text:
https://royalcbd.com/faq/ No Further a Mystery
Frequently Asked Questions (Frequently Asked Question).
General CBD Questions.
1. What is CBD?
CBD stands for cannabidiol. It's one of over 113 normally occurring cannabinoid compounds in the Cannabis plant. The blossoms of cannabis (both hemp as well as cannabis) secrete a sticky material plentiful in cannabinoids like CBD, in addition to terpenes and also different other phytochemicals.
Contrasted to THC-- the other primary cannabinoid in the marijuana plant-- CBD is non-psychoactive. This indicates that despite just how much you take, it's not going to create the particular "high" from cannabis.
CBD has actually also just recently been categorized as a nutritional supplement in the USA-- giving the general public open access to CBD supplements.
2. Exactly How Does CBD Work?
CBD engages with the endocannabinoid system-- an intricate network of receptors and also natural chemicals spread out throughout the entire body. This system is tasked with managing the equilibrium of numerous body organ systems.
The best wealth of these receptors lies in the brain, the main nervous system, and also the body immune system. Practically every organ in your body, consisting of the skin and gastrointestinal tract, comes with cannabinoid receptors.
The endocannabinoid system regulates a plethora of major organic features, consisting of:.
Immune response.
Sensory assumption.
Stress and anxiety management.
Cognitive performance.
CBD overcomes the endocannabinoid system by boosting our normally happening endocannabinoids-- thus aiding the body keep balance.
3. What's the Distinction In Between Hemp as well as Marijuana?
From a scientific viewpoint, both hemp as well as marijuana are the same varieties-- Cannabis sativa.
The distinction is in their chemical make-up.
Anything with less than 0.3% THC by dried weight is classified as hemp (non-psychoactive), while anything over this is cannabis (psychoactive).
Hemp is extra frequently made use of for making food (hemp seeds), rope, paper, building materials, natural plastics, and most significantly-- organic supplements such as CBD oil.
Marijuana is utilized recreationally, mentally, or for clinical purposes as a result of its psychedelic results.
At Royal CBD, we just use hemp plants that have been specifically bred for their high CBD content as well as lack of psychedelic THC.
4. Hemp vs. Marijuana Derived Cannabinoids: Are They the Same?
Yes. CBD is CBD regardless of if it comes from a hemp plant or a cannabis plant.
Nonetheless, the proportion of cannabinoids will be really different from a hemp plant and a cannabis plant. The most obvious distinction is THC as well as CBD-- cannabis has high THC, as well as typically, a reduced CBD degree, while hemp is high in CBD, and reduced in THC. The various other cannabinoids will vary a great deal between these 2 plants too.
Terpenes likewise differ a whole lot in between hemp and marijuana-- but this truly depends on the pressures being made use of and also can differ a whole lot from one plant to the following.
Our partnering farmers make use of selectively-bred hemp stress that contain higher terpene content than regular hemp plants.
5. What's one of the most Reliable Method to Take CBD?
There are many different means you can take CBD-- and also none are a lot more efficient than the others.
One of the most prominent approach is using the mouth or sublingually with the help of oil, capsules, or our favored-- CBD-infused gummies.
CBD oils are the most versatile, as you can take your dose directly into the mouth, blend it in with food, or hold it under your tongue to speed up absorption.
6. Just How Much CBD Should I Take and also How Frequently?
The recommended day-to-day offering size of our CBD oil is 5-50 mg CBD daily relying on your weight, and also just how CBD impacts you separately.
We suggest starting at 1-6 mg of CBD for every single 10 pounds of body weight and also enhancing that by just a couple of mg every day till you obtain the preferred results.
If you're utilizing our 250 mg oil, there are an overall of 30 decreases (servings) with 8 mg dosage per decline, while our 500 mg oil has around 16mg per drop with 30 declines per bottle.
7. What Are one of the most Vital Aspects to Think About When Buying CBD Products?
When selecting in between our different CBD-rich essences, focus on 2 main aspects:.
1. The Strength/Potency.
Because the dose of CBD can vary so much from a single person to the following, it aids to buy the best potency (strength) to match your individual dosage. For example, if you require a high dosage of CBD oil, it makes even more sense to get a high-potency oil. In order to achieve a high dosage with the reduced strength oils, you will require to take a lot of oil each time.
If you need only a tiny dose of CBD to obtain the preferred results, a low strength oil is a much better option due to the fact that it's more difficult to go overboard on your CBD dosage with these oils.
2. The Type of CBD Item.
CBD is available in all forms-- oils, pills, gummies, teas, and even topical salves or ointments.
