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

Summary

A laser speckle contrast imaging system was used to monitor the changes in renal microvascular blood flow during unilateral renal ischemia-reperfusion after the resection of the right kidney in mice.

Abstract

Laser speckle contrast imaging (LSCI) is a non-invasive technique for measuring superficial blood perfusion over large areas. With its high spatiotemporal resolution, LSCI semi-quantifies heterogeneous blood flow changes across different regions within the imaged area. Here, we demonstrate the technique of LSCI to record blood flow during unilateral renal ischemia-reperfusion injury (IRI) in mice. Following right nephrectomy, the position of the mouse and the focal point of the objective of the LSCI system were adjusted to ensure the left kidney was in the field of view. LSCI scans were performed before, during, and after the ischemia of the kidney. Furthermore, by selecting regions of interest (ROIs), blood flow in the regions was simultaneously monitored. The method of LSCI visualizes microvascular perfusion of the kidney and allows for a real-time mapping of blood flow. It may be utilized to monitor the microvascular blood flow and evaluate the effect of an intervention on the reperfusion kinetics and spatial homogeneity during mouse renal IRI or other similar animal models.

Introduction

Ischemia-reperfusion injury (IRI) is among the leading causes of acute kidney injury (AKI), which is associated with high morbidity and high mortality in patients^1,2. The change of renal hemodynamics is one of the critical factors of IR-induced AKI¹. IR-evoked microcirculatory dysfunction is a major reason for the development of injury, leading to oxygen deprivation and mitochondrial dysfunction of cells^3,4. Therefore, monitoring blood flow changes in real time is an effective method for ensuring the successful ischemia or reperfusion of the kidney and assessing the degree of IRI in animal models for the disorder.

There are multiple techniques to measure the microvascular perfusion of tissues, including laser Doppler flowmetry (LDF)⁵, nicotinamide adenine dinucleotide dehydrogenase (NADH) autofluorescence imaging^6,7,8, infrared imaging⁹, and indocyanine green (ICG) angiography¹⁰. Among them, infrared imaging and NADH autofluorescence imaging indirectly measure the microvascular perfusion. LDF, using an optical probe, only monitors regional blood flow⁵, and scanning LDF is not quick enough to monitor the real-time changes of tissue microvascular perfusion under I/R conditions¹¹. ICG angiography can also identify renal blocked vessels¹⁰ but requires the administration of a contrast agent, which restricts its use to a limited number of measurements¹². Laser speckle contrast imaging (LSCI) is an efficient non-invasive method for monitoring renal microperfusion in real-time¹³. It has been used to generate spatially resolved information related to blood flow dynamics. As a coherent beam is shone onto a rough surface, the reflected or scattered light waves will interfere with each other due to minute path differences, forming a random distribution of bright and dark spot patterns, known as "speckle". LSCI maps the speed of blood flow in superficial tissues by analyzing the degree of blurriness of the "dynamic speckle" produced when laser light irradiates the tissue^12,14,15. In this study, the LSCI system operated at a laser wavelength of 785 nm with a spatial resolution of 3 µm, a field of view adjustable between 6.3 mm × 4.7 mm and 42 mm × 31 mm, and an imaging speed of up to 120 frames per second, enabling high-resolution, real-time mapping of cortical microvascular perfusion. In light of these advantages, LSCI has been applied in a range of experimental and clinical contexts for blood flow monitoring, includingburn wounds¹⁶, retinal perfusion¹⁷, cerebral blood flow¹⁸, esophagus¹⁹, and the intestine²⁰.

It should be noted that LSCI primarily assesses perfusion in superficial tissue, provides relative rather than absolute flow values, and can be influenced by tissue optical properties and motion artifacts. These inherent characteristics are considered when interpreting the imaging data.

This work, using a model involving right nephrectomy to facilitate exposure and imaging of the contralateral kidney, monitored the changes in microvascular perfusion before, during, and after the ischemia in a unilateral renal I/R mouse model using LSCI and demonstrated the spatial heterogeneity of blood flow on the surface of the kidney.

Protocol

All animal procedures complied with the NIH Guide for the Care and Use of Laboratory Animals and the Guide for the Care and Use of Laboratory Animals, and were approved by the Institutional Animal Care and Use Committee of Shantou University Medical College. To eliminate gender and strain-based differences in IRI effects, only male C57BL/6N mice were used in the study. All mice were matched in age and weight to produce comparable results. A sample size of n = 5 was used, consistent with prior methodological studies utilizing LSCI for technical validation²¹. The animals received humane care and were euthanized after the experiments by CO₂ inhalation. The reagents and the equipment used are listed in the Table of Materials.

Before beginning the surgical procedure, turn on the laser speckle contrast imaging system. The system used in this study operates with a 785 nm laser. Set the working distance to approximately 15-20 cm above the surgical field. The system calculates blood flow in Laser Speckle Perfusion Units (LSPU) based on the spatial contrast algorithm. Ensure the instrument is warmed up and stable before proceeding.

1. Animal preparation and laparotomy

1. Sterilize the surgical table before each procedure. Prepare and place all required materials (sterilized instruments and cotton swabs, gauze, pre-diluted anesthetics, heating pad, vascular clamp, saline solution, depilatory cream, cotton balls, stitches, skin antiseptics, and suture) on the surgical table.
2. Administer meloxicam (0.2 mg/kg body weight; subcutaneous injection) to the mouse 30 min in advance to alleviate pain.
3. Pour a certain amount of sterilized saline into a clean container. Then take a piece of gauze, and cut out a suitable opening in the middle.
4. Anesthetize a mouse by intraperitoneal injection of 2.5% Tribromoethanol (400 mg/kg body weight; previously diluted in sterile saline).
   NOTE: Tribromoethanol was used as an anesthetic during the process of right nephrectomy. And 1% isoflurane was used for anesthesia during prolonged procedures of monitoring blood flow.
5. Place the mouse onto the heating pad with tape and use depilatory cream to remove the hair from the area on the mouse's abdomen. Throughout surgery, use the heating pad to maintain rectal temperature at 37 °C ± 0.5 °C.
6. Disinfect the skin in the surgical area with 75% alcohol first and then with iodophor using a cotton swab.
7. Use forceps to pinch the mouse's toe. Once the mouse shows no pain response, make a vertical incision along the midline of the abdomen using scissors. Gently separate the skin from the peritoneum, then cut open the peritoneum and enter the abdominal cavity.
8. Apply a retractor to the mouse to optimize visibility and cover the operative field with gauze, while aligning the gap on the gauze with the abdominal cavity, and moisten it with saline.

2. Right nephrectomy

1. Use a cotton swab to gently push the intestine towards one side of the gauze, exposing the right kidney and the surrounding tissues. Fold the gauze to cover the intestine and moisten it with normal saline to prevent the intestine from drying out.
2. Use angled forceps to lift the right ureter. Pass two 6-0 suture threads through the ureter and perform ligation at two positions.
3. Transect the ureter between the two ligatures using microscissors.
4. After moistening the cotton balls with normal saline, use fine forceps to pick up the cotton balls, gently push the liver upwards, and fix it with the cotton balls, to expose the kidneys and the blood vessels on their right side.
5. Use angled forceps to bluntly separate the perinephric fat pads along the side of the right kidney until the renal artery and vein are fully exposed.
   NOTE: Simultaneously separate the mass of adrenal tissue located above the kidney.
6. Carefully slide angled forceps underneath the renal artery and vein to create a channel. Then, pass a 6-0 silk braided suture around the vessels to ligate them together.
7. Cut the occluded renal artery and vein located close to the kidney. Remove the kidney along with the remaining adherent tissue.
8. Remove the cotton ball used to hold the liver.

3. Monitoring renal blood flow with Laser Speckle Contrast Imaging (LSCI) during ischemia-reperfusion

1. Turn on the LSCI system and the instrument's dedicated software.
2. Click on Original Image and Preview to view the raw, unprocessed image of the surgical field.
3. Change the position of the mice, maintain anesthesia using 1% isoflurane delivered at 600 mL/min via a nose cone, and adjust the camera's magnification while observing the original image to achieve a clear view of the target kidney.
4. Click on Simultaneous Display to view the real-time blood flow image. Set magnification to 2.5× and select Automatic Blood Flow Adjustment to optimize the image. Then, lock the Blood Flow Parameters. Besides, set up to record one frame of the picture every 5000 ms.
   NOTE: Wet cotton balls can be used to ensure the stable position of the kidney and keep the surrounding organs and tissues from it, enhancing the distinction between the kidney area and the background.
5. Set the size of the square area parameters to 112 and arrange the 5 square areas uniformly in a fixed orientation on the kidney view. Click on the Real-time Image Registration and Record to begin recording blood flow data.
   NOTE: The machine will automatically record the laser speckle perfusion units (LSPU), which represent the real-time blood flow units in the ROIs and can be used for analyses. During the detection process, breathing-related motion or environmental changes may cause the field of view to shift. If the system indicates this in the detection area, the current recording should be stopped. Then click on Real-time Image Registration and Record again.
6. After recording a 30 min baseline, separate the adipose tissue surrounding the left kidney vessels. Use the hemostatic forceps to grasp a microclamp and place it across the left renal artery and vein near the hilum. Continue real-time monitoring for another 30 min.
7. Release the micro-clamp to initiate reperfusion. Monitor and record the real-time recovery of renal microvascular blood flow.
8. Once the blood flow reperfusion is monitored for at least 60 min, stop monitoring and suture the abdominal incision with a 7-0 silk braided suture.

