Microvascular Perfusion Monitored by Laser Speckle Contrast Imaging during Renal Ischemia-Reperfusion Injury in Mice

In this study, we use the lasers speckle contrast imaging systems to monitor the microperfusions in the guinea during the ischemia reperfusion. This technology can be applied to research-related to changes in blood flow, like solid organ transplantations, drug efficacy, and ischemia studies. To begin disinfect the surgical table before each procedure.

Arrange sterilized instruments, cotton swabs, gauze, pre-diluted anesthetics, and all other required items neatly on the table. Administer meloxicam at 0.2 milligram per kilogram body weight by subcutaneous injection 30 minutes before surgery to alleviate pain. Pour sterilized saline into a clean container.

Using scissors, cut a suitable opening in the center of a piece of gauze. Place an anesthetized mouse on the heating pad and secure it with tape. Apply depilatory cream to remove abdominal hair.

Using the heating pad, maintain rectal temperature at 37 degrees Celsius throughout surgery. With a cotton swab, disinfect the surgical area first with 75%alcohol, and then with iodophor, and repeat the procedure two more times. Then use forceps to pinch the mouse toe to confirm absence of pain response.

With a pair of scissors, make a vertical midline abdominal incision. Gently separate the skin from the peritoneum and then incise the peritoneum to enter the abdominal cavity. Apply a retractor to improve visibility.

Cover the operative field with gauze, aligning the opening with the abdominal cavity. Moisten the gauze with saline. With a cotton swab, gently move the intestine to one side to expose the right kidney and surrounding tissues.

Fold the gauze to cover the intestine, then moisten it with saline to prevent drying. Next, use a pair of angled forceps to lift the right ureter. Pass two 6-0 suture threads through the ureter and ligate at two positions.

Using microscissors, transect the ureter between the two ligatures. Then use saline moistened cotton balls held with fine forceps to gently push the liver upward. Fix the liver with the cotton balls to expose the kidneys and right sided vessels.

Next, use angled forceps to bluntly separate the perinephric fat pads until the renal artery and vein are fully exposed.Carefully. Slide the forceps underneath the renal artery and vein to create a channel Pass a 6-0 silk braided suture around both vessels and ligate them together. Cut the occluded renal artery and vein near the kidney.

Remove the kidney with adherent tissue. Then remove the cotton ball used to hold the liver. Turn on the laser speckle contrast imaging system and launch the dedicated software.

Click original image and then preview to display the raw surgical field image. Reposition the mouse. Adjust camera magnification while viewing the original image to obtain a clear view of the target kidney.

Then click simultaneous display to view real-time blood flow imaging. Set magnification to 2.5X and select automatic blood flow adjustment. Then lock the blood flow parameters.

Set recording to capture one frame every 5, 000 milliseconds. Next, set the square area size parameter to 112 and uniformly position five square regions on the kidney image. Click realtime image registration and record to begin blood flow recording.

After recording a 30 minute baseline, bluntly separate adipose tissue around the left kidney vessels. Using hemostatic forceps, place a microclamp across the left renal artery and vein near the hilum. Continue real-time monitoring for another 30 minutes.

Release the microclamp to initiate reperfusion. Monitor and record real-time recovery of renal microvascular blood flow. After monitoring reperfusion for at least 60 minutes, stop recording and close the abdominal incision.

Administer one milliliter saline by intraperitoneal injection to prevent dehydration. Place the mouse on the heating pad and monitor until it regains consciousness and can right itself. During the initial 30 minutes, renal microvascular blood flow gradually became stable at baseline.

Heterogeneity in perfusion levels among different regions of interest was observed before ischemia. Following clamping of the renal artery and vein, blood flow dropped sharply and the renal microcirculation entered a no perfusion state. Upon release of the clamp, blood flow surged instantly to a peak and then decreased before gradually recovering to a comparatively stable level.

Microvascular perfusion during 60 minutes of reperfusion was much lower than that before ischemia. We can obtain the renal microperfusion's blood flow through the laser speckle contrast imaging systems. Thereby verifying the process of ischemia and the reperfusion.

To obtain a clear kidney with good contrast, we need to be patient and spend time separating the kidney from the surrounding resource and the tissues. In the future, additional indicators of pathophysiology can be incorporated and this technology can be used to monitor the effect of drugs on renal ischemia.