Choosing the appropriate sort of CBD depends upon what you're utilizing it for, as well as exactly how important comfort is in your CBD program.
CBD oils are one of the most well-rounded, as you can utilize them both inside as well as topically-- yet aren't as straightforward to dosage if you've never ever used CBD prior to. Other kinds of CBD, such as gummies come with enhancements in the taste, as well as makes application much simpler, but aren't as all-around as oils and casts.
8. Is Hemp Seed Oil the Same as CBD Hemp Oil?
Many individuals have a tendency to confuse CBD-rich hemp removes with the hemp seed oil you can find at their neighborhood supermarket. In reality, these 2 oils are globes apart.
Typical hemp seed oil is made by cold pressing the seeds of the hemp plant. The oil that's generated is abundant in nutrients like gamma-linolenic acid (GLA), amino acids, and trace minerals. It produces an excellent resource of nutrition-- however doesn't consist of CBD or any one of the other cannabinoids.
CBD hemp oils, on the other hand, are made from the resinous flowers of the women hemp plants (without seeds). The material is drawn out utilizing state-of-the-art extraction methods to collect the cannabinoids, and also terpenes normally found in the plant material. This is the source of CBD.
Royal CBD Item Questions.
1. How Much CBD Is in Royal Products?
Our hemp extracts include differing amounts of CBD, varying from 250 mg up to 2500 mg.
Right here's a summary of our hemp items alongside their cannabinoids per fluid ounce:.
A) 250 mg Full-Spectrum CBD Oil.
Our 250 mg Full-Spectrum CBD Oil has 250 mg of pure cannabinoids per bottle. This indicates that there's about 8.33 mg of CBD and trace cannabinoids per 1 mL of oil.
The cannabinoid profiles we use mostly consists of CBD, but also includes other cannabinoids like CBC, CBG, as well as CBN-- this is what makes them full-spectrum.
Find out more about Royal CBD 250mg Full-Spectrum CBD Oil.
B) 500mg Full-Spectrum CBD Oil.
This is our more powerful CBD oil-- containing two times as much CBD as our 250 mg oil.
With 500 mg of cannabinoids, this oil works out to including roughly 17 mg of CBD and also trace cannabinoids per 1 mL of oil.
Find out more regarding Royal CBD 500 mg full-spectrum CBD oil.
C) Royal CBD 10 mg Gummies.
Our isolate-based CBD gummies include provide 300 mg per container, for a total of 10 mg per private gummy.
Unlike our oils, we've picked to make use of CBD isolate to give a pure dose of CBD with each gummy.
The isolate we utilize to make Royal CBD 10 mg gummies has no THC, terpenes, or any other compounds-- it's just pure cannabidiol.
Find out more about Royal CBD 10 mg Gummies.
D) Royal CBD 25 mg Gummies.
A more powerful variation of our THC-free vegan CBD gummies packs 25 mg of pure CBD into each gummy. The whole bottle includes 750 mg of CBD.
Learn more about Royal CBD 25 mg Gummies.
2. Where Do You Source Your Hemp and CBD From?
Our items are sourced from natural, top quality Industrial Hemp cultivated on environmentally aware and also sustainable land in Colorado.
3. If a Hemp Essence Includes 40% CBD, What's in the Continuing to be 60%?
Our CBD-rich hemp essences are sourced from the Cannabis Sativa plant. The drawn out oil contains normally occurring phytocannabinoids such as CBD along with some minor cannabinoids like CBC, CBN, as well as CBG.
Royal CBD hemp extracts consist of over 100 various phytocannabinoids.
The primary active substance in our items is CBD, that makes up the bulk of the extract's structure-- the rest is made up with these other cannabinoids, terpenes, polyphenols, other phytochemicals from the cannabis plant, and components like MCT oil.
Other Components In Royal CBD Oils:.
Amino acids.
Essential fatty acids (including omega 3 & 6).
Trace minerals (calcium, iron, magnesium, potassium, zinc).
Vitamins (B1, B2, B6, D).
MCT oil.
4. What Type of Quality Screening Do Do YouPerform on Your Products?
All our products are third event lab checked to make sure the THC degree is below 0.3%.
5. What Removal Methods Do You Use?
All of our CBD oils and also gummies are made from hemp essences with cutting-edge supercritical CARBON DIOXIDE extraction methods.
By intricately managing the temperature level and pressure of carbon dioxide gas, we can maintain it in a state described as supercritical-- it's not a gas, nor a liquid, however behaves as both.
When we rinse our hemp samples with this supercritical CO2, it strips away all the resinous materials as well as cannabinoids from the plant, leaving the fibers, healthy proteins, and also various other unnecessary elements.
As quickly as the stress is released, the CO2 vaporizes immediately, leaving a cleansed, CBD-rich resin.
This method of extraction is extremely tidy-- particularly compared to the older solvent-based extraction that usually left toxic natural solvents in the final product.
6. Are Your Hemp Extracts Safe? What Are the Prospective Adverse Effects?
All our hemp extracts are third-party examined to confirm there are no impurities such as heavy metals, pesticides, herbicides, or raw material like mold and mildew or bacteria.
The FDA has not yet consisted of CBD in their list of supplements that are Usually Considered As Safe (GRAS). However, the compound is in the procedure of gaining this title by the FDA.
Present research study on the compound suggests minimal side effects and a reduced capacity for danger.
7. Will your CBD products cause favorable outcomes on a medication test for THC?
We make use of full-spectrum CBD in all of our products (except gummies as well as topicals), suggesting that they may contain trace amounts (less than 0.3%) of THC and also there may be a mild possibility that it will certainly show on positive for THC on a medicine test.
For more information concerning why we use full spectrum CBD oil, as well as the advantages of complete range CBD contrasted to CBD isolate, please see this article.
We would certainly advise talking to your employer prior to an examination being carried out to allow them find out about the scenario if you 'd like to use our complete range products. As we're not medical professionals, it's highly suggested that you talk to a certified physician before taking CBD items.
We are a team of CBD supporters with the vision of a much better globe, the globe where people can boost the top quality of their lives in all-natural ways, without risking their health and wellness.
We understand just how hard can it reach find a trusted CBD distributor-- we have actually all existed at some point. This is why you can read us like a book. Our choice of CBD products includes only pure as well as potent CBD oil, sourced from organic hemp, removed with ethical approaches, and also laboratory examined for top quality.
Long story short, with Royal CBD, you obtain the most effective of all worlds.
You Are Worthy Of the Top-Quality CBD Oil, As Well As We Know It.
We really feel obliged to deliver the gold-standard degree of quality to make sure that you might easily forget all those not-so-pleasant experiences with other CBD oil distributors in the past.
By choosing Royal CBD, not just do you obtain access to completely natural, full-spectrum CBD removes, but you can likewise rest popular that we will not leave you with any unanswered concerns. You see, the cannabis community is about more than just an organisation.
Whenever you want to ask us regarding the dosage, suggested usage techniques, or which CBD oil will work best for you, simply give us a call, send us a message, or connect with our social media sites networks-- this is why it's called customer support.
Why Choose Royal CBD?
Sourced from 100% Organic Products.
Hemp is a bio-accumulator, indicating that it draws all chemicals from the dirt, We source our CBD from totally organic, all-natural, GMO-free commercial hemp that is cultivated without using chemicals or other unsafe compounds during the farming.
Extracted With Treatment.
Forget any solvent residue in your oil. We make use of CO2 extraction systems as our main tool for drawing out all energetic substances from our hemp plants. This is a heatless and also solventless approach that ensures the optimum strength as well as https://royalcbd.com/faq/ purity of the final product.
Full-Spectrum CBD.
With Royal CBD, you can embrace the advantages given by the harmony accomplished by all cannabinoids as well as terpenes in your CBD extracts. Various studies have actually discovered that full-spectrum marijuana products can aid individuals maximize the results of CBD; by doing this, you can individualize your experience with Cannabidiol.
Lab Checked for Effectiveness and Purity.
Know what you're acquiring before you even put the hands on your bottle of CBD oil. Each of our CBD items is examined in 3rd-party labs for the entire cannabinoid material, along with the visibility of solvent, toxins, and heavy metals. Did we discuss that you can read us like a book?
Do Not Go Ordinary, Go Royal.
You are not a target or a number on a consumer spread sheet. You are an individual who seeks to live a better, much healthier life, as well as you made a decision to put your count on us. We appreciate that, which is why you will be cared for from more than business point of view.
Your contentment is our top objective, so whenever you have actually any type of uncertainties associated with making use of CBD oil, we encourage you to send us a message, as well as we will certainly be happy to aid you out.
Surf our selection of CBD-infused products and also make the last action to transform the ordinary CBD experience right into an imperial one!


text:
The Wedding Ringer
Canadian Netflix subscription is required to watch on Netflix
Date Added: 28th January 2020
Want to keep forever? Try looking for 'The Wedding Ringer' on Amazon.ca (paid link)
12/01/2017: Added to Netflix
12/01/2020: Removed from Netflix
28/01/2020: Streaming Again
A tech entrepreneur engaged to a gorgeous girl is afraid to admit he has no friends, so he hires Best Man Inc. to get him the groomsmen he needs.
Certificate: Crude and sexual content, language throughout, some drug use and brief graphic nudity
Year: 2015
Duration: 1hr 41m
Audio: English [Original], French
Subtitles: French
Director: Jeremy Garelick
Cast: Kevin Hart, Josh Gad, Affion Crockett, Kaley Cuoco, Jorge Garcia, Dan Gill, Corey Holcomb, Ken Howard, Colin Kane, Cloris Leachman
Login or register to get email updates...
This title is available on Netflix in Canada

subset: commoncrawl


text:
You Could Make Your Roof Awesome (Again).
Top notch Shingle Roofer Company in Navasota Texas is a call away!
Call Today!
Call 979-599-7533!
No matter if you require a roof for a new building, or a fix to an already existing rooftop, [[ sitename ]] guarantees to leave your home much more attractive than we found it.
This focus upon details sets us aside from various other roofing professionals considering that all the ability in the world doesn't replace talent and taste.
Naturally our roofing contractors are technical specialists ready to take on any type of roofing system, yet they also have the aesthetic expertise and also attention to detail necessary to elevate the overall look of your house.
{Why Need to You Pick College Station Roofing for Shingle Roofer Company| You should opt for College Station Roofing for Shingle Roofer Company because:
- Guarantee that work will be finished on schedule!
- Certified and insured personnel!
- Top quality materials to offer a durable roofing system!


text:
Elegant and sexy, this Short Sleeve Sleepwear will make you will feel unique and special. Enjoy your night! Hurry up and get it while supplies last!
Gender: WomenItem Type: NightgownsDecoration: LaceMaterial: SilkDresses Length: Above Knee, MiniCollar: Square CollarSleeve Length: Shortcolor: 4 colorssize: L/XLstyle: sexy and charming
Be the first to ask a question about this.


text:
Boston MA Real Estate & Homes for Rent
1,923 Properties Found
The median home value in Boston, MA is $710,000.
This is
higher than
the county median home value of $610,000.
The national median home value is $232,276.
The average price of homes sold in Boston, MA is $710,000.
Approximately 34.07% of Boston homes are owned,
compared to 59.47% rented, while
6.33% are vacant.
Boston real estate listings include condos, townhomes, and single family homes for sale.
Commercial properties are also available.
If you like to see a property, contact Boston real estate agent to arrange a tour today!
Sort Results:
1–25 of 1,923 properties displayed
Courtesy: Compass The Shorey Realty Group
REDUCED RENT $1800 FIRST 3 MONTHS. Rent will increase to market value at $3100/month thereafter. Renovated/modern 2 bed 1 bath condo overlooking historic Worcester Sq. Private deck & parking! This bow-front 3rd floor unit has front-facing windows, bright open living/dining area w/ fireplace, exposed brick & fabulous kitchen w/ newer granite counter/stainless steel appliances, plenty of counter/cabinet space w/neat pass-through. Highlights include central air, in unit laundry, gas fireplace & cooking, exposed brick, wood flooring & common storage. Enjoy your own parking space w/ direct access to the building. Conveniently close to Route 93, silver & orange lines, Boston Medical Center & Boston Univ Medical & Dental schools, Northeastern Univ, Symphony Hall & steps to the area's finest restaurants, quaint boutiques, shops and everything else this historic neighborhood has to offer! WalkScore of 96 which is considered "A Walker's Paradise", walk to over 100 shops, cafes & restaurants.
Courtesy: Matahari Real Estate LLC Irene Novia Loka
Live in one of Boston most prestigious condominium building. The Ritz Residence features all the luxury amenities you would expect with a building this caliber: 24hr. Concierge, Doorman & Porter service, 1 VALET GARAGE PARKING, Ritz Hotel services, and many more. The unit features spacious living & dining rooms with high ceilings, marble bathrooms, modern kitchen with stainless appliance and open style layout, in-unit washer dryer, Brazilian cherry hardwood floor, and private balcony. Located within walking distance to all major MBTA Train Lines, movie theatre, fine dining, and Boston Common Public Garden. EASY TO SHOW!
Courtesy: Compass 617Living
BROKER FEE PAID BY LANDLORD | ROOFTOP POOL -- **VIDEO TOUR AVAILABLE** Welcome to the ultimate residential experience. This luxurious apartment offers floor to ceilings windows with hardwood flooring throughout. The stylish kitchen features quