MOUSE :: HMDP :: Vascular injury response

M.        Hybrid Mouse Diversity Panel:  Vascular injury response

  1. Mice (R. LeBoeuf)
  2. Phenotyping (R. LeBoeuf)
  3. Tissues, weights (R. LeBoeuf)
  4. Expression arrays (R. LeBoeuf)

 

 

1.         Mice (R. LeBoeuf)

Male mice only are used for this project.  Briefly, mice arrive from The Jackson Laboratories (JAX) at 5-6 wk of age and are maintained on normal pelleted rodent chow (PicoLab Rodent Diet 20 #5053, LabDiet; www.labdiet.com).  Mice undergo left carotid tie-off injury at 8 wk of age (see below for methods) and then are killed 28 days later (see below for necropsy).  Both carotid arteries are removed, pictures are taken under a dissection microscope, and then carotids are formalin fixed, paraffin embedded and sectioned (described below).  Appropriate tissue sections are stained using Movat’s pentachrome and assayed for a variety of vascular endpoints including intima areas and volumes (see below).  Our goal (based on power calculations that can be provided) is to obtain n=8 of injured carotid arteries and the non-injured contralateral arteries.  In addition, we collect n=4 carotid arteries (left side) from ‘sham’ treated mice (no injury).  Mice are housed n=4/cage.     

Of note is that mice from the AKXL RI strain set have been re-derived at the JAX labs for this project and a colony of these animals is now housed at the University of Washington (LeBoeuf lab).  Thus, this set of RI strains as well as parental controls may experience some environmental differences from the remaining panel which are used directly from JAX.  We include AKR/J and C57L/J mice from JAX and from our in-house panel (AKR/J/UW and C57L/J/UW) for comparison.

 


2.         Phenotyping (R. LeBoeuf)

a.  Carotid tie-off procedures:

There is no preoperative food or water restriction.   Animal preparation consists of administering anesthetic (ketamine/xylazine [mix 0.65 ml ketamine [100 mg/ml] and 0.22 ml xylazine [20 mg/ml] with 9.13 ml of sterile saline to yield a 6.9 mg/ml solution for a final dose of 0.02 ml/g body weight) by IP injection, shaving incision area, wiping down incision area with Betadyne, and application of eye lubricant.  (Note: The strains we work with are new to us so when administering anesthesia we use caution and do not give the mouse the full dose right away.)

Surgery involves a midline incision on ventral neck, cleaning connective tissue and nerve from contact with distal portion of carotid artery, placement of suture loops (Silk Suture 6/0, George Tiemann & Company) around carotid just below the external/internal bifurcation and complete ligation of the common carotid artery.  Skin incisions are closed with wound clips.

We use autoclaved instruments at the start of each day, cleaning with soap and water then hot glass bead sterilizing instruments between animals.  Surgical field is draped with sterile gauze.

The surgeon wears a facemask, hair bonnet, and a sterile surgical gown along with sterile gloves.  Surgeon’s hands are washed with soap prior to putting on the sterile gloves.  Surgery is performed in a SPF approved surgical procedure room.  Additional anesthetic cocktail is given IP as needed.  Surgery takes 20 minutes generally.

Post-surgical care includes placing animal on warming pad until fully awake before returning to its cage.  Depending on the time of day, animals are checked later the same day, or the next morning, and again the following day for any sign of bleeding, swelling, scruffy fur, hunched back, lethargy, and lack of eating.   It is our policy to euthanize any animal that appears to be in pain or not recovering as expected after surgery.  After surgery when mouse is fully awake and no longer showing any signs of drowsiness from anesthesia, pain medication is given subcutaneously. The pain medication we use is buprenophrine mix (Dilute 0.1mL of full strength Buprenex [0.3mg/mL] to 0.9mL of sterile water to yield 0.03 mg/mL solution for final dose of 0.05mg/kg).   Surgical clips are removed at 14 days.

b.  Removal and treatment of carotid arteries:

At 28 days, mice are euthanized by IP injection with Beuthanasia-D (390mg/mL). When mouse is dead, body cavity and neck is wiped with 70% EtOH.  An incision is made from mouse’s abdomen to the neck.  Rib cage and neck bone are cut and tissues are removed from neck to reveal carotids.  With sharp fine pointed spring scissors (Fine Science Tools 15003-08), we make a small nick below the suture loops on the distal end of left carotid. We cut the abdominal aorta and carefully perfuse with 10-15 mL of 1xPBS.  Use surgical forceps (Aesculap FD281R) to clear away fat and connective tissues away from carotids.  Remove the left carotid, leaving the suture attached to the distal end of the injured carotid and a small piece of aorta attached to the proximal end of the injured carotid.  Remove the right carotid, leaving the bifurfication and innomate artery attached to identify the ends of the carotid.  Place the carotids into tubes containing 1mL of 10% buffered formalin. Fix carotids in 10% buffered formalin for 24 hours.  After 24 hours, put the carotids in 70% EtOH for storage before paraffin embedding.

c.  Pictures taken under dissecting scope.

Before paraffin embedding, carotids are cleaned further under the dissecting microscope.  Any fat or connective tissues are removed at this point.  On the distal end of the carotid, the sutures are trimmed and on the proximal end, the aortic tissues are trimmed off.  A photo of the injured carotid is taken under a dissecting microscope (Wild Heerbrug Plan1x) with a Nikon CoolPix990 camera attached.  Carotids are placed on black wax, pinned down with dissecting pins, and imaged with a 1 cm ruler.  

d.  Paraffin embedding.

Carotids are processed in 70% EtOH (minimum of 25 minutes), 95% EtOH (25 minutes), 3X with 100% EtOH (25 minutes each time), 3X with SlideBrite (35 minutes each time), 2X with Paraffin (1 hour first time, 1.5 hour 2nd time).  Carotids are embedded upright in paraffin wax.  Up to 8 carotids are placed in one paraffin block in an “L” formation.  A very small amount of paraffin is place in a mold to create a paraffin film on the bottom of the mold that allows the carotids to stick.  With fine pointed forceps, carotids are carefully positioned proximal end pointed down one at a time in the “L” formation.  After all the carotids are placed in the mold, hot paraffin wax is carefully added into mold.  At this point, check that all carotids are still upright and readjust quickly if needed.  Carefully place block onto cooling plate.  Store blocks in 4°C refrigerator for sectioning or at room temperature for long term storage.

Carotid sections are cut with a Leica Manual Microtome.  Sections are cut at eight microns, three sections are placed on one slide, every two consecutive sections are skipped.  Slide Scheme:  save 1 section à toss 2 sections à save 1 section à toss 2 sections à save 1 section, continue for next slide until carotid ends. Slides are air dried overnight, then baked for one hour at 60°C.

e.  Quantification of carotid artery injury (Phenotyping):

Serial sections 8 mm thick were cut and a subset of 15 - 17 sections from each animal spanning approximately 4.5 mm of the artery were stained with Movat’s stain (Ref 1) (see below for Movat’s stain protocol) for morphometric analysis.  Digitized images of these sections were analyzed by the ImageJ software for Windows (NIH, version 1.41).   Four measurements were made by tracing along 1) the luminal surface, 2) the luminal surface of the internal elastic lamina (IEL), 3) the perimeter of the IEL, and 4) the perimeter of the external elastic lamina (EEL).  The luminal area was calculated from the circumference of the lumen (measurement 1).  The neointimal area was calculated by subtracting the measurement 1 from measurement 2.  The medial area was calculated by subtracting measurement 3 from measurement 4.  Each measure was averaged over all sections analyzed.

Ref 1.  Movat HZ. Demonstration of all connective tissue components in a single section. Arch Pathol. 1955;60:289-95.

 

f.   Movat’s Pentachrome Stain:  Details

Staining Results:     Nuclei and elastic fibers                             Black
Ground substance (proteoglycan)                        Blue   
Muscle                                                           Red
Collagen                                                        Yellow           
Fibrinoid and fibrin                                      Intense red

1.       Deparaffinize and hydrate to distilled water*
2.       Preheat Alkaline Alcohol in 60 degree oven
3.       Mordant in Bouin’s in microwave, covered, for 1 minute (reagent should be ~50°).  Let sit for 10 minutes.
4.       Wash in running water for 10 minutes.
5.       5% sodium thiosulfate for 5 minutes.
6.       Wash in running water for 5 minutes.
7.       Stain in 1% Alcian Blue for 20 minutes.
8.       Running water for 2-5 minutes.
9.       Pre-heated Alkaline Alcohol (60°) for 10 minutes.
10.     Running water for 2-5 minutes.
11.     Stain in Movat’s Weigerts for 40 minutes
12.     Wash briefly in running water, rinse well in distilled water.
13.     Stain in Crocien Scarlet/Acid Fuchsin for 1 minute.
14.     Rinse in distilled water.
15.     5% Phosphotungstic acid for 5 minutes.
16.     Transfer directly to 1% acetic acid for 5 minutes.
17.     Rinse well in distilled water.
18.     Dehydrate – 1 x 95% EtOH x 1 minute
2 x 100% EtOH x 1 minute.
19.     Stain in alcoholic saffron for 10 minutes.
20.     Rinse – 2 x 100% EtOH x 1 minute.
21.     Clear in xylenes or histoclear 3 x 5 minutes.
22.     Coverslip with cytoseal mount.

* Take slides through xylenes or histoclear (3 x 2 min), 100% EtOH (3 x 2 min), 95% EtOH (3 x 2 min), 70% EtOH (1 x 2 min), water (1 x 2 min).  Ready to begin.

Reagents and Stains

Alkaline Alcohol
95% EtOH                                         45 ml
conc. Ammonium hydroxide            5 ml

1% Alcian Blue
Alcian blue                                            1 g
Distilled water                                   100 ml

STOCK Weigert’s Hematoxylin

            2% Alcoholic hematoxylin
Weigert’s hematoxylin         10 g
95%EtOH                              500 ml
Store in dark or covered bottle

            Ferric Chloride
Ferric chloride hexahydrate             12.4 g
Distilled water                                   500 ml
Conc. HCl                                             5 ml

            Iodine Solution
Iodine                                       10 g
Potassium iodide                   20 g
Distilled water                       500 ml
Dissolve the potassium iodide in a small amount of distilled water (can take 12 –24 hours).  Add the iodine.  When completely dissolved, add the remainder of the distilled water.  Store in dark or covered bottle

WORKING Weigert’s solution

            2% Alcoholic hematoxylin              30 ml
Ferric chloride                                  20 ml


Iodine solution                                 10 ml
Mix in order.  Use immediately.  Discard after use.

STOCK Crocein Scarlett
Crocein scarlett MOO 3B                  1 g
Distilled water                                   99.5 ml
Conc. glacial acetic acid                   0.5 ml

STOCK Acid Fuchsin
Acid fuchsin                                       0.1 g
Distilled water                                   99.5 ml
Conc. Glacial acetic acid                  0.5 ml
Filter and let stand 24 - 48 hours.

 

WORKING Crocein Scarlett / Acid Fuchsin

            Stock Crocein Scarlett                    40 ml


Stock Acid Fuchsin                         10 ml

5% Phosphotungstic Acid
Phosphotungstic acid                         5 g
Distilled water                                   100 ml

1% Glacial Acetic Acid
Conc. Glacial acetic acid                  1 ml
Distilled water                                   99 ml

Alcoholic Saffron
Saffron,spanish                                   6 g
100% EtOH                                       100 ml
Place in air tight bottle in 55 – 60° oven for 48 hours to extract saffron.  Cool the extract to room temperature.  Filter through coarse filter paper in a covered funnel.  Store in a dark, tightly-stoppered container.

5% Sodium Thiosulfate
Sodium thiosulfate                              5 g
Distilled water                                   100 ml

 


3.         Tissues, weights (R. LeBoeuf)

At 28 days post-ligation, mice are weighed and plasma taken and stored at -80oC until analyses.  Tissues collected are, left and right external carotid arteries, aorta, liver and pancreas.  Liver, pancreas, and aorta (after removal of fat and adventitia) are immediately frozen in liquid N2 and stored at -80oC until analyses.  Livers will be weighed in the future as needed.  Pancreas will be used to quantify total insulin content.  Aortas will be used for RNAseq or other analyses as needed.

In addition, we are now collecting circulating monocytes from the blood collected during plasma preparation. 

 


4.         Expression arrays (R. LeBoeuf)

None so far, but we anticipate performing expression arrays on mouse aortas if needed.  In addition, we are planning to perform microRNA analysis on selected tissues, but this has not yet been decided (as of February 2012).