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Dec 2019

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Live Intravital Intestine with Blood Flow Visualization in Neonatal Mice Using Two-photon Laser Scanning Microscopy
用双光子激光扫描显微镜观察新生小鼠活体肠内血流   

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Abstract

This protocol describes a novel technique to investigate the microcirculation dynamics underlying the pathology in the small intestine of neonatal mice using two-photon laser-scanning microscopy (TPLSM). Recent technological advances in multi-photon microscopy allow intravital analysis of different organs such as the liver, brain and intestine. Despite these advances, live visualization and analysis of the small intestine in neonatal rodents remain technically challenging. We herein provide a detailed description of a novel method to capture high resolution and stable images of the small intestine in neonatal mice as early as postnatal day 0. This imaging technique allows a comprehensive understanding of the development and blood flow dynamics in small intestine microcirculation.

Keywords: Intravital imaging (活体成像), In vivo imaging (体内成像), Two-photon laser scanning microscopy (TPLSM) (双光子激光扫描显微镜), Necrotizing enterocolitis (NEC) (坏死性小肠结肠炎), Neonatal mouse imaging (新生小鼠成像)

Background

Development of in vivo real-time imaging

Recent technological advances are overcoming the limitations of conventional histological analysis by enabling live imaging on experimental animals. In contrast to conventional histological microscopy techniques, the intravital approach provides insight into previously unknown morphogenetic and functional processes in live tissues. Furthermore, this information can be acquired in real time lapses, whereas alternative techniques are limited to a snapshot in time. The alternative techniques have the added requirement of determining the best timing to acquire images during a specific process in an experimental protocol.


Two-photon laser-scanning microscopy (TPLSM), compared to confocal microscopy, offers in vivo imaging that is superior for deep optical sectioning of living tissue (Pittet and Weissleder, 2011). The higher resolution and reduced phototoxicity of this method allow longer time periods of continuous real-time imaging on intact organs. We have standardized in vivo real-time imaging of intra-abdominal organs through TPLSM and used it to study the contribution of abnormal intestinal microcirculation in pathophysiological processes. For example, we have used this technology to study bacterial translocation in dextran sodium sulfate (DSS)-induced colitis (Toiyama et al., 2010), neutrophil extracellular traps (Tanaka et al., 2014a), thrombus formation in laser-induced endothelium injury (Koike et al., 2011), visualization of chemotherapy responses of colorectal liver metastases to the tumor microenvironment (Tanaka et al., 2014b), and the dynamics of circulating free DNA in a model of DSS-induced colitis (Koike et al., 2014). Additionally, previous reports have shown the application of intravital multiphoton microscopy to study pathophysiological processes in other abdominal organs including the liver (Honda et al., 2013; Lu et al., 2014; Liang et al., 2015), pancreas (Coppieters et al., 2010; Martinic and von Herrath, 2008), spleen (Ferrer et al., 2012), and kidneys (Peti-Peterdi et al., 2012; Hackl et al., 2013; Devi et al., 2013; Schiessl and Castrop, 2013).


In recent years TPLSM has become increasingly popular in in vivo research. However, intravital imaging of the small intestine in neonatal mice has been challenging due to their small body size and fragility of the intestinal wall. We have developed a novel application of TPLSM to visualize and study the small intestine of neonatal mice in vivo.


Advantages of the method

Two of the key advances of TPLSM are deeper tissue penetration and reduced photobleaching. These advances have facilitated the study of dynamic processes in vivo while minimizing injury to the organ or cell under investigation. Obtaining intravital stable images of the gastrointestinal tract has been difficult due to the movements caused by heartbeat, respiration, and intestinal peristalsis. To reduce the effects of these movements, we and others have purposefully designed devices and equipment that improve live image stability of the gastrointestinal tract (Watson et al., 2005; McDole et al., 2012; Ritsma et al., 2013). However, setting up such devices is time consuming, costly, and present technical challenges that require extensive trial and error. Current systems of choice for stabilizing intravital imaging are: a viewing window with a vacuum chamber (Looney et al., 2011), a microstage device (Cao et al., 2012), and our previously designed organ stabilizer (Japanese Patent No. 5268282) (Toiyama et al., 2010). These methods have been used for imaging the small intestine in adult mice, however, none of them have been applied to the imaging of the neonatal small intestine. One reason for this is that these devices are too large and limit the working space for the neonatal small intestine. To limit the movements induced by heartbeat and breathing during imaging, it is important that the organ stabilizer is detached from the neonatal abdominal wall. In addition, fixation maneuvers required when using previously developed organ stabilizing devices can cause injury or affect the neonatal intestinal microcirculation.


Our method allows for direct microvascular blood flow analysis. Stappenbeck TS et al. (2002) reported a method to analyze intestinal microcirculation indirectly from intestinal tissue samples harvested immediately after injecting fluorescein isothiocyanate-labeled dextran into the heart (Yu et al., 2009; Watkins and Besner, 2013; Yazji et al., 2013). However, thisand other similar methods for intravital imaging of the adult mouse intestine are not functional in blood flow dynamics. Our method allows for blood flow dynamics analysis, and facilitates investigation of intestinal villi development and establishment of the capillary network complexity (Stappenbeck et al., 2002). This protocol will allow the investigation of pathological processes associated with intestinal blood flow dynamics in vivo, thus promoting translational research.


The method we describe in this protocol is simple, overcomes the limitations of previous systems, and allows for stable live imaging of the neonatal mouse small intestine. This method interferes minimally with the microcirculation and enables high resolution intravital imaging of the small intestine for long periods of time. This protocol allows for unprecedented stability of intravital imaging of the neonatal small intestine.


Applications of the method

The TPLSM imaging method described here can be easily applied to investigate different physiological processes in the neonatal intestine in multiple mouse models. For example, we are using this method to study blood flow dynamics and inflammatory responses in necrotizing enterocolitis (NEC), intestinal epithelium and micro-vasculature development in short bowel syndrome (SBS), inflammatory and immunological status in inflammatory bowel disease (IBD), and ischemia-reperfusion injury in midgut volvulus. Previous studies investigated neonatal intestinal microcirculation in experimental NEC ex vivo, however, these studies did not consider the effects of blood flow dynamics in capillary-vessels in the villi. Our new method allowed us to measure neonatal intestinal microcirculation from movies of live blood flow and to derive blood flow velocity, vessel diameter and inflammation, and assess irrigation of the serosal and mucosal layers. Moreover, this technique is being used to visualize and quantify live blood flow dynamics during reperfusion and ischemia in experimental midgut volvulus, which will be useful to identify the primary intestinal tissues affected.


Here, we provide a step-by-step methodology to set up the neonatal mouse small intestine using a simple stabilizing device to evaluate the intestinal microcirculation by TPLSM.


Limitation of the approach

The proximal jejunum close to the ligament of Treitz is difficult to study due to its proximity and attachment to the abdominal wall. The described technique allows for analysis of the small intestine between the anterior superior iliac spine and the xiphoid process transversely, and between the sternum and the posterior abdominal wall longitudinally. Analyzing the intestine outside these marking points (for instance, in portions of the colon) may lead to potential bleeding from the mesentery due to excessive stretching applied on the intestine for appropriate positioning. Additionally, the device is limited to areas of the intestine that are naturally close to the abdominal wall without the need for heavy manipulation to avoid potential intestinal damage. The organ stabilizing device should not be in direct contact with the mouse abdominal wall to avoid image instability caused by movements due to breathing and heartbeat.


