High frequency electrical field-ultrashort wave therapy for treatment of cerebral ischemia/reperfusion injury in rats☆Histopathological evaluation

BACKGROUND: Ultrashortwave (USW) therapy may be a new method for treatment of ischemic cerebrovascular diseases. It is necessary to study its treatment time window.OBJECTIVE: To observe the effect of USW on reperfusion injury after occlusion of the middle cerebral artery (MCAO) in rats and discuss its acting mechanisms and best occasion. DESIGN: Randomized controlled observation, animal experiment. SETTING: Laboratory of Department of Rehabilitation Medicine, First Hospital Affiliated to China Medical University.MATERIALS: Sixty-six healthy Wistar rats of either gender and of clean grade, aged 18–20 weeks, weighing from 250 to 300 g, were provided by the Experimental Animal Center of China Medical University. An USW device (Shanghai Electrical Device Company) with the frequency of 40.68 MHz and the maximum output power of 40 W, and the first channel power controlled at about 11 W was used in this study. Output power was determined by photometry.METHODS: Sixty-six rats were randomly divided into 3 groups: Sham-operation group (n =6): The suture was inserted only 1.0 depth during operation, which did not cause MACO; Model group (n =12): The USW treatment procedure was performed with the power off on the model rats; USW treatment group (n =48): The 48 rats were randomly divided into modeling 0, 6, 12 and 18 hours 4 subgroups. USW therapy without heat was used on the head of rats for 10 minutes at each time point. Twelve rats in USW treatment group were decapitated following treatment at each time point, and then their brain tissues were harvested. The rat brain tissues in other groups were harvested by decapitation at 24 hours after modeling. When the rats were awake, the neurologic deficit was scored by Zea-Longa five-point scale (a score of 0 indicated no neurologic deficit, a score of 1 indicated failure to extend left paw fully, a score of 2 indicated circling to the left, and a score of 3 indicated falling to the left, and rats with a score of 4 did not walk spontaneously and has a depressed level of consciousness.) Rats which still survived at 24 hours and was scored 1 and 2 on the neurologic scoring were involved in the analysis. ① Determination of cerebral water content: Cerebral water contents of healthy and injured hemisphere were determined by wet/dry weighing method. Cerebral water content (100%) =(1–dry/wet weight)×100%.②Infarction volume: The brain tissue was sliced into 2 mm sections and each section was stained with 20 g/L 2,3,5-triphenyltetrazolium chloride (TTC) by TTC staining technique for 30 minutes in a water bath at 37 ℃.Then, the section was fixed in 100 g/L formaldehyde for 10 minutes .The infarction volume was analyzed by using an imaging analyzer.③ Preparation of light microscopic sample: The rat brain tissue fixed by 100 g/L neutral formaldehyde and stained with TTC, were gradiently dehydrated with alcoholic, embedded with paraffin, sliced and stained by HE, finally, the sections were observed under the light microscope.MAIN OUTCOME MEASURES: Cerebral water content, cerebral infarction volume and cerebral histomorphology of rats in each group.RESULTS: Sixty-six rats were involved in the final analysis. ①Cerebral water content: There were no significant differences of cerebral water content in healthy hemisphere among groups (P > 0.05). Cerebral water content of injured hemisphere in the model group and at modeling 0, 6, 12 and 18 hours in the USW treatment group was (81.50±0.74) %, (81.02±0.83) %, (79.78±0.70) %, (79.74±0.84) %, (79.39±1.06) %, respectively, which was significantly higher than that in the sham-operation group [(78.09±0.52) %, P < 0.05]. At modeling 0, 6 and 12 hours, the cerebral water content in the injured hemisphere in the USW treatment group was significantly lower than that in the model group, respectively (P < 0.05). It indicated that USW treatment given at 6, 12 and 18 hours after ischemia/reperfusion can lessen brain edema. ② Cerebral infarction volume: At modeling 18 hours, cerebral infarction volume in the injured hemisphere of USW treatment group was smaller than that in the model group [（191.62±121.45），（362.03±142.01）mm3，t =2.23，P < 0.05]. ③ Cerebral histomorphological observation: No swelling was found in the brain tissue section of rats in the sham-operation group. In the model group, the size of infarction hemisphere was obviously increased, gyrus became flattened, cortical sulci was shallow, the color at infarct focus obviously became light, and the tissue was fragile and brittle. In the sham-operation group, it was found under the microscope that mesenchyma was highly swelled, neuronal peripheral interspace was obviously broadened, neurons presented triangle, nucleoli were reduced, condensed even disappeared, and neutrophils in the vascular cavity were obviously increased. In the USW treatment group, pathological injury was not obviously lessened at 0 hour, moderate or mild edema could be found in the injured hemisphere of USW treatment group at modeling 6,12 and 18 hours, and at this time, neutrophils in vascular cavity were increased slightly, and pathological injuries were lessened.CONCLUSION: USW may play a protective effect on cerebral ischemia/reperfusion injury by decreasing brain edema and/or cerebral infarction volume. The treatment action of USW may start at 6 hours after reperfusion, and the best occasion of application may be at 18 hours after reperfusion.

High frequency electrical field-ultrashort wave therapy for treatment of cerebral ischemia/reperfusion injury in rats Histopathological evaluation

BACKGROUND: Ultrashortwave (USW) therapy may be a new method for treatment of ischemic cerebrovascular diseases. It is necessary to study its treatment time window.OBJECTIVE: To observe the effect of USW on reperfusion injury after occlusion of the middle cerebral artery (MCAO) in rats and discuss its acting mechanisms and best occasion. DESIGN: Randomized controlled observation, animal experiment. SETTING: Laboratory of Department of Rehabilitation Medicine, First Hospital Affiliated to China Medical University.MATERIALS: Sixty-six healthy Wistar rats of either gender and of clean grade, aged 18-20 weeks, weighing from 250 to 300 g, were provided by the Experimental Animal Center of China Medical University. An USW device (Shanghai Electrical Device Company) with the frequency of 40.68 MHz and the maximum output power of 40 W, and the first channel power controlled at about 11 W was used in this study. Output power was determined by photometry. METHODS: Sixty-six rats were randomly divided into 3 groups: Sham-operation group (n =6): The suture was inserted only 1.0 depth during operation, which did not cause MACO; Model group (n =12): The USW treatment procedure was performed with the power off on the model rats; USW treatment group (n =48): The 48 rats were randomly divided into modeling 0, 6, 12 and 18 hours 4 subgroups. USW therapy without heat was used on the head of rats for 10 minutes at each time point. Twelve rats in USW treatment group were decapitated following treatment at each time point, and then their brain tissues were harvested. The rat brain tissues in other groups were harvested by decapitation at 24 hours after modeling. When the rats were awake, the neurologic deficit was scored by Zea-Longa five-point scale (a score of 0 indicated no neurologic deficit, a score of 1 indicated failure to extend left paw fully, a score of 2 indicated circling to the left, and a score of 3 indicated falling to the left, and rats with a score of 4 did not walk spontaneously and has a depressed level of consciousness.) Rats which still survived at 24 hours and was scored 1 and 2 on the neurologic scoring were involved in the analysis. ①Determination of cerebral water content: Cerebral water contents of healthy and injured hemisphere were determined by wet/dry weighing method. Cerebral water content (100%) =(1 - dry/wet weight) ×100%.②Infarction volume: The brain tissue was sliced into 2 mm sections and each section was stained with 20 g/L 2,3,5- triphenyltetrazolium chloride (TTC) by TTC staining technique for 30 minutes in a water bath at 37 ℃.Then, the section was fixed in 100 g/L formaldehyde for 10 minutes .The infarction volume was analyzed by using an imaging analyzer. ③Preparation of light microscopic sample: The rat brain tissue fixed by 100 g/L neutral formaldehyde and stained with TTC, were gradiently dehydrated with alcoholic, embedded with paraffin, sliced and stained by HE, finally, the sections were observed under the light microscope.MAIN OUTCOME MEASURES: Cerebral water content, cerebral infarction volume and cerebral histomorphplogy of rats in each group.RESULTS: Sixty-six rats were involved in the final analysis. ①Cerebral water content: There were no significant differences of cerebral water content in healthy hemisphere among groups (P ＞ 0.05). Cerebral water content of injured hemisphere in the model group and at modeling 0, 6, 12 and 18 hours in the USW treatment group was (81.50±0.74) %, (81.02±0.83) %, (79.78±0.70) %, (79.74±0.84) %, (79.39± 1.06) %, respectively, which was significantly higher than that in the sham-operation group [(78.09±0.52) %, P ＜ 0.05]. At modeling 0, 6 and 12 hours, the cerebral water content in the injured hemisphere in the USW treatment group was significantly lower than that in the model group, respectively (P ＜ 0.05). It indicated that USW treatment given at 6, 12 and 18 hours after ischemia/reperfusion can lessen brain edema. ②Cerebral infarction volume: At modeling 18 hours, cerebral infarction volume in the injured hemisphere of USW treatment group was smaller than that in the model group [ (191.62±121.45) , (362.03±142.01) mm3, t=2.23, P ＜ 0.05]. ③Cerebral histomorphological observation: No swelling was found in the brain tissue section of rats in the sham-operation group. In the model group, the size of infarction hemisphere was obviously increased, gyrus became flattened, cortical sulci was shallow, the color at infarct focus obviously became light, and the tissue was fragile and brittle. In the sham-operation group, it was found under the microscope that mesenchyma was highly swelled, neuronal peripheral interspace was obviously broadened, neurons presented triangle, nucleoli were reduced, condensed even disappeared, and neutrophils in the vascular cavity were obviously increased. In the USW treatment group, pathological injury was not obviously lessened at 0 hour, moderate or mild edema could be found in the injured hemisphere of USW treatment group at modeling 6,12 and 18 hours, and at this time, neutrophils in vascular cavity were increased slightly, and pathological injuries were lessened.CONCLUSION: USW may play a protective effect on BACKGROUND: Ultrashortwave (USW) therapy may be a new method for treatment of ischemic cerebrovascular diseases. It is necessary to study its treatment time window.OBJECTIVE: To observe the effect of USW on reperfusion injury after occlusion of the middle cerebral artery (MCAO) in rats and discuss its acting mechanisms and best occasion. DESIGN: Randomized controlled observation, animal experiment. SETTING: Laboratory of Department of Rehabilitation Medicine, First Hospital Affiliated to China Medical University.MATERIALS: Sixty-six healthy Wistar rats of either gender and of clean grade, aged 18-20 weeks, weighing from 250 to 300 g, were provided by the Experimental Animal Center of China Medical University. An USW device (Shanghai Electrical Device Company) with the frequency of 40.68 MHz and the maximum output power of 40 W, and the first channel power controlled at about 11 W was used in this study. Output power was determined by photometry. METHODS: Sixty-six rats were randomly divided into 3 groups: Sham-operation group (n =6): The suture was inserted only 1.0 depth during operation, which did not cause MACO; Model group (n =12): The USW treatment procedure was performed with the power off on the model rats; USW treatment group (n =48): The 48 rats were randomly divided into modeling 0, 6, 12 and 18 hours 4 subgroups. USW therapy without heat was used on the head of rats for 10 minutes at each time point. Twelve rats in USW treatment group were decapitated following treatment at each time point, and then their brain tissues were harvested. The rat brain tissues in other groups were harvested by decapitation at 24 hours after modeling. When the rats were awake, the neurologic deficit was scored by Zea-Longa five-point scale (a score of 0 indicated no neurologic deficit, a score of 1 indicated failure to extend left paw fully, a score of 2 indicated circling to the left, and a score of 3 indicated falling to the left, and rats with a score of 4 did not walk spontaneously and has a depressed level of consciousness.) Rats which still survived at 24 hours and was scored 1 and 2 on the neurologic scoring were involved in the analysis. ①Determination of cerebral water content: Cerebral water contents of healthy and injured hemisphere were determined by wet/dry weighing method. Cerebral water content (100%) =(1 - dry/wet weight) ×100%.②Infarction volume: The brain tissue was sliced into 2 mm sections and each section was stained with 20 g/L 2,3,5- triphenyltetrazolium chloride (TTC) by TTC staining technique for 30 minutes in a water bath at 37 ℃.Then, the section was fixed in 100 g/L formaldehyde for 10 minutes .The infarction volume was analyzed by using an imaging analyzer. ③Preparation of light microscopic sample: The rat brain tissue fixed by 100 g/L neutral formaldehyde and stained with TTC, were gradiently dehydrated with alcoholic, embedded with paraffin, sliced and stained by HE, finally, the sections were observed under the light microscope.MAIN OUTCOME MEASURES: Cerebral water content, cerebral infarction volume and cerebral histomorphplogy of rats in each group.RESULTS: Sixty-six rats were involved in the final analysis. ①Cerebral water content: There were no significant differences of cerebral water content in healthy hemisphere among groups (P ＞ 0.05). Cerebral water content of injured hemisphere in the model group and at modeling 0, 6, 12 and 18 hours in the USW treatment group was (81.50±0.74) %, (81.02±0.83) %, (79.78±0.70) %, (79.74±0.84) %, (79.39± 1.06) %, respectively, which was significantly higher than that in the sham-operation group [(78.09±0.52) %, P ＜ 0.05]. At modeling 0, 6 and 12 hours, the cerebral water content in the injured hemisphere in the USW treatment group was significantly lower than that in the model group, respectively (P ＜ 0.05). It indicated that USW treatment given at 6, 12 and 18 hours after ischemia/reperfusion can lessen brain edema. ②Cerebral infarction volume: At modeling 18 hours, cerebral infarction volume in the injured hemisphere of USW treatment group was smaller than that in the model group [ (191.62±121.45) , (362.03±142.01) mm3, / =2.23, P ＜ 0.05]. ③Cerebral histomorphological observation: No swelling was found in the brain tissue section of rats in the sham-operation group. In the model group, the size of infarction hemisphere was obviously increased, gyrus became flattened, cortical sulci was shallow, the color at infarct focus obviously became light, and the tissue was fragile and brittle. In the sham-operation group, it was found under the microscope that mesenchyma was highly swelled, neuronal peripheral interspace was obviously broadened, neurons presented triangle, nucleoli were reduced, condensed even disappeared, and neutrophils in the vascular cavity were obviously increased. In the USW treatment group, pathological injury was not obviously lessened at 0 hour, moderate or mild edema could be found in the injured hemisphere of USW treatment group at modeling 6,12 and 18 hours, and at this time, neutrophils in vascular cavity were increased slightly, and pathological injuries were lessened.CONCLUSION: USW may play a protective effect on cerebral ischemia/reperfusion injury by decreasing brain edema and/or cerebral infarction volume. The treatment action of USW may start at 6 hours after reperfusion, and the best occasion of application may be at 18 hours after reperfusion.