Bioluminescence Imaging of Heme Oxygenase-1 Upregulation in the Gua Sha Procedure
Kenneth K. Kwong1, 2, Lenuta Kloetzer1, 2, 3, 4, Kelvin K. Wong5, 6, Jia-Qian Ren1, 2, Braden Kuo1, 2, 3, 4, Yan Jiang7, Y. Iris Chen1, 2, Suk-Tak Chan8, 1, 2, Geoffrey S. Young9, Stephen T.C. Wong5, 6
1Department of Radiology, Massachusetts General Hospital, Harvard Medical School, 2Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital, Harvard Medical School, 3Gastrointestinal Unit, Massachusetts General Hospital, Harvard Medical School, 4Department of Medicine, Massachusetts General Hospital, Harvard Medical School, 5Center for biotechnology and Informatics, The Methodist Hospital Research Institute, 6Department of Radiology, The Methodist Hospital, Weill Cornell Medical College, 7Bejing University of Chinese Medicine, 8Department of Health Technology and Informatics, The Hong Kong Polytechnic University, 9Department of Radiology, Brigham and Women’s Hospital, Harvard Medical School
Gua Sha is a traditional Chinese folk therapy that employs skin scraping to cause subcutaneous microvascular blood extravasation and bruises. The protocol for bioluminescent optical imaging of HO-1-luciferase transgenic mice reported in this manuscript provides a rapid in vivo assay of the upregulation of the heme oxygenase-1 (HO-1) gene expression in response to the Gua Sha procedure. HO-1 has long been known to provide cytoprotection against oxidative stress. The upregulation of HO-1, assessed by the bioluminescence output, is thought to represent an antioxidative response to circulating hemoglobin products released by Gua Sha. Gua Sha was administered by repeated strokes of a smooth spoon edge over lubricated skin on the back or other targeted body part of the transgenic mouse until petechiae (splinter hemorrhages) or ecchymosis (bruises) indicative of extravasation of blood from subcutaneous capillaries was observed. After Gua Sha, bioluminescence imaging sessions were carried out daily for several days to follow the dynamics of HO-1 expression in multiple internal organs.
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- Female HO-1-luciferase transgenic mice (age of 4-6 weeks) can be purchased from Taconic Farms, Inc. Upon arrival, mice are placed in an animal housing facility for at least 4 days to allow accommodation.
- Preparation for bioluminescence imaging:
- Weigh each HO-1 mouse. Animal weight is required for calculating the dosage of luciferin.
- Hair removal: Use a cotton swap to apply hair remover Nair® over the abdomen and/or back of the animal. Waiting for 5 to 10 seconds. Use a clean cotton swap to wipe off hair. Dip a piece of tissue in distilled water to wipe clean remaining hair.
- Place a non-fluorescent black paper (Strathmore Artagain® black paper) on the imaging platform of an IVIS 100 station to reduce background noise.
- Prepare luciferin (from Xenogen Corporation) solution at concentration of 7.5 mg/ml (dissolved in sterile H2O). The dose of luciferin is 65.5 mg/kg body weight. Thus, a 20 gram mouse requires intraperitoneal injection of 0.175 ml luciferin solution. To achieve this, a single injection volume of luciferin solution (0.175ml) is preloaded to a syringe with a 26-gauge needle
- Gua Sha procedure: Gua Sha is applied only once before running the bioluminescence imaging protocol. Since Gua Sha is not painful, the mouse needs only to be briefly anesthetized by isoflurane to stay calm. Gua Sha procedure includes the following steps:
- Apply cooking oil or distilled water to lubricate skin areas targeted by Gua Sha a few times during the Gua Sha procedure.
- Repetitively scrape the hair-free region of the back of the mouse in gentle but firm force using a ceramic soup spoon or a plastic spoon.
- The scraping continues until the back skin turns red, which is a sign of subcutaneous blood extravasation, usually achieved within 2 to 3 minutes.
- Bioluminescence imaging may be started immediately or several hours after Gua Sha as HO-1 upregulation is built up slowly over a few hours. The procedure for bioluminescence imaging is:
- Anesthetize the mouse in an anesthesia induction chamber filled with a mixture of isoflurane (1.5%) and medical grade air.
- Once the mouse is anesthetized, move the mouse to the imaging chamber on the IVIS 100 optical imaging station. Position the mouse in a supine position (abdomen up). The imaging chamber is continuously infused with 1.5% of isoflurane. The imaging platform is heated at 37°C to keep the mouse warm.
- Set the imaging acquisition at "medium binning" and set exposure time to be 30 seconds. Start to acquire images. Set the machine to repeat the imaging acquisition every three minutes, either manually or automatically. After the acquisition of the first image, a region of interest (ROI) is drawn to cover the abdominal, chest, and head area. This ROI is then copied and pasted to the following images using "Living Image®", software accompanying the IVIS 100 station.
- Signal intensity is measured in photons per second. Mark time for the signal intensity to reach its peak value and keep on imaging the mouse for about 5 to 10 minutes after the peak time.
- When the imaging acquisition at the supine position is done, flip the mouse over and lay it in the prone position (back up). Continue imaging the animal for 5 to 10 minutes with the ROI now drawn to cover the back and the head area in the use of the software "Living Image®". The choice of imaging the prone position after the supine position is arbitrary. One can choose to start with the prone position, depending on the primary organs of interest. Another option is to image the supine and prone positions at separate experiments.
- When the imaging acquisition at the prone position is finished, turn off isoflurane and move mouse from the imaging chamber to the induction chamber to recover. The induction chamber is now infused with medical air only and the mouse usually wakes up in less than one minu