Abstract
The protective effect and mechanism of low-intensity laser acupoint irradiation on focal cerebral ischemia-reperfusion (CIR) injury in rats were investigated. Male Sprague-Dawley rats were randomly divided into a sham group, a CIR model (model) group, and a model plus laser irradiation (laser) group. The focal CIR model was induced by middle cerebral artery occlusion in all except the rats in the sham group. After modeling, the Baihui, Mingmen, and left Zusanli points of the rats in the laser group were irradiated with 15 mW using a semiconductor laser, and each point was irradiated for 15 min once a day for 7 d. The treatments used in the sham and model groups were the same as in the laser group except that the laser output power was zero. After treatment, the expressions of serum superoxide dismutase (SOD) activity and serum malonaldehyde (MDA) content, the expression of growth-associated protein (GAP-43), the activities of succinic dehydrogenase and lactic dehydrogenase in brain tissue, were measured. The results showed that acupoint irradiation with a semiconductor laser can improve energy metabolism, enhance the expression of GAP-43, increase the levels of expression of serum SOD, and decrease the serum MDA content in a rat model of focal CIR, suggesting the mechanism for reduction of CIR injury.
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1. Introduction
The mechanisms of cerebral ischemia-reperfusion (CIR) injury are complex, which include free radical damage, inflammatory reactions, apoptosis, and overloading of Ca2+. Lipid peroxidation generates large numbers of free radicals, playing a very important role in CIR injury. Therefore, the activation of the antioxidant system against oxidative stress is an important mechanism to alleviate ischemia-reperfusion injury. The growth-associated protein (GAP-43) is a general intrinsic determinant of anatomical plasticity. Its expression in neurons correlates closely with axonal elongation, synaptogenesis, and nerve sprouting during development and in the adult. Disordered energy metabolism after CIR is an important factor in the induction of CIR injury [1]. This paper aimed to observe the protective effects of low-intensity laser acupoint irradiation of Zusanli, Mingmen, and Baihui points on focal CIR injury in rats. The mechanism of the protective effect of low-intensity laser acupoint irradiation on CIR injury was investigated.
2. Materials and methods
2.1. Animal grouping and model preparation
Adult male, specific pathogen-free Sprague-Dawley rats, weighing 220–260 g, were obtained from the Experimental Animal Center of Henan University of Science and Technology. Before surgery, the rats were kept at room temperature (24 ± 1 °C), with free access to food and water.
The rats were randomly divided into a sham group, a CIR model (model) group, and a model plus laser irradiation (laser) group, with 15 rats per group. With the exception of the sham group, the cerebral ischemia rat models were established using right middle cerebral artery occlusion for 90 min, followed by reperfusion, with the thread embolism method [2]. The rats in the sham group were treated the same as those in the model group except that the thread was not inserted. Rats in each group were released into their usual cage after waking and were free to eat and drink.
2.2. Treatment
A SUNDOM-300IB/216 semiconductor laser therapeutic apparatus was used (Beijing SUNDOM Medical Equipment Co. Ltd, China). This is a GaAlAs semiconductor dual-wavelength laser, with an output wavelength of 650/810 nm and an output power of 0–500 mW, which could be adjusted continuously, and a laser irradiation area of 50 mm2.
After confirming the success of modeling, the Baihui, Mingmen, and left Zusanli points of the rats in the laser group were irradiated with the semiconductor laser (output power at 15 mW); each point was irradiated for 15 min, once a day for 7 d, for a total of 7 irradiation treatments. The treatments used in the sham and model groups were the same as in the laser group except that the laser output power was zero.
2.3. Outcome measures
2.3.1. Detection of the rats serum superoxide dismutase (SOD) activity and malonaldehyde (MDA) levels.
Seven days after laser treatment, 3 rats were randomly selected from each group to be anesthetized by intraperitoneal injection, their chests were opened, and the blood samples were obtained from the abdominal aorta. The blood samples were placed at room temperature for 2 h. The specimens were then centrifugated for 20 min, separating the serum, which were then packed and kept in the refrigerator at −80 °C.After refrigeration, the Xanthine oxidase method was used to detect the serum SOD activity, and TBA method was used to detect the serum MDA content of the specimens.
2.3.2. Detection of the expression of GAP-43 around the cerebral infarction area.
Seven days after laser treatment, other 3 rats were randomly selected from each group to be anesthetized by intraperitoneal injection and then decapitated. The brain tissue in the ischemic zone was fixed for 24 h in 4% paraformaldehyde at 4 °C; the samples were paraffin -embedded using routine methods, and successive coronal sections of 5 µm were made. The expression of GAP-43 in the peripheral area of cerebral infarction of the specimens was measured by streptomycin avidin peroxidase immunohistochemistry as previously described. The sections were placed under light microscopy for photography and observation at 400× . Three slices of the cerebral infarction region were taken from each specimen, and 5 high power fields of view from each slice were chosen randomly to count the number of GAP-43 positive cells by Image-Pro plus image analysis software.
2.3.3. Detection of the activities of succinate dehydrogenase (SDH), Na+-K+-ATPase, and lactic dehydrogenase (LDH) in the brain tissue.
After the last treatment, other 6 rats were decapitated immediately and the brains were placed in a refrigerator at −80 °C. The brains were removed from the refrigerator for testing and accurately weighed after thawing, using a weight: volume ratio of 1:9. Then, 4 °C normal saline was added to prepare a 10% brain homogenate, which was centrifuged at low temperature. The activities of succinate dehydrogenase (SDH), Na+-K+-ATPase, and lactic dehydrogenase (LDH) in the supernatant liquid were determined by colorimetry. Kits and reagents were purchased from Nanjing Jiancheng Bioengineering Institute, China, and testing was performed in strict accordance with reagent instructions.
2.3.4. Detection of the volume of cerebral infarction.
Seven days after laser treatment, the remaining 3 rats in each group were a decapitated and the entire brainwas removed. Four blocks of coronal brain tissue were cut and frozen quickly on dry ice, and then coronal sections of 18 µm were made with a constant cold box slicer. They were placed in 4% paraformaldehyde for 7 min, then placed in the dye cresyl violet for 2 min, then they were dehydrated with ethanol in stepwise increasing concentrations, cleared in xylene, and finally sealed in neutral resin. The infarct volume in each group was calculated by Image J software.
2.4. Statistical methods
Statistical analysis was performed using SPSS17.0 software and the data were reported as the mean and standard deviation (x ± s). One-way analysis of variance was used to compare the measurement data among the groups. P < 0.05 was defined as a statistically significant difference.
3. Results
3.1. Comparison of the serum SOD activity and MDA content in rats for each group
The serum SOD activity in rats of the model group was significantly lower than those of the sham and laser groups. The serum MDA content of the rats in the model group was significantly increased compared with those of the sham and laser groups. The results showed that the laser acupoint irradiation had a significant effect on increasing the levels of SOD and decreasing the levels of MDA, as seen in table 1.
Table 1. The serum SOD activity and MDA content in the rats of each group (x ± s).
| Group | N | SOD (U ml−1) | MDA (nmol ml−1) |
|---|---|---|---|
| The sham group | 3 | 127.89 ± 0.56 |
4.12 ± 0.04 |
| The model group | 3 | 99.32 ± 1.96 |
5.08 ± 0.02 |
| The laser group | 3 | 105.43 ± 2.53 |
4.67 ± 0.03 |
aCompared with the sham group P < 0.05. bCompared with the model group P <0.05. cCompared with laser group P < 0.05.
3.2. Comparison of the expression of GAP-43 positive cells and cerebral infarction volume
The expression of GAP-43 in the positive cells in the cerebral cortex and striatum of the cerebral infarction area in the laser group was brownish yellow, and the granules were large, dark and densely distributed. The number of GAP-43 positive cells in the laser group was significantly higher than that in the sham and the model groups (P < 0.05). Compared with the sham group, the cerebral infarction volume in the model group was significantly larger (P < 0.05). Compared with the model group, the cerebral infarction volume in the laser group decreased significantly (P < 0.05), as shown in table 2.
Table 2. The expression of GAP-43 and cerebral infarction volume of each group (x ± s).
| Group | N | The number of GAP-43 positive cells per high-power field | Cerebral infarction volume (mm3) |
|---|---|---|---|
| The sham group | 3 | 0 | 0 |
| The model group | 3 | 49.87 ± 1.42 |
298.1 ± 32.1 |
| The laser group | 3 | 69.62 ± 2.69 |
124 ± 24.5 |
aCompared with the sham and the model group P < 0.05. bCompared with the sham group P < 0.05.
3.3. Comparison of the activities of SDH, Na+-K+-ATPase, and LDH in the brain tissue
Compared with the sham group, the activity of the LDH increased in the brain tissue of the model group rats and the activity of Na+-K+-ATPase decreased (P < 0.05), but there was no significant difference in the activity of SDH (P > 0.05). Compared with the model group, the activity of LDH in the brain tissue of rats in the laser group was decreased, and the activities of SDH and Na+-K+-ATPase increased (P < 0.05). As seen in table 3.
Table 3. The activities of LDH, SDH, and Na+-K+-ATPase in each group (x ± s, U mg−1 protein).
| Group | N | LDH | SDH | Na+-K+-ATP |
|---|---|---|---|---|
| Sham group | 6 | 18.9 ± 1.5 | 4.4 ± 0.4 | 4.2 ± 0.3 |
| Model group | 6 | 23.1 ± 2.3 |
4.5 ± 0.6 | 3.2 ± 0.6 |
| Laser group | 6 | 18.6 ± 2.1 |
5.8 ± 0.6 |
4.1 ± 0.4 |
aCompared with the sham group P < 0.05. bCompared with the model group P < 0.05.
4. Discussion
Normally, the oxidation and antioxidant system of brain tissue are in equilibrium. However, after CIR injury, these systems are imbalanced, leading to a decrease of SOD activity, the up regulation of MDA expression, a decreased scavenging free radical capacity, and a decrease in antioxidation activity. This further results in a disorder of the energy metabolism caused by oxidative lipid damage, and changes in the cell membrane permeability, eventually leading to tissue damage. In the present study, the effects of serum SOD activity and serum MDA content in rats using semiconductor laser acupoint irradiation were observed. The experimental results suggested that the level of expression of serum SOD was up-regulated, but the serum MDA content was significantly down-regulated in the rats of the laser group. Therefore, laser acupoint irradiation can eliminate oxygen free radicals, inhibit lipid peroxidation and reduce reperfusion injury.
GAP-43 is believed to be a molecular marker of neural plasticity in nerve growth, development, and damage repair, and a molecular marker of axonal regeneration. Our study found that expression of GAP-43 in peripheral areas of cerebral infarction was significantly enhanced after cerebral ischemia in rats, suggesting that after cerebral ischemia causing neuronal damage, the body starts to repair the mechanisms of the nerves. Low intensity semiconductor laser acupoint irradiation can further enhance the expression of GAP-43, suggesting that low intensity semiconductor laser acupoint irradiation can promote nerve growth.
Na+-K+-ATPase is a protease present in the cell membrane and mitochondrial inner membrane, and is involved in the transport of substances and energy conversion, and plays an important role in information transmission. A decrease in ATPase activity can lead to increased Na+ and calcium in brain cells, and can activate a variety of enzymes involved in the production of free radicals, which aggravate brain damage [3, 4]. LDH is also an important enzyme in energy metabolism. LDH is mainly distributed in cytoplasm, and is involved in aerobic oxidation of sugars and anaerobic glycolysis. Changes in LDH levels directly affect energy metabolism [5]. SDH is a rate-limiting enzyme of the tricarboxylic acid cycle, and a marker enzyme of mitochondria. It is mainly distributed in the mitochondrial inner membrane. SDH activity reflects the strength of the tricarboxylic acid cycle, and indirectly reflects the function of mitochondria [6].
During CIR, a large number of free radicals are produced, resulting in stress reactions in mitochondria, destruction of enzyme complexes in the respiratory chain, and loss of integrity of the intima. This leads to a decrease in ATP levels. Disordered energy metabolism can lead to a series of pathophysiological changes related to CIR injury, such as intracellular calcium overload, increased release of excitatory amino acids, increased free radical production, and aerobic oxidation disorders.
Acupuncture is a unique treatment method of traditional Chinese medicine. According to the theory of acupuncture, which describes meridians and acupoints, acupuncture at the Baihui and Zusanli points has a positive regulatory effect on ischemic cerebrovascular disease [7]. Previous research has investigated the efficacy of acupuncture in the treatment of CIR injury in rats [8]. A semiconductor laser has the required characteristics for laser acupoint irradiation therapy: deep tissue penetration, and is therefore the preferred choice for this therapy [9, 10]. Transcranial low-level light therapy has gained interest as a non-invasive, inexpensive and safe method of modulating neurological and psychological functions in recent years [11].
The results showed that LDH activity in brain tissue increased significantly after CIR in rats, but there is no obvious change in SDH activity, and the activity of Na+-K+-ATPase decreased. The decrease in Na+-K+-ATPase activity and the increase in LDH activity indicate that mitochondrial function is destroyed in many pathways. Mitochondrial dysfunction leads to an increase in anaerobic glycolysis. Low-intensity semiconductor laser irradiation at the Baihui, Mingmen, and left Zusanli points inhibited LDH activity in rat brain tissue and increased Na+-K+-ATPase activity. This suggested that laser irradiation could improve energy metabolism in rats with CIR injury.
5. Conclusion
The results of this study show that low-intensity semiconductor laser acupoint irradiation at the Baihui, Mingmen, and Zusanli points can improve energy metabolism, enhance the expression of GAP-43, increase the levels of expression of serum SOD, and decrease the serum MDA content in a rat model of focal CIR, suggesting the mechanism for reduction of CIR injury.