4. Post-operative recovery and care

1. Administer fluids with an intraperitoneal injection of 1 mL saline to prevent dehydration following the surgery.
2. Keep the mice on the heat pad. Carefully monitor mice until they have recovered consciousness, appear alert, and are able to right themselves.

Results

This article aimed to detect changes in renal blood flow during IR-induced AKI. Therefore, we employed a common mouse model that involves right nephrectomy (Figure 1A) followed by an IR procedure on the contralateral kidney (Figure 1B). Because real-time monitoring of blood flow during IRI was required, all manipulations on the mice were performed at the instrument region throughout the entire process (Figure 1C).

Discussion

This work describes the application of an LSCI system for the real-time, high-resolution visualization of renal microvascular blood flow dynamics during IRI in mice. The protocol offers a significant advantage over traditional endpoint measurements or methods with limited spatial resolution, such as LDF, by providing a two-dimensional perfusion map that captures regional heterogeneity within the kidney microcirculation. The efficiency and usability of LSCI for intraoperative real-time monitoring are key strengths of this...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Absorber canister (for anesthesia)	Shenzhen Laiyue Biotech Co., Ltd, China	10-0210
Alcohol, medical (75%)	HENGLIYA, Xinjiang, China	N/A	Skin disinfectant
Anesthetic vaporizer	Midmark	https://www.midmark.com/animal-health/products/anesthesia/detail/matrx-vip-3000-veterinary-vaporizer
Cotton swabs	Qingdao Hainuo Biological Engineering Co., Ltd, China	https://www.hainuocn.com/index/detail/448.html
Depilatory cream	VEET	https://www.veet.com.cn/jingchun/whx
Gauze bandage	Winner Medical Co., Ltd, Shenzhen, China	https://www.winnermedical.com/gauze-bandage.html
Heating pad	Renqiu Dingsheng Heating Equipment Co., Ltd, China	HD-1902	For maintaining body temperature
Iodophor solution	Taixinkang, Jinxian, China	https://www.11467.com/nanchang/co/120661.htm	Skin antiseptic
Isoflurane	RWD Life Science Co., Ltd, China	R510-22-10	Inhalation anesthetic
Laser speckle contrast imaging (LSCI) system	Hubei Xunwei Optoelectronic Technology Co., Ltd, China	SIM BFI HR PRO	MoorFLPI-2 equivalent system
Meloxicam	Qilu Animal Health Products Co., Ltd, China	https://en.qiludb.com/companion_animal/500.html	0.2 mg per kg of body weight; analgesic
Micro-clamp	Shanghai Medical Instrument(Group) Co., Ltd, China	W40140	Vascular clamp
Normal saline (0.9% sodium chloride)	Cisen Pharmaceutical Co., Ltd, China	https://www.cisen-pharma.com/index/shows?catid=42&id=62
Silk braided suture (6-0)	Yangzhou Fuda Medical Devices Co., Ltd, China	http://www.fdma.com.cn/products_detail/12.html	For vessel ligation
Silk braided suture (7-0)	Wego Terumo (Weihai) Medical Products Co., Ltd, China	https://www.weigaoholding.com/	For wound closure, non-absorbable surgical sutures with the curved needle
Syringe (1 mL)	Shandong Ande Healthcare Apparatus Co., Ltd, China	https://www.andehealthcare.com/html/chanpinzhanshi/Medication-Delivery/syringes/131.html	With 26 G needle
Tribromoethanol	Sigma-Aldrich	T48402-100G	2.5% solution; injectable anesthetic