatigue on the selected MCAO rats.
In this experiment, a Doppler ultrasound blood flow meter (BV 520, LG Biotech, Shanghai, China) was used to measure the MCAO rat carotid artery blood flow, including mean arterial blood flow velocity measurements (MN) and the maximum spectral peak (PK). First, the selected MCAO rats’ hair was shaved from the dorsal neck, and then the Doppler ultrasound on carotid blood flow (DUCBF) was measured and recorded for PK and MN (Figure
Hair shaved rats on the dorsal neck; (a) Doppler ultrasound measurement performed on carotid blood flow (b).
Primary cultures of rat cortical neurons (provided by Professor Yi Hsuan Lee of the Department of Physiology, Taipei Medical University, Taiwan) were prepared from the cerebral cortex of 14-day-old rat foetuses. The cerebral cortices of foetuses obtained under sterile conditions were dissected and dissociated mechanically, by pipetting 10 times with 10 mL of DMEM (Gibco Invitrogen Corporation, Barcelona, Spain). The cell suspension was filtered through nylon mesh with a pore size of 90
The cells are separated as control and bioceramic irradiation groups, while the bioceramic irradiation group received bioceramic irradiation for 10 minutes. The cells and source of the bioceramic irradiation were separated by plastic culture discs, without direct contact (Figure
In vivo experimental model of bioceramic irradiation on astrocytes of rats, in addition to NO concentration measurement.
All cell dishes were then stained with DAF-FM diacetate for fluorescence measurements for NO. All cells were analyzed by a fluorescence-activated cell sorter (FACS) and flow cytometry at the single-cell level. All the data were acquired and analyzed, and the mean fluorescence intensities of intracellular NO production in astrocytes of rats were determined.
All experiments were done at least three times, each time with two or more independent observations. Statistical analysis was performed by paired
After the MCAO procedure on ten rats, three of the rats expired within 24 hours of the surgery. Although the surviving rats initially expressed transient neurological deficits on motor behavior, all of the MCAO rats were nearly recovered in general appearance. In order to select the brain damaged rats after the MCAO procedure, the rats underwent tunnel maze tests. Three out of the seven rats exhibited poor performance on the tunnel maze tests, taking more than 200 seconds, and were selected for PLB treatments (Figure
Comparison of MCAO rats on tunnel maze performance, with and without PLB.
Our data showed improvements in the required time for the three selected MCAO rats (number 3, number 4, and number 8) to complete the tunnel maze after PLB treatments (
Based on statistical results of the selected MCAO rats from the second round of treadmill exercise to assess muscular fatigue restoration, it was found that PLB irradiation could enhance the completion rate and average speed (Figures
(a) The performance of number 3 rat with and without PLB, during running distance (meter) on treadmill under different speeds (cm/sec). (b) The performance of number 4 rat with and without PLB, during running distance (meter) on treadmill under different speeds (cm/sec). (c) The performance of number 8 rat with and without PLB, during running distance (meter) on treadmill under different speeds (cm/sec).
In the beginning, the MCAO rats failed to finish the exercise at the maximum speed setting. After PLB irradiation, it was found that number 3, number 4, and number 8 MCAO rats completed all the second-round treadmill exercises at the maximum speed setting by an average speeds rate of
(a) The performance of number 3 rat with and without PLB for restoration of muscular fatigue, by running distance (meter) on treadmill under different speeds (cm/sec). (b) The performance of number 4 rat with and without PLB for restoration of muscular fatigue, by running distance (meter) on treadmill under different speeds (cm/sec). (c) The performance of number 8 rat with and without PLB for restoration of muscular fatigue, by running distance (meter) on treadmill under different speeds (cm/sec).
In the beginning, we predicted significant differences of MN and PK by DUCBF measurement of PLB irradiation before and after treadmill exercise. In comparing PLB irradiation before and after treadmill exercise, however, the results of DUCBF measurements showed no significant differences on PK and MN before and after PLB irradiation with
Results of selected MCAO rats for carotid Doppler ultrasound experiment with and without PLB irradiation (
Number of MCAO rat | Timing of DUCBF measurement | DUCBF measurement without PLB | DUCBF measurement with PLB | Data subtraction of measurement without PLB from that with PLB |
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Number 3 | Preexercise PK | 0.378 | 0.284 |
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Postexercise PK | 0.336 | 0.473 |
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Preexercise MN | 0.176 | 0.162 |
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Postexercise MN | 0.183 | 0.217 |
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Number 4 | Preexercise PK | 0.432 | 0.444 |
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Postexercise PK | 0.84 | 0.835 |
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Preexercise MN | 0.217 | 0.254 |
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Postexercise MN | 0.34 | 0.59 |
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Number 8 | Preexercise PK | 0.434 | 0.738 |
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Postexercise PK | 1.163 | 0.7 |
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Preexercise MN | 0.296 | 0.538 |
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Postexercise MN | 0.75 | 0.573 |
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MN: mean arterial blood flow velocity measurements; PK: maximum spectral peak.
Levels of intracellular NO synthesis of astrocytes of rats in the control and bioceramic irradiation groups are shown in Figure
By cytometric analysis, NO production in bioceramic irradiation group within astrocytes of rats is statistically significantly increased (
Stroke is a leading cause of adult motor disability, and the corresponding recovery of motor function from stroke is usually a slow and frustrating process [
In this study, PLB irradiation externally on chronic stroke rats is a new kind of cortical stimulation, without applying magnetism and strong electricity. We had previously demonstrated that bioceramic materials treatment was able to promote microcirculation in patients [
We found that PLB irradiation was able to improve different running speeds of the selected MCAO rats, particularly in maximum speed treadmill running and second-round exercise. We suggest that cortical brain stimulation of microcirculation and enhancement of peripheral muscular activity are the two main causes for better exercise performance in MCAO rats by PLB. In the future, medical instrument development for clinical PLB trials is necessary for chronic cerebral infarction. The above findings can be of important reference for future medical device development. Further research of human trial on the correlations among regional cerebral flow, neuromuscular performance, and motor restoration is necessary.
The authors declare that they have no competing interests.
Lei Zhang and Paul Chan contributed equally to this study.
This study was funded by Shanghai Science and Technology Committee of China (Grant no. 15DZ0503500). The study was also assisted by Professor Yi Hsuan Lee.