Considerations for intestinal preparation

The neonatal mouse should be fasted for at least 4 hours before microscopic observation since food residue within the intestinal lumen could affect the imaging results by affecting intestinal blood flow and/or potential for development of ischemia. Some reports have shown that intestinal blood flow varies by gestational age and feeding time (Pezzati et al., 2004; Watkins and Besner, 2013; Thompson et al., 2014; Morgan et al., 2014), suggesting that feeding tolerance should also be considered in the study protocol. Therefore, for analyzing the small intestine microcirculation using this method, a fixed fasting duration and gestational age between for all mice being used should be considered before starting the experiment to allow for appropriate comparison. The ideal fasting time to use will vary depending on the specific parameters to be measured in the neonatal small intestine. For example, 6-hour fasting allows proper imaging of the neonatal ileum in 5-day old pups subjected to experimental NEC, but this may vary if examining a different disease or mice of a different age. The varying effects on blood circulation secondary to feeding should be minimized by maintaining a homogenous feeding schedule and fasting duration across all mice being examined.


Procedure for NEC induction

NEC is induced by gavage feeding a hyperosmolar formula gavage, exposure to temporal hypoxia and oral administration of lipopolysaccharide (LPS) (Zani et al., 2008). Gavage feeding is given 3 times a day, using a 1.9-Fr silicon catheter (Vygon UK Ltd, Gloucestershire, UK). The hyperosmolar formula is prepared with 15 g of SMA Gold (SMA Nutrition, Berkshire, UK) in 75 ml of Esbilac canine supplement (Pet-Ag Inc., Hampshire, IL) (Barlow et al., 1974). Pups are exposed to hypoxia before each feed by placing them in a hypoxic chamber containing a gas mixture of 5% O2 and 95% N2 for 10 min, monitored with an O2 gas detector (BW O2 Gas Alert Clip Extreme, Rockall Confined & Safety, Cardiff, UK). LPS is administered on the 1st and 2nd day after NEC induction; mice are gavaged with 4 mg/kg/day LPS (lipopolysaccharide from Escherichia coli 0111:B4, Sigma-Aldrich Company Ltd., Dorset, UK) mixed within the formula feed. During the whole experiment, mice are kept in a neonatal incubator to maintain temperature (30 °C) and humidity (40%).

Materials and Reagents

Note: All reagents may be substituted with appropriate alternatives from other manufacturers.

Consumables

  1. Sterile 1.0 ml syringe with 26 Gauge needle or smaller (VWR, catalog number: 309597)

  2. Sterile gauze (VWR, catalog number: CA95041-740)

  3. Soft absorbable pad (VWR, catalog number: 95057-862)

  4. Rubber grove

  5. Kimwipes (VWR, catalog number: 102097-615)

  6. Microscope slides (VWR, catalog number: 48311-703, 1.0 mm thickness)

  7. Microscope cover glass (VWR, catalog number: 48393-172, 0.13-0.17 mm thickness)

  8. Falcon tube (15 ml) (VWR, catalog number: CA60819-761)

  9. Adhesive tape (VWR, catalog number: 89097-912)


Animals

Neonatal ROSAmT/mG;Tie2-Cre mice of both sexes were used to visualize the hemodynamics of small intestinal microcirculation and leukocyte movement. Table 1 shows a comprehensive list of transgenic mouse lines carrying fluorescent reporters that could be used in this protocol. Alternative transgenic reporter lines expressing fluorescent proteins can also be used in this method.

!Caution: Please note that all animal experiments should be performed following ethical guidelines and regulations of both the animal and imaging facilities. Research should not begin until use live animals is approved by the facility’s Animal Care Committee.


Table 1. Fluorescent positive mouse lines

Name Strain Type Promoter Specificity
GFP mouse C57BL/6 Tg(CAG-EGFP) Chicken β-Actin and cytomegalovirus enhancer All tissues except for erythrocytes and hair appear green under excitation light.
mTmG mouse C57BL/6 RosamTmG Chicken β-Actin/pCA These mice possess loxP sites on either side of a membrane-targeted tdTomato (mT) cassette and express strong red fluorescence in all tissues and cell types examined. When bred to Cre recombinase expressing mice, the resulting offspring have the mT cassette deleted in the Cre expressing tissue(s), allowing expression of the membrane-targeted EGFP (mG) cassette located just downstream.
Tie2 Cre (C57BL/6 x SJL)F1 Tek-cre Tek, endothelial-specific receptor tyrosine kinase These transgenic mice express Cre recombinase under the control of a mouse Tek promoter and enhancer. This promoter is active in endothelial cells.


Reagents

  1. Sterile Phosphate-buffered saline (PBS, 1×, pH 7.2)

  2. Ultra-purified water

  3. Disinfectant: 70% ethanol solution (70 ml of 100% ethanol added to 30 ml of water)

Equipment

Note: All equipment may be substituted with appropriate alternatives from other manufacturers.

General Equipment

  1. Appropriate microscope stage (Zeiss LSM710 motorized X, Y stage with Z focus) with heating pad (FHC Inc., catalog number: 40-90-2-07, Bowdoin, ME, USA)

  2. Gas anesthesia vaporizer (IsoTec4; Datex-Ohmeda GE Healthcare, Waukesha, WI, USA)

  3. Oxygen gas

  4. Hair removal cream (Nair® hair remover cream for face)

  5. Curved blunt forceps (VWR, catalog number 76319-850)

  6. Fine forceps (VWR, catalog number 82027-408)

  7. Scissors

    !Caution: Only use the tip of sharp scissors when conducting fine neonatal mouse surgery. This will allow for more precise work with less tissue damage.

  8. Solder lug terminal; 0.3 mm; M4 (Manufacturer OSTERRATH, manufacturer part number 60-2814-51/0030, Figure2A)

  9. Holding devices for both the cover glass and solder lug terminal (TEKTON 7521 Helping Hand with Magnifier, Figure 2C)


Microscope
  1. Inverted two-photon laser scanning microscope (TPLSM, e.g., Zeiss LSM710)

  2. Laser: Ti:Sapphire Chameleon Vision (Coherent)

  3. Objectives: 20× (Water immersion lens, e.g., Zeiss W Plan APOCHROMAT, 1.0 DIC (UV) VIS-IR∞/0.17)

  4. Software application: ZEN (Zeiss, imaging software)

Note: Magnifiers are not needed in this study and can be removed from the device.


Anesthesia

  1. Portable anesthesia machine

  2. Isoflurane vaporizer

  3. Anesthesia breathing circuit

  4. O2 gas flow regulator for E-cylinders (Praxair)

  5. O2 tank (E-cylinder)

  6. Anesthesia breathing circuit and nose cone

Procedure

Stage setup

  1. Make sure the heating pad of the stage is plugged in and that the stage is at 37 °C before starting the animal procedure.


Microscope setup
  1. Turn on the laser source, microscope and start the imaging applications, and all imaging components (data analysis workstation/computer, monitor, laser sources, camera, raster scanning unit, and detectors).

    !CAUTION: Prepare all microscopic settings before subjecting the animals to anesthesia.


Anesthetic cone setup
  1. Uncouple the normal sized anesthetic cone from the anesthetic breathing circuit and cover the end with a cut-out finger from a rubber glove. Cut a small hole at the tip of the rubber finger to fit the size of the neonatal mouse head (Figure 1A).

    !CAUTION: The hole size of the rubber glove finger should fit tightly around the head of the neonate mouse head, otherwise the isoflurane gas might leak.


Mice and surgery for image preparation
  1. A neonatal ROSAmT/mG;Tie2-Cre mouse of either sex is used in this protocol to visualize the hemodynamics of the small intestinal microcirculation.

  2. Place the mouse in the chamber for anesthesia with 2% isoflurane in oxygen. Confirm appropriate anesthesia by the absence of the withdrawal reflex after a toe pinch, as well as physiological responses including reduced respiratory and heartbeat rate. Once the mouse is anesthetized, reduce the isoflurane gas flow to 1-1.5%.

  3. Put the mouse on the preheated heating pad on a diaper pad, and the mouse head into the end of the anesthetic breathing circuit, with the mouse’s upper extremities fixed to the rubber finger with tape (Figure 1B).

    !CAUTION: To ensure that the animal is unconscious during the procedure, isoflurane is delivered continuously via the end of the anesthetic breathing circuit with the rubber finger covering the nose.

  4. Tape the lower extremities of the mouse to fix the body on the stage of the microscope and position it with a downward slope towards the tail (Figure 1C).

  5. The level of the downward slope should be such that it makes a triangular space on the neonatal abdomen from a lateral view. The angle θ from 30 to 45 degree is the best range for this experiment (Figure 1D, see also Table 2).

    ▲ CRITICAL STEP: Making this triangular space is critical to stabilize the image or movie. The top side of this triangle should be aligned at the same height as the sternum, and the bottom of the angle should be positioned above the Anterior Superior Iliac Spine (Figure 1D).


    Table 2. Troubleshooting table

    Step Problem Possible reason Solution
    8 Can not make the triangle space The level of downward slope is not enough Put a small back pillow made by Kimwipe or a small folded cloth between the soft absorbable pad and mouse's back. See also Figure 1D.
    12 Bleeding from the abdominal wall Incision line may have transversed the inferior epigastric artery and vein Make a muscle incision vertically at the middle of abdominal wall. You can distinguish the vertical abdominal midline from other muscle lesion because only the midline can see through to the inside of the abdominal wall clearly.
    13 Bleeding from the intestine or mesenteric lesion Too much force was used to pulled the intestine outside of the abdominal cavity During picking up of the intestine and putting it back into the abdominal cavity, be careful not to pull the intestine lower than the superior interior iliac spine level to avoid the intestinal and mesentric injury induced by tensile force.
    17 Having a difficulty making the gentle curve of the solder lug Fine manual work could be difficult by hand Use two fine pliers and gently make the curve.
    23 The fixed intestinal color is getting dark after putting the cover glass on it The pressure on the fixed intestine is too high Remove the cover glass quickly and release the compressed intestine. Re-check the diameter of both the intestine and the space created by the curved solder lug, and regulate the shape of the solder lug to fit the intestinal diameter.
    24 Can not keep the optimal intestinal observation position without intestinal physical injury. Pulling up the intestine over the heart level or pulling down the intestine over the superior anterior iliac spine level. Re-check the anesthetic mouse body condition. Make sure that there is a enough trianglular square under the horizontal line of sternum. See also step 5 troubleshooting.
    27 Noisy background signal Laser power is too high Too high of a laser power can cause not only photobleaching or photo-damage to the observed intestine but also non-specific autofluorescence. Decreasing the laser power (regulation of both laser intensity and fluorescent gain) is necessary, and then keep the imaging lesion clear as long as possible when long observation time is needed.
    Difficulty focusing the images Focusing area is outside of the contact area Control the x and y axes and find the appropriate contact region. Possible intestinal scan area is limited in the attached lesion with coverglass. See also step 20.


  6. Disinfect the whole abdominal skin area with 70% ethanol. Note that normally, mice have no hair in the neonatal period. If the mice have the hair on the operational skin area, you should use the hair remover gel to remove the hair softly with a small cotton swab.

  7. Put a sterile gauze on top of the disinfected abdominal area. The sterile gauze should have a small hole in the middle large enough to allow making a small incision.

  8. Make a vertical skin incision at the lower to middle abdominal area. This incision will expose the intestine and urinary bladder through the abdominal muscle layer.

  9. Cut the abdominal muscle and peritoneal layer vertically in the middle (Figure 1E, see also Table 2).

    !CAUTION: The length of the abdominal muscle incision should be shorter than the length of the skin incision to prevent intestinal protrusion and allow the mouse’s body to maintain its position throughout the imaging.

  10. Push gently on the lateral sides of the abdominal wall to allow a portion of the intestine to be completely externalized from the abdominal cavity. Using the cecum as a landmark, locate the segment of the small intestine that is of interest to your study, and place the rest of the intestinal length back into the abdominal cavity (Figure 1F, see also Table 2).

    ▲CRITICAL STEP: It is not recommended to use forceps to locate the cecum because even with gentle maneuvers, picking with forceps could easily injure the neonatal intestine. Additionally, it is possible that with an intestinal disease model, the microcirculation of the target area is already compromised, making the intestine and blood vessels even more delicate. With confirmation of the location of the cecum, which allows to distinguish the small intestine from the colon, the intestine should be carefully handled from a region away from the target area. The rest of the intestine should be put back into the abdominal cavity using blunt forceps.

    !CAUTION: Gentle maneuvers are necessary while moving the intestine around. Using blunt forceps, try to move the intestine by grasping tissue away from target region. Avoid unnecessary picking of the intestine with the forceps to reduce the number of contact points and to prevent puncturing/injuring the intestine. Avoid grasping the mesentery when moving the intestine.

  11. For the small intestine section that is selected for observation, use blunt forceps to gently pull out a small portion of the intestine that you will be observing. It is important to do this gently and allow the tissue to be under no tension to prevent potential ischemia or damage. Place one drop of PBS on the intestine to avoid drying.



    Figure 1. Neonatal mouse preparation for the TPLSM analysis. (A-D) Mouse anesthesia and appropriate positioning. (E, F) Intestine exposure.


Preparation of custom fixing device

  1. Bend the solder lug to a gentle curve, creating a space to sandwich the intestine between the ring of the solder lug and the cover glass (Figures 2A, 2B).

  2. Attach the other end of the bent solder lug to the holding device with tape (Figure 2C).

  3. Put the cover glass on the end of the solder lug that is attached to the holding device and clip it in place using the holding device. Ensure that there is space between the ring of the solder lug and the cover glass. The thickness of the space between the cover glass and the ring of the solder lug should be exactly the same as the maximum diameter of the intestine (Figure 2D, see also Table 2).

    !CAUTION: Maintaining a fixed space between the solder lug and cover glass according to the actual diameter of the intestine is crucial to keep the intestine in place while allowing proper intestinal blood circulation.

  4. Bend the ring of the solder lug to a complete horizontal plane parallel to the cover glass (Figure 2E, 2F).

    ▲CRITICAL STEP: Precise bending of the solder lug to sandwich the intestine between the ring of the solder lug and the cover glass is crucial to keep the small intestine in position during microscopic observation.

  5. After creating the appropriate space between the bent solder lug and cover glass and orienting the solder lug ring parallel to the cover glass on a horizontal plane, remove the cover glass.



    Figure 2. Fixing device setup for intestinal microscopic observation. (A, B) Solder lug. (C, D). Solder lug attached to holding device with appropriate spacing, the spacing should be the same as the diameter of the intestine being used (generally between 1 and 3 mm). (E, F) Cover glass attached on top of the solder lug and clipped with the holding device.


Preparation of the microscopic stage

  1. Transfer the customized fixing device to the microscopic stage where the mouse is mounted.

  2. Put the ‘U’ shaped intestine on the ring of the solder lug. Wet the intestine with a drop of PBS and place the cover glass directly on top of the area, fixing the cover glass with the clip of the holding device. The bottom area of the ‘U’ shaped intestine should be slightly over the solder lug (Figure 3A).

    CRITICAL STEP: If the bottom area of ‘U’ shaped intestine is not over the solder lug, the position of the intestine should be moved, as miss positioning may prevent stable intravital imaging.



    Figure 3. Neonatal mouse intestine exposed for TPLSM observation. (A-C) Mouse intestine preparation and areas of observation. The yellow marking or alternatively in the red dotted area shows the contact lesion area between small intestinal wall and the cover glass. The green dashed line (under image) shows the cover glass level (the top picture in C: view from above the cover glass, the bottom picture in C: view from the frontal view. The observable area is between the green dotted line and the yellow line from the frontal view. The red dashed circle represents the outer intestinal wall line from the frontal view. (D) Illustration of mouse intestine for microscopic observation.


  3. Put one drop of distilled water on the cover glass where the intestine is touching the cover glass. Bring down the objective lens immersion into the waterdrop dome and begin the microscopic observation (Figure 3B).

  4. Figure 3C shows the ideal set up of the customized holding device, the microscope, and the mouse. After putting the cover glass on top of the intestine, only a small portion of the intestine will be in contact with the cover glass (shown in Figure 3C inside the yellow marking or alternatively in the red dotted area). This area will be the best location to observe the whole intestinal wall from the serosa level to the villi. When viewing the set up from the side, the segment of intestine sandwiched between the solder lug and the cover glass should look slightly oval shaped (Figure 3C red dotted circles). This positioning of the intestine is essential to prevent ischemia (see also Table 2).

    ▲CRITICAL STEP: Make sure that the intestine is not damaged after fixation on the device and putting on the coverglass. Damaged tissue can be identified by increased redness. If the color of the intestine turns dark soon after fixation, remove the cover glass immediately and release the compression to restore intestinal blood flow circulation.

    !CAUTION: Placing of the cover glass and securing it with the clip must be done very carefully. Applying too much pressure with the clip can break the cover glass.

  5. Once the intestine is fixed, ensure that any parts of the device are not in direct contact with the mouse body (Figure 3D). Additionally, confirm that the fixed intestine that is being observed lies between the sternum and the Anterior Superior Iliac Spine in position (see also Table 2).

    !CAUTION: Do not pull the intestine above the level of the heart (towards the head) to avoid both the potential positional effects on blood pressure and the potential effects on blood flow from stretching the tissue. As well, do not pull the intestine below the level of the Anterior Superior Iliac Spine, this would impose too much tension and cause ischemia.


Intravital imaging
  1. Intravital observation can now be performed using a Zeiss LPM710 inverted microscope (Zeiss) with ×20 water immersion objective lens (W Plan-Apochromat 20×/1.0 DIC, VIS-IR M27 75mm). TPLSM images are acquired at 512 × 512 pixels of spatial resolution from a 386.45 μm field of view. The excitation wavelength to detect GFP is 910 nm. The scan speed should be set at 1.27 μs/pixel. Two-photon fluorescence signals are collected by an internal detector (non-descanned detection method) at an excitation wavelength.

    !CAUTION: The laser power is adjusted according to the imaging depth and intestinal diameter. When imaging at greater depths, the laser power level should be increased (up to 100%) manually using the laser power level controller. The laser power should be adjusted slowly from a low to high level to avoid photo-bleaching of the target area, which may occur at higher laser power. If the observation area is photo-bleached the stage must be moved to focus on another area.

  2. Each area of interest is scanned at a high magnification (water-immersion objective 20× or higher if desired) by manually setting the X/Y plane and adjusting the Z axis to obtain high-resolution, clear TPLSM images.

  3. In our experimental setting, the imaging depth ranged from 100 μm to 400 μm. Optimal high-resolution images were obtained from the tissue surface up to 200 μm in depth. For optimal simultaneous imaging of EGFP and tdTOMATO, detection gain should be adjusted for EGFP (to 500-570) or tdTOMATO (to 580-650)

    !CAUTION: The range of gain value should be changed according to each intestinal region. A higher gain produces a stronger signal; however, it also produces more background.

    ▲CRITICAL STEP: The combination of laser power and gain value determines the intensity of the laser signal. The settings of these two values depend on the quality of image focus and on the length of time of continuous imaging (see also Table 2).

  4. Start the observation and recording by TPLSM and save the acquired data. Saving the data as a czi file is recommended because czi files can be used to analyze the blood flow dynamics or make Z-stack movies/3-dimentional images using ZEN 2 lite software after imaging.

    !CAUTION: For the analysis of blood flow dynamics, including blood flow velocity, blood flow volume, and leukocyte rolling speed, we recommend capturing 80-100 frames within 30 s.

  5. After microscopic intravital observations, euthanize the mouse under general anesthesia according to approved protocols.

Data analysis

Processing of image data

  1. Launch the ZEN 2.0 lite software (freely available from the ZEISS company website after registration).

  2. Open the saved czi files and place a properly sized scale bar. The czi file contains information about each imaging setting, as well as scanned area size and time. To create image data from sequential time course images, choose one image and select the ‘export/import’ button from the file tag. Then, select ‘export’ and choose the appropriate file type (e.g., JPEG, TIFF, PNG etc.). To create a movie from the data, select the ‘movie export’ function and choose the appropriate file type (e.g., AVI, WMF, MOV etc.). The czi files can be used for the analysis.


Blood flow velocity

The blood flow velocity (V) is calculated as described in several studies that quantified the blood flow in vessels of different caliber, from arteries to capillaries (Tang et al., 2015). In one movie (20-30 s), select a sequence of the four to six most clear images of a blood vessel. Measure the tangent length that is parallel to the blood flow direction (ΔX) and the cosine length of the strand that is vertical to the blood flow direction (ΔT). The blood flow velocity is calculated using the following formula (Figures 4A, 4B and Figure 5):

V [µm/ms] = ΔX/ΔT


Velocity can be defined as a mean value calculated from four or six strands selected from the movie.



Figure 4. Dynamic intestinal microcirculation in neonatal mice. A. Intestinal microcirculation image obtained by TPLSM. B. Blood flow volume calculation. C. Blood vessel wall shear rate calculation. D. Leukocyte rolling speed calculation.



Blood flow volume

Blood flow volume (FV) is calculated using the following formula (Figure 4B and Figure 5):


FV [µm3/ms] = π(d/2)2ΔX/ΔT


[d] is the maximum diameter of the blood vessel


Shear rate of blood vessel wall

Blood vessel wall shear rate (W) is calculated in a 100-µm segment of the vessel using the formula based on the Newtonian definition (Russell et al., 2003) (Figure 4C and Figure 5):


W [1/ms] = 8V/d



Figure 5. Example of experimental result and data analysis. A. One still/flame image cutting from the recorded video (one flame scan speed: 380 ms). From this find a straight line of the platelet. B. Measure the tangent length of the straight line that is parallel to the blood flow direction (ΔX), scan time (ΔT), and blood vessel maximum diameter (d). C. Calculate the velocity, flow volume, and shear rate using the specific formula.


Number of adherent leukocytes

Adherent leukocytes can be defined in each vessel segment as cells that do not move or detach from the endothelial lining within a specified observation period of 5-10 s. Quantification is done by counting the number of adherent leukocytes sticking to the endothelial surface within a 100-µm length of a single vessel (Kubes et al., 2003; Nakagawa et al., 2006).


Leukocytes rolling speed

Leukocytes’ rolling phenomenon is observed mainly in the post capillary venule. The post capillary venule is a V1 or V2 level vein that is located in the submucosal level, made from the combined branches of the capillary vessels (Yu et al., 2009). The maximum diameter of this venule in the neonatal mouse intestine is approximately 20-60 µm. Choose a frame in which there is a consistent maximum diameter of post-capillary venules and select a single rolling leukocyte to follow as it rolls on the inner venule wall. Measure the distance travelled by the leukocyte and record the length of travel time, as determined by the time from the first frame observed to the last frame observed. Leukocyte rolling speed [µm/ms] is calculated in [µm/ms] (Figure 4D):


Leukocyte rolling speed = [µm/ms]


Where: leukocyte traveled distance length [µm]/ the scan time span between the two selected time frames [ms].


Anticipated results

By using this method of TPLSM imaging of the neonatal intestine, we are able to observe and analyze the microcirculation and blood flow dynamics of the neonatal small intestine in detail (Video 1). Video 1 starts at tips of the villi and travels downwards towards the based of the villi and then through the underlying vascular supply and smooth musculature at the end. Moreover, acquisition of z-stack and x-y data allows creating three-dimensional images of the entire structure of the small intestine, from the top of the villi to the bottom, including the submucosal area. Administration of additional fluorescent dyes such as SYTOX green or red prior to imaging enables the localization of affected cells in a disease model (Video 2). Using this approach, we revealed that the top of the villi is the main area affected by necrosis in the experimental NEC mouse model (Video 2). This approach is also useful for long-term analysis of the effects of drugs and other procedures on the neonatal small intestine. Furthermore, this approach allows the user to visualize changes in intestinal microcirculation in a target area.

In summary, the combination of the organ stabilizing method for TPLSM described in this protocol yields reliable results for studying neonatal intestine development and intestinal pathophysiology.


Video 1. Microcirculation of the neonatal small intestine


Video 2. 3-D image of the whole villi in a control neonatal mouse and the necrotizing enterocolitis mouse model


Acknowledgments

BL is the recipient of Restracomp Fellowship, The Hospital for Sick Children and Early Career Award Program grant from Thrasher Research Fund (14503). PDO is supported by the Canadian Institutes of Health Research (CIHR) (162208 and 149046), the Heart and Stroke Foundation of Canada (G-17- 0018613), and the Natural Sciences and Engineering Research Council of Canada (NSERC) (500865). AP is the recipient of a Canadian Institutes of Health Research (CIHR) Foundation Grant 353857.

Author contributions: Y.K., B.L., Y.C., N.G., M.A., H.M., C.L., A.H., and R.W.: Conception and design, Collection and/or assembly of data, Data analysis and interpretation, Manuscript writing, Final approval of manuscript. K.U., M.I., and P.D.O. Conception and design, Final approval of manuscript. A. P.: Conception and design, Financial support, Final approval of manuscript.

Competing interests

The authors have no conflict of interest to declare.

Ethics

All procedures described in this protocol were approved by the Institutional Animal Care Committee at the Toronto Medical Discovery Tower (No.4886.0), and by the Advanced Optical Microscopy Facility.

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  • 简介

    [摘要]该协议描述了一种使用双光子激光扫描显微镜(TPLSM)研究新生小鼠小肠病理基础的微循环动力学的新技术。多光子显微镜技术的最新技术进步允许对不同器官(例如肝,脑和肠)进行活体分析。尽管取得了这些进步,但对新生啮齿类动物的小肠进行实时可视化和分析在技术上仍然具有挑战性。我们在此提供了一种新方法的详细描述,该方法最早可在出生后第0天捕获新生小鼠中小肠的高分辨率和稳定图像。这种成像技术可以全面了解小肠微循环中的发育和血流动力学。



    [背景]

    体内实时成像的发展

    通过允许在实验动物上进行实时成像,最近的技术进步克服了常规组织学分析的局限性。与传统的组织学显微镜技术相比,活体检查方法可洞悉活组织中以前未知的形态发生和功能过程。此外,该信息可以实时获取,而替代技术仅限于时间快照。替代技术对确定在实验方案中的特定过程中获取图像的最佳时机具有额外的要求。

    Ť WO光子激光扫描显微术(TPLSM),相比共聚焦显微镜,优惠体内成像,它优于用于活组织(Pittet和Weissleder的深光学切片,2011)。在该方法的更高的分辨率和减小的光毒性允许连续实时成像的完整上器官更长的时间段。我们已经通过TPLSM标准化了腹腔内器官的体内实时成像,并用于研究异常肠道微循环在病理生理过程中的作用。例如,我们已使用这项技术研究了硫酸右旋糖酐(DSS)引起的结肠炎中细菌的移位(Toiyama等人,2010),嗜中性白细胞胞外陷阱(Tanaka等人,2014 a ),激光中血栓形成的情况。诱导的内皮损伤(Koike等,2011),可视化大肠肝转移瘤对肿瘤微环境的化学反应(Tanaka等,2014 b ),以及DSS诱导的结肠炎模型中循环游离DNA的动力学(Koike等人,2014年)。此外,先前的报道显示,活体多光子显微镜技术可用于研究其他腹部器官包括肝脏的病理生理过程(Honda等,2013 ;Lu等,2014;Liang等,2015 ),胰腺(Coppieters等)。等人,2010;马丁尼克和冯·赫拉特,2008),脾脏(费雷尔等人,2012)和肾脏(Peti-Peterdi等人,2012 ;Hackl等人,2013; Devi等人,2013;Schiessl)和Castrop ,2013年)。

    近年来,TPLSM在体内研究中变得越来越流行。然而,由于新生小鼠的小体型和肠壁的脆弱性,其对小肠的活体成像非常具有挑战性。我们已经开发了TPLSM的一种新应用,以可视化和研究体内新生小鼠的小肠。



    该方法的优点

    TPLSM的两个关键进步是更深的组织渗透和减少的光漂白。这些进展促进了体内动态过程的研究,同时使对所研究的器官或细胞的伤害最小化。由于心跳,呼吸和肠蠕动引起的运动,很难获得胃肠道的活体内稳定图像。为了减少这些运动的影响,我们和其他人专门设计了可以改善胃肠道实时图像稳定性的设备(Watson等,2005;McDole等,2012;Ritsma等,2013 )。但是,设置此类设备非常耗时,成本高昂,并且存在技术挑战,需要大量的反复试验。当前选择的用于稳定活体内成像的系统是:带有真空室的观察窗(Looney等,2011),微型载物台装置(Cao等,2012)和我们先前设计的器官稳定器(日本专利5268282) )(Toiyama et al。,2010)。这些方法已经被用于成年小鼠的小肠成像,但是,它们都没有被应用于新生儿小肠的成像。原因之一是这些设备太大,并且限制了霓虹膜小肠的工作空间。为了限制在成像过程中由心跳和呼吸引起的运动,重要的是将器官稳定器与新生儿腹壁分离。在此外,固定机动使用先前开发的器官稳定装置可以在需要时造成伤害或影响肠道新生儿微循环。

    我们的方法可以直接进行微血管血流分析。Stappenbeck TS等。(2002)报道了一种方法,该方法间接地分析了将荧光素异硫氰酸酯标记的右旋糖酐注射入心脏后立即采集的肠道组织样本中的肠道微循环(Yu等人,2009; Watkins和Besner ,2013; Yazji等人,2013)。但是,这种和其他类似的方法对成年小鼠小肠进行活体内成像在血流动力学中不起作用。我们的方法可以进行血流动力学分析,并有助于调查肠绒毛的发育和建立毛细血管网络的复杂性(Stappenbeck等,2002)。该协议将允许研究与体内肠道血流动力学相关的病理过程,从而促进翻译研究。

    我们在此协议中描述的方法很简单,克服了以前系统的局限性,并允许对新生小鼠小肠进行稳定的实时成像。该方法对微循环的干扰最小,并且可以长时间对小肠进行高分辨率的活体成像。该协议允许新生儿小肠的活体成像的前所未有的稳定性。



    该方法的应用

    此处描述的TPLSM成像方法可以轻松应用于多种小鼠模型中的新生儿肠道中不同的生理过程。例如,我们正在使用这种方法研究坏死性小肠结肠炎(NEC)的血流动力学和炎症反应,短肠综合征(SBS)的肠上皮和微脉管系统发育,炎性肠病(IBD)的炎症和免疫状态,和肠中肠扭转缺血再灌注损伤。以前的研究调查了离体NEC实验中新生儿肠道微循环,但是,这些研究并未考虑绒毛中毛细血管中血流动力学的影响。我们的新方法使我们能够从活血流电影中测量新生儿肠道微循环,并得出血流速度,血管直径和炎症,并评估浆膜和粘膜层的冲洗率。此外,该技术被用于可视化和量化实验中肠扭转中缺血和再灌注过程中的活血流动动态,这将有助于识别受影响的主要肠道组织。

    在这里,我们提供了使用简单的稳定装置通过TPLSM评估肠道微循环的分步方法来建立新生小鼠小肠。



    方法的局限性

    由于Treitz韧带附近并与腹壁相连,因此难以研究靠近Treitz韧带的近端空肠。所描述的技术允许横向分析of前上棘和剑突之间的小肠,纵向分析胸骨和后腹壁之间的小肠。在这些标记点之外(例如,在结肠的某些部位)分析肠子可能会导致肠系膜潜在的出血,这是由于在肠子上过度拉伸以进行适当定位所致。另外,该装置限于自然地靠近腹壁的肠区域,而无需进行繁重的操作以避免潜在的肠损伤。器官稳定装置不应直接与小鼠腹壁接触,以免由于呼吸和心跳引起的运动而导致图像不稳定。



    肠道准备的注意事项

    显微镜观察之前,新生小鼠应禁食至少4小时,因为肠腔内的食物残留可能会通过影响肠血流量和/或缺血发展潜力而影响成像结果。一些报告表明,肠血流量随胎龄和进食时间的不同而变化(Pezzati等,2004 ;Watkins和Besner ,2013 ;Thompson等,2014; Morgan等,2014),这表明摄食耐受性也应在研究方案中考虑。因此,为了使用此方法分析小肠微循环,在开始实验之前,应考虑所有正在使用的小鼠的固定空腹持续时间和胎龄,以进行适当的比较。理想的禁食时间将根据新生儿小肠中要测量的特定参数而有所不同。例如,禁食6小时可以对经过实验NEC的5天大的幼仔进行正确的新生儿回肠成像,但是如果检查不同的疾病或不同年龄的小鼠,这可能会有所不同。应当通过在所有接受检查的小鼠上保持均一的进食时间表和禁食持续时间,将进食后对血液循环的各种影响减至最小。



    NEC诱导程序

    通过饲喂高渗配方奶管饲管,暴露于暂时性缺氧和口服脂多糖(LPS)来诱导NEC (Zani et al。,2008)。使用1.9-Fr的硅导管(英国格洛斯特郡的Vygon UK Ltd)每天进行3次强饲。用在75ml Esbilac犬补充剂(Pet-Ag Inc.,Hampshire,IL)中的15g SMA Gold(SMA Nutrition,Berkshire,UK)制备高渗配方(Barlow等,1974)。在每次喂食之前,将幼犬置于缺氧的小室内,使其处于低氧状态,将其置于含有5%O 2和95%N 2的混合气体中10分钟,并用O 2气体检测仪(BW O 2气体警报夹Extreme,Rockall)进行监测密闭和安全,英国加的夫)。在NEC诱导后的第1天和第2天给予LPS;用配方饲料中混合的4 mg / kg /天的LPS(来自大肠杆菌0111:B4的脂多糖,Sigma-Aldrich Company Ltd.,Dorset,英国)管饲小鼠。在整个实验过程中,将小鼠饲养在新生儿恒温箱中以保持体温(30 °C)和湿度(40%)。

    关键字:活体成像, 体内成像, 双光子激光扫描显微镜, 坏死性小肠结肠炎, 新生小鼠成像




    材料和试剂



    注意:所有试剂都可以用其他制造商的适当替代品代替。


    耗材小号


    1.0毫升无菌注射器,针头为26或更小号(VWR ,目录号:309597)
    无菌纱布(VWR ,目录号:CA95041-740)
    软吸收垫(VWR ,目录Ñ棕土:95057-862)
    橡胶林
    Kimwipes(VWR ,目录号:102097-615)
    显微镜载玻片(VWR ,目录号:48311-703,厚度1.0 mm)
    显微镜盖玻片(VWR ,目录号:48393-172,厚度0.13-0.17 mm)
    猎鹰管(15 ml)(VWR ,目录号:CA60819-761)
    胶带(VWR ,目录号:89097-912)


    动物


    分别使用新生儿ROSA mT / mG ;两只性别的Tie2-Cre小鼠观察小肠微循环和白细胞运动的血流动力学。表1列出了可在此方案中使用的携带荧光报告基因的转基因小鼠品系的完整列表。表达荧光蛋白的替代性转基因报告基因系也可用于该方法。


    !注意:请注意,所有动物实验均应遵守动物和影像设备的道德准则和规定。只有在设施的动物保育委员会批准使用活体动物后,才可以开始研究。



    表1 。荧光阳性小鼠系



    试剂种类


    无菌磷酸盐缓冲盐水(PBS,1 × ,p H 7.2)
    超纯净水
    消毒剂:70%乙醇溶液(将70 ml 100%乙醇添加到30 ml水中)


    设备



    注意:所有设备都可以用其他制造商的适当替代品代替。


    通用设备


    带有加热垫的合适的显微镜载物台(Zeiss LSM710电动X,Y载物台,Z焦点),带有加热垫(FHC Inc. ,目录号:40-90-2-07,美国缅因州Bowdoin )
    气体麻醉蒸发器(IsoTec4;美国威斯康星州沃克斯夏的Datex-Ohmeda GE Healthcare)
    氧气
    脱毛霜(奈尔®面部毛发去除霜)
    弯曲钝钳(VWR ,货号76319-850)
    细钳(VWR ,目录号82027-408)
    剪刀
    !警告:进行精细的新生鼠手术时,请仅使用锋利的剪刀的尖端。这将使工作更精确,组织损伤更少。


    焊片端子;0.3毫米;M4(制造商OSTERRATH ,制造商零件编号60-2814-51 / 0030,图2A )
    盖玻片和焊片端子的固定装置(TEKTON 7521放大镜助手,图2C )


    显微镜


    倒置两光子激光扫描显微镜(TPLSM,例如Zeiss LSM710)
    激光:钛:蓝宝石变色龙视觉(相干)
    物镜:20 × (水浸镜头,例如,蔡司W Plan APOCHROMAT,1.0 DIC(UV)VIS-IR∞/ 0.17 )
    软件应用程序:ZEN(Zeiss,成像软件)
    注意:本研究不需要放大镜,可以从设备中将其删除。



    麻醉


    便携式麻醉机
    异氟烷气化器
    麻醉呼吸回路
    用于E气瓶(Praxair)的O 2气体流量调节器
    O 2油箱(E缸)
    麻醉呼吸回路和鼻锥


    程序



    舞台设置


    在开始动物操作之前,请确保已插入平台的加热垫且平台处于37 °C 。


    显微镜设置


    打开激光源,显微镜并启动成像应用程序以及所有成像组件(数据分析工作站/计算机,监视器,激光源,照相机,光栅扫描单元和检测器)。
    !注意:在对动物进行麻醉之前,请准备所有微观设置。



    麻醉锥的设置


    从麻醉呼吸回路上拆下正常尺寸的麻醉锥,并用橡胶手套切开的手指遮住末端。在橡胶手指的顶端切一个小孔,以适合新生儿鼠标头的大小(图1A)。
    !小心:橡胶手套的手指的孔尺寸应紧紧贴在新生鼠头的头部周围,否则异氟醚气体可能会泄漏。



    小鼠和外科手术准备图像


    在此方案中,使用任一性别的新生儿ROSA mT / mG ; Tie2-Cre小鼠来可视化小肠微循环的血流动力学。
    将鼠标放入含2%异氟烷的氧气中进行麻醉。脚趾捏合后没有撤退反射,以及包括呼吸和心跳频率降低在内的生理反应,以确认适当的麻醉。一旦小鼠被麻醉时,异氟烷气体流降低到1 - 1.5%。
    将鼠标放在尿布垫上预热的加热垫上,并将鼠标头插入麻醉呼吸回路的末端,并用胶带将鼠标的上肢固定在橡胶手指上(图1B)。
    !注意:为确保动物在此过程中保持清醒状态,异氟醚通过麻醉呼吸回路的末端连续输送,橡胶手指覆盖鼻子。


    用胶带粘住鼠标的下肢,将其身体固定在显微镜的载物台上,并将其向下倾斜,使其朝向尾巴放置(图1C)。
    向下倾斜的水平应使得从侧面看在新生儿腹部上形成一个三角形的空间。从30至45度的角θ是该实验的最佳范围(图1D,也参见Ť能够2)。
    ▲关键步骤:形成此三角形空间对于稳定图像或影片至关重要。该三角形的顶边应与胸骨高度对齐,并且该角的底部应位于I前上棘的上方(图1D)。


                                                         





    表2 。故障排除表



    用70%乙醇消毒整个腹部皮肤区域。请注意,正常情况下,小鼠在新生儿时期没有毛发。如果小鼠的手术皮肤上有头发,则应使用除毛凝胶,用小棉签将其柔软地去除。
    在消毒的腹部上方放一块无菌纱布。无菌纱布的中间应有一个小孔,该孔应足够大以允许切开一个小切口。
    在腹部的下至中部做一个垂直的皮肤切口。该切口将通过腹肌层暴露肠道和膀胱。
    切开腹部肌肉和腹膜层垂直地在中间(图1E,也参见Ť能够2)。
    !注意:腹肌切口的长度应短于皮肤切口的长度,以防止肠突出并在整个成像过程中使小鼠的身体保持其位置。


    轻轻推动腹壁的侧面,以使一部分肠腔完全从腹腔向外突出。我们荷兰国际集团盲肠作为地标,找到小肠是关心你的学习的部分,并把肠道长度放回腹腔的其余部分(图1F,又见牛逼能2)。
    ▲关键步骤:不建议使用镊子定位盲肠,因为即使进行轻柔的操作,用镊子采摘也很容易伤害新生儿肠。另外,在肠道疾病模型中,目标区域的微循环可能已经受到损害,从而使肠道和血管变得更加脆弱。在确认盲肠的位置后,可以将小肠与结肠区分开,应在远离目标区域的区域小心处理肠。其余的肠子应用钝镊子放回腹腔。


    !小心:在移动肠子时,必须进行轻柔的操作。使用钝钳,尝试通过抓住组织远离目标区域来移动肠道。避免用镊子不必要地摘取肠子,以减少接触点的数量,并防止穿刺/伤害肠子。移动肠子时避免抓住肠系膜。


    对于选择进行观察的小肠部分,请使用钝钳轻轻拉出要观察的一小部分肠子。轻柔地进行此操作并让组织处于无张力状态以防止潜在的局部缺血或损伤,这一点很重要。将一滴PBS放在肠子上以避免干燥。




    图1.用于TPLSM分析的新生小鼠制备。(AD)鼠标麻醉和适当的位置。(E,F)肠暴露。



    定制定影装置的准备


    弯曲焊耳到平缓曲线,创造一个空间以夹住之间的肠的焊料凸块的环和盖玻璃(图小号2A,2 B)。
    用胶带将弯曲的焊片的另一端连接到固定装置上(图2C)。
    将盖玻片放在连接到固定装置的焊片的末端,然后使用固定装置将其固定在适当的位置。确保焊片环和盖板玻璃之间有空间。盖玻璃和焊料凸块的环之间的空间的厚度应该是完全一样的肠的最大直径(图2D,也参见Ť能够2)。
    !小心:根据小肠的实际直径,在焊片和盖玻片之间保持固定的空间对于保持小肠在适当的位置,同时允许小肠的血液循环至关重要。



    将焊片的环弯曲到与防护玻璃平行的完整水平面(图2E,2 F)。
    ▲关键步骤:准确地将焊片弯曲以将小肠夹在焊片的环和盖板玻璃之间,这对于在显微镜观察期间将小肠保持在适当位置至关重要。


    在弯曲的焊片和防护玻璃之间留出适当的空间,并在水平面上将焊片环与防护玻璃平行地对准后,取下防护玻璃。




    图2.用于肠镜观察的固定设备设置。(A,B)焊片。(C,D)。焊片应以适当的间距连接到固定装置,间距应与所用肠子的直径相同(通常在1到3毫米之间)。(E,F)盖上安装在焊片顶部并用固定装置夹住的玻璃。



    微观阶段的准备


    将定制的固定设备转移到安装鼠标的微观平台上。
    将“ U”形的肠子放在焊片的环上。用一滴PBS润湿肠子,然后将盖玻片直接放在该区域的顶部,用固定装置的夹子固定盖玻片。“ U”形肠的底部区域应略微超过焊片(图3A)。
    ▲关键步骤:如果“ U”形小肠的底部区域不在焊片上方,则应移动小肠的位置,因为定位不当可能会妨碍稳定的活体成像。







    图3 。暴露的新生小鼠肠道用于TPLSM观察。(AC)小鼠肠的准备和观察区域。黄色标记或红色虚线区域表示小肠壁和盖玻片之间的接触病变区域。绿色虚线(下图)显示了防护玻璃水平仪(C的顶部图片:从防护玻璃上方观察,C的底部图片:从正面观察)。可观察区域在绿色虚线和从正面看是黄色的线,红色虚线圆圈是从正面看的外部肠壁线。



    将一滴蒸馏水放在肠子接触盖玻片的盖玻片上。将物镜浸入水滴圆顶中并开始显微镜观察(图3B)。
    图3C显示了定制的固定设备,显微镜和鼠标的理想设置。将盖玻片放在肠顶上后,只有一小部分肠会与盖玻片接触(图3C中黄色标记内或红色虚线区域内)。该区域是观察从浆膜层到绒毛的整个肠壁的最佳位置。从侧面看时,夹在焊片和盖玻片之间的那部分肠应看起来略呈椭圆形(图3C中的红色虚线圆圈)。肠道的这种定位对于预防缺血至关重要(另见表2)。
    ▲关键步骤:固定在设备上并戴上防护玻璃后,请确保肠未受损。受损的组织可以通过增加的发红来识别。如果固定后肠子的颜色很快变黑,请立即取下盖玻片并释放压力,以恢复肠子的血液循环。


    !注意:必须非常小心地放置好玻璃盖并用夹子固定。用夹子施加太大的压力可能会损坏盖板玻璃。


    固定肠子后,请确保设备的任何部分均未与鼠标主体直接接触(图3D)。另外,确认观察到的固定肠位于胸骨和I上前棘之间的位置(另请参见表2)。
    !注意:请勿将肠子拉到心脏上方(朝向头部),以免对血压产生潜在的位置影响,也避免因拉伸组织而对血流产生潜在影响。同样,不要将肠子拉到I前上棘的水平以下,这会产生过大的压力并引起局部缺血。



