An In Vivo Model to Study Shear-Dependent Bacterial Adhesion

الملخص

Source: Claes, J., et al, In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions. J. Vis. Exp. (2015).

This video demonstrates an in vivo method for analyzing shear-dependent bacterial adhesion in a live mouse model. Following surgical preparation and catheterization, the mesenteric vasculature is exposed for real-time imaging under a fluorescence microscope. Perfusion with an ionophore and fluorescently labeled bacteria reveals how shear stress triggers von Willebrand factor (VWF) strand formation and bacterial aggregation on endothelial surfaces.

البروتوكول

1. In Vivo Mesenteric Perfusion Model
   1. Preparation/surgery of the mouse
      1. Fast the mouse the night before the experiment in order to limit bowel movement.
      2. Give a 6-8 week old mouse (C57Bl/6) pre-operative analgesia by a subcutaneous injection of buprenorphine (0.1 mg/kg body weight (BW)) 20-30 min prior to the surgery.
      3. Anesthetize the mouse by intraperitoneal injection of ketamine (125 mg/kg BW) and xylazine (12.5 mg/kg BW). Check by pedal reflex. Apply vet ointment to prevent dryness.
      4. Place the mouse on a thermo-controlled heating pad at 37 °C on a microscope tray. Since this is a terminal procedure, there is no need for strict aseptic procedures. Make an incision near the jugular vein, gently remove the right side of the cervical muscle, and isolate the jugular vein from the surrounding tissue.
      5. Insert a 2 French intravenous catheter into the right jugular vein for infusion of fluorescently labeled bacteria or other solutions. Open the peritoneal cavity via a midline abdominal incision and use cotton swabs to spread the mesenterium and to visualize the mesenteric arteriolar and venular circulation.
      6. Place the mouse on the right side of a transparent plate and secure the cannula with tape. Use a hot pack to prevent hypothermia. To prevent dehydration of the tissue, drop 500 µl 0.9 % Sodium chloride (NaCl) on the intestines.
2. Fluorescence microscopy of bacterial adhesion to the mesenteric circulation
   1. Work in a dark room. Use cotton swabs to immobilize the vessels and visualize them under an inverted microscope.
   2. Topically apply 5 µl of a 10 mM solution of the Ca²⁺-ionophore A23187 dissolved in dimethyl sulfoxide (DMSO). After 10 sec, inject 100 µl labeled bacteria through the jugular catheter. Take time-lapse images. After the experiment is finished, euthanize the mouse according to institutional-approved guidelines.
3. Image Analysis
   1. Obtain live images using an inverted fluorescence microscope, captured using a black and white camera, and developed using any imaging software. Apply automated exposure time and contrast optimization specific to the equipment used.
   2. Acquire time-lapse images using the ‘Acquisition’ tool in the toolbar (Multidimensional Acquisitions – Time) using 40 cycles of 1,000 images/sec. Save the images in an appropriate image file format.
   3. Process images using ImageJ analysis software to measure the area of fluorescent signal per image. Define the threshold to set lower and upper threshold values, segmenting gray-scale images into features of interest. Identify the region of interest (blood vessel) and measure the area limited to the threshold and the region of interest. Compare bacterial adhesion, expressed as fluorescent area, using any statistical or graphing software (Figure 1).

النتائج

Figure 1. A schematic overview of the in vivo mesenteric perfusion model. A right jugular vein catheter (yellow line) is inserted for the administration of fluorescently labeled bacteria (orange dots), additional anesthetics, or other components such a...

المواد

قائمة المواد المستخدمة في هذه المقالة

الاسم	الشركة	رقم فهرسي	التعليقات
Temgesic	Reckitt Benckiser	283716	bruprenorphine
Anesketin (Ketamin hydrochloride 115 mg/ml (100 mg/ml ketaminum))	Eurovet	BE-V136516	ketamin
XYL-M 2% (xylazine hydrochloride 23.32 mg/ml (20 mg/ml xylazine))	VMD Arendonk	BE-V170581	xylazine
2 French intravenous catheter green	Portex	200/300/010
0,9% Sodium chloride (NaCl)	Baxter Healthcare	W7124
Cotton swabs	International Medical Product	300230
Ca2+-ionophore solution A23187	Sigma-Aldrich	C7522-10 MG
26 Gauge 1 ml syringe	BD Plastipak	300013
26 Gauge 1 ml syringe with needle	BD Plastipak	300015	intra-peritoneal injection
Centrifuge 5810-R	Eppendorf	5811 000.320
Glass cover slips (24x50)	VWR	BB02405A11	Thickness No, 1
PHD 2000 Infusion	Harvard Apparatus	702100	High-accuracy Harvard infusion pump
Axio-observer DI	Carl-Zeiss	Inverted fluorescence microscope
ImageJ	National Institute of Health	Analysis software
Graphpad Prism 5,0	Graphpad Software	Analysis software
AxioCam MRm	Carl-Zeiss	Black and white camera