| Objective:To clarify the correlation between yawning (drowsiness) and sleep, and to study effect of Sinisource lyophilized powder intervention on yawning (drowsiness).Methods:1. To replicate the yawning model of ratsMale SD rats were randomly divided into control group and model group. The microinjection catheters were implanted into the PVN region of rat brain under anesthetized and stereotactic fixed. The animals were allowed 2 days of recovery from surgery. The model group, each rat was injected 0.3 μL of Ringer’s solution containing 50 ng NMDA into PVN region by microinjector at 9:00-13:00 o’clock, and the same volumes of Ringer’s solution for control group. Prolonged injection for 1 minute at rate of 0.3 μL/min and maintain the needle for 1 min after injection. Then, remove the microsyringe at the end. After dosing, recording yawning times of both groups in 1h by high-definition video were performed. To replicated yawning model of rats.2. Study of the correlation between yawning and sleep2.1 The influence of yawning on Fos protein expression of neurons in VLPO area of rat brains.Male SD rats were randomly divided into control group and model group. The animals were given distilled water (10 mL/kg) by orally for 7 days. At the fifth day, animals were anesthetized and stereotactic fixed, followed by microinjection catheter implanted into the PVN region of the brains. After surgery all animals were allowed to recover for another 2 days, followed by injection of 50 ng NMDA in 0.3 μL Ringer’s solution into PVN region of model group at 9:00-13:00 o’clock, and the same volumes of Ringer’s solution were injected for control group animals. Injection prolonged to 1 minute at the rate of 0.3 μL/min, followed by maintain the needle for 1 min after injection and removed at the end. After dosing, yawning times recording of the control and model groups for 1h by high-definition video were performed. After the recording was completed, two animal groups were perfused immediately with normal saline and 4% paraformaldehyde. The rat brains were removed and post fixed in 4% paraformaldehyde, dehydration (in 5%,10%, and 20% sucrose solution), embedded in OCT. The rat brains were sectioned by freezing microtome. Brain coronal sections were taken and subjected to standard c-Fos immunocytochemistry and were assessed for Fos protein expression level in VLPO area.2.2 The effect of yawning on the sleep-related factors in rat brainsMale SD rats were randomized and divided into control group and model group. Animals were orally given by distilled water (10 mL/kg) for 7 days. On the fifth day, microinjection catheters were implanted into PVN region of the brains while animals were under deep anesthetized and fixed. The animals were allowed 2 days of recovery from surgery, and followed by microinjector injection of 50 ng NMDA in 0.3 μL Ringer’s solutiong into PVN region of model group at 9:00-13:00 o’clock. Control group animals were injected the same volumes of Ringer’s solution. Injection prolonged to 1 minute at the rate of 0.3 μL/min, followed by maintain the needle for 1 min after injection, and removed at the end. After dosing, yawning times recording of the control and model groups for 1h by high-definition video were performed. After, the recording was completed, fresh brains were removed immediately, grinded in ice saline, followed by low temperature centrifuging (3000rpm, 10min), and collected of supernatant which was measured by enzyme-linked immunosorbent assay (ELISA) to assess nitric oxide synthase (NOS) and interleukin-2 (IL-2) content, and to detect nitric oxide (NO) content by nitrate reductase assay.3. Study the effect of Sinisource lyophilized powder intervention on the correlation between yawning and sleep3.1 The effect of Sinicource Lyophilized Powder on the Fos protein expression of VLPO area neurons in the yawning rat brain.Male SD rats were set as treatment group. Animals were orally received Sinisource lyophilized powder (10 mL/kg) for 7 days. In the fifth day, animals were narcotized and fixed for implanting the microinjection catheter into PVN region of the animals’ brains. Two days later, the operated animals have been recovered, then injected 0.3 μL Ringer’s solution that contain 50 ng NMDA into PVN region of administration group animals’brains with microinjector at 9:00-13:00 o’clock. Injection was prolonged for 1 min in the rate of 0.3μL/min, maintained the needle for 1 min after injection, and removed the micro syringe. After dosing, yawning times recording of the treatment group rats for 1 h by high-definition video. After the recording was completed, animals were immediately perfused with saline and 4% paraformaldehyde to take brains. The brains were fixed (4% paraformaldehyde), followed by dehydration (5%,10% and 20% sucrose solution), and embedded by OCT. After taking the rats’ brains slices by freezing microtome, measured the Fos protein expression level of VLPO area neurons by immunohistochemistry technique (SP).3.2 The influence of Sinisource Lyophilized Powder on sleep-related factors in yawning rats’ brainsMale SD rats were set as administration group. The animals were orally given the Sinisource lyophilized powder (10 mL/kg) for 7 days. In the fifth day, animals were anesthetized and stereotactic fixed, the microinjection catheter was implanted into PVN region of the animals’ brains. Two days later, after surgery animals have been recovered, injected 0.3 μL Ringer’s solution containing 50 ng NMDA into PVN region of model group by microinjector at 9:00-13.00 o’ clock, and injected the same volumes of Ringer’s solution into control group animals. Injection prolonged 1 min at the rate of 0.3μL/min, and maintained the needle for 1 min after injection. Finally, the micro syringe was removed. After dosing, recorded the yawning times of the control and model group in 1 h by high-definition video. After the recording was completed, taken the rats’fresh brains immediately. Grinding brains in ice saline, and centrifuged (3000r/min) for 10 min in the low temperature. After centrifugation, collected the supernatant which was measured by enzyme-linked immunosorbent sorbent assay (ELISA) to detect nitric oxide synthase (NOS) and interleukin-2 (IL-2) content in the brains and to detect nitric oxide (NO) content using nitrate reductase assay.Results:1. Copying the yawning model of ratsThe results revealed that the yawning time between control and model group significantly increased which was statistically difference between both groups (P<0.01).2. Study on the correlation between yawning and sleeping2.1 The influence of yawning on Fos protein expression in neurons of VLPO area in rats’brainsThe results revealed that the yawning time between control and model group significantly increased which was statistically difference between both groups (P<0.01). The results also revealed that Fos protein expression in neurons of VLPO area was significantly increased (P<0.01) after injecting NMDA into PVN in 1 h when compared between control and treatment group.2.2 The effect of yawning on the sleep-related factors in rats’brainsThe results showed that NO content in the model group rats’brains significantly increased when compared to control group (P<0.01). In addition, NOS content in the model group rats’brains was no significant change, there was no significant change between both groups (P>0.05); IL-2 content in the group rats’brains significantly decreased which was a very significantly difference between the two groups (P<0.01).3. To study the intervention effect of Sinisource lyophilized powder on the correlation between yawning and sleep3.1 The effect of Sinicource Lyophilized Powder on the Fos protein expression of VLPO area neurons in the yawning rats’brainsThe results exhibited that the yawning times of treatment group significantly increased when compared to the model group (P<0.05). Furthermore, the results also showed that Fos protein expression in neurons of VLPO area significantly increased after injection of NMDA into PVN in 1 h, there was a significant difference between the two groups (P<0.05) when compared the model group against administration group.3.2 The influence of Sinisource Lyophilized Powder on sleep-related factors in yawning rats’brainsThe results showed that NO content in the administration group rats’ brains significantly increased when compared with the model group. There was a significant difference between the two groups (P<0.05). Moreover, NOS content in the administration group significantly increased, there was a statistically significant change between both groups (P<0.01), but IL-2 content in the administration group rats was no significant change between the two groups (P> 0.05).Conclusion:1. There is a correlation between yawning (drowsiness) and sleeping. Its relevance is as follows:yawning (drowsiness) can not only increase the activity of neurons in the sleep center (VLPO area), but increase the sleep-related factor (NO) content which plays an important role in the induction of sleelp. It can also reduce the IL-2 content in brains.2. Sinisource lyophilized powder cannot only increase yawning (drowsiness) significantly and improve the activity of neurons in the sleep center (VLPO area), but also increase sleep-related factors (NO, NOS) content which hid no effect on the IL-2 content in brains. |
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