    活体成像


    现在,可以使用配备× 20浸水物镜(W Plan-Apochromat 20 ×/ 1.0 DIC,VIS-IR M27 75mm)的Zeiss LPM710倒置显微镜(Zeiss)进行玻璃体内观察。从386.45μm视场中,以512 × 512像素的空间分辨率获取TPLSM图像。检测GFP的激发波长是910nm 。扫描速度应设置为1.27μs /像素。通过内部检测器(非去扫描检测方法)以激发波长收集双光子荧光信号。
    !注意:根据成像深度和肠直径调整激光功率。当在更深的深度成像时,应使用激光功率水平控制器手动提高激光功率水平(最高100%)。激光功率应从低到高缓慢调节,以免目标区域发生光漂白,这可能在较高的激光功率下发生。如果观察区域是光漂白的,则必须移动平台以将焦点对准另一个区域。


    通过手动设置X / Y平面并调整Z轴,可以以高倍率(如果需要,将水浸物镜达到20倍或更高)扫描每个感兴趣的区域,以获得高分辨率,清晰的TPLSM图像。
    在我们的实验环境中,成像深度范围为100μm至400μm。从组织表面获得深度达200μm的最佳高分辨率图像。对于EGFP和tdTOMATO的最佳同时成像,检测增益应该被调整EGFP(500 - 570)或tdTOMATO(580 - 650)
    !注意:增益值的范围应根据每个肠道区域而变化。增益越高,产生的信号越强;但是,它也会产生更多的背景。


    ▲关键步骤:激光功率和增益值的组合决定了激光信号的强度。这两个值的设置取决于图像聚焦质量和上的连续成像的时间长度(还参见Ť能够2) 。


    通过TPLSM开始观察和记录,并保存获取的数据。建议将数据另存为czi文件,因为czi文件可用于分析血流动力学或在成像后使用ZEN 2 lite软件制作Z-stack电影/ 3维图像。
    !注意:为了分析血流动力学,包括血流速度,血流量和白细胞滚动速度,我们建议在30 s内捕获80-100帧。


    活体显微镜下观察后,根据批准的方案在全身麻醉下对小鼠实施安乐死。


    数据分析



    图像数据处理


    1.启动ZEN 2.0 lite软件(注册后可从ZEISS公司网站免费获得)。     

    2.打开保存的czi文件,并放置适当大小的比例尺。czi文件包含有关每个成像设置以及扫描区域大小和时间的信息。要从连续的时程图像创建图像数据,请选择一张图像,然后从文件标签中选择“导出/导入”按钮。然后,选择“导出”,然后选择相应的文件类型(例如,JPEG,TIFF,PNG等)。要创建一个电影从数据中,选择“导出电影”功能,然后选择相应的文件类型(例如,AVI,WMF,MOV等)。czi文件可用于分析。     



    血流速度


    血流速度(V)的计算方法已在多项研究中进行了描述,这些研究对从动脉到毛细血管的不同口径血管中的血流进行了量化(Tang等人,2015)。在一个电影(20 - 30秒)中,选择一个血管的四到六个最清晰的图像的序列。测量平行于血液流动方向(ΔX)的切线长度和垂直于血液流动方向(ΔT)的股线的余弦长度。使用以下公式计算血流速度(图s 4A,4B和图5):



    V [µm / ms] =ΔX/ΔT



    速度可以定义为从电影中选择的四或六股线中计算出的平均值。





    图4.新生小鼠的肠道动态微循环。一。TPLSM获得的肠道微循环图像。乙。血流量的计算。Ç 。血管壁剪切率的计算。d 。白细胞滚动速度的计算。



    血流量


    使用以下公式计算血流量(FV)(图4B和图5):



    FV [微米3 / MS] = π(d / 2)2 ΔX/ΔT



    [d]是血管的最大直径



    血管壁剪切率


    使用基于牛顿定义的公式(Russell等,2003),以100 µm的血管节段计算血管壁剪切速率(W )(图4C和图5):



    W [1 / ms] = 8V / d





    图5.实验结果和数据分析的示例。A.从录制的视频中剪切一个静止/火焰图像(一个火焰扫描速度:380毫秒)。从中找到血小板的直线。B.测量切线长度的直线平行于血液流动方向(的Δ X),扫描时间(Δ T),和血管最大直径(d)。C.使用特定公式计算速度,流量和剪切速率。                                                       



    粘附白细胞数


    粘附的白细胞可以在每个血管段中定义为在指定的5到10 s的观察期内不会从内皮层移动或脱离的细胞。通过计算单个血管在100 µm长度内粘附在内皮表面的粘附白细胞的数量来进行定量(Kubes等,2003 ;Nakagawa等,2006)。



    白细胞滚动速度


    白细胞的滚动现象主要在毛细血管后静脉中观察到。毛细血管后静脉是位于粘膜下层的V1或V2级静脉,由毛细血管的合并分支制成(Yu等,2009)。新生小鼠小肠中该小静脉的最大直径约为20-60 µm。选择一个框架,该框架中的毛细血管后小静脉的最大直径是一致的,并选择一个滚动的白血球在小静脉内壁上滚动。测量白细胞行进的距离并记录行进时间的长度,该时间由从观察到的第一帧到观察到的最后一帧的时间确定。白细胞滚动速度[µm / ms]以[µm / ms]计算(图4D):



    白细胞滚动速度= [µm / ms]



    其中:白细胞行进距离长度[μm] /两个选定时间范围之间的扫描时间跨度[ms]。



    预期结果


    通过使用新生儿肠TPLSM成像的该方法中,我们能够观察和肛门YZE的新生儿小肠的微循环和血流动力学详细(视频1)。视频1从绒毛的尖端开始,然后向下延伸至绒毛的底部,然后最后穿过基础的血管供应和平滑的肌肉组织。此外,获取z-stack和xy数据可以创建小肠整个结构的三维图像,从绒毛的顶部到底部(包括粘膜下区域)。在成像之前施用其他荧光染料,例如SYTOX绿色或红色,可以在疾病模型中定位受影响的细胞(视频2)。使用这种方法,我们发现在实验NEC小鼠模型中,绒毛的顶部是受到坏死影响的主要区域(视频2 )。这种方法对于长期分析药物和其他程序对新生儿小肠的作用也很有用。此外,该方法允许用户可视化目标区域中肠道微循环的变化。


    总之,本协议中所述的用于TPLSM的器官稳定方法的组合可为研究新生儿肠道发育和肠道病理生理学提供可靠的结果。





    视频1.新生儿小肠的微循环





    视频2.对照新生小鼠和坏死性小肠结肠炎小鼠模型中整个绒毛的3-D图像



    致谢



    BL是Thrasher研究基金(14503)的Restracomp奖学金,病童医院和早期职业奖计划的获得者。PDO得到了加拿大卫生研究院(CIHR)(162208和149046),加拿大心脏和中风基金会(G-17- 0018613)和加拿大自然科学与工程研究理事会(NSERC)(500865)的支持。 。AP是加拿大卫生研究院(CIHR)基金会353857的获得者。


    作者贡献:YK,BL,YC,NG,MA,HM,CL,AH和RW:概念和设计,数据收集和/或组装,数据分析和解释,手稿撰写,手稿最终批准。KU,MI和PDO概念和设计,手稿的最终批准。AP:构思和设计,财务支持,稿件的最终批准。



    利益争夺



    作者没有利益冲突要声明。



    伦理



    该规程中描述的所有程序均已获得多伦多医学发现塔(No.4886.0)的机构动物护理委员会的批准,并获得了高级光学显微镜设施的批准。






    参考



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    引用:Koike, Y., Li, B., Chen, Y., Ganji, N., Alganabi, M., Miyake, H., Lee, C., Hock, A., Wu, R., Uchida, K., Inoue, M., Delgado Olguin, P. and Pierro, A. (2021). Live Intravital Intestine with Blood Flow Visualization in Neonatal Mice Using Two-photon Laser Scanning Microscopy. Bio-protocol 11(5): e3937. DOI: 10.21769/BioProtoc.3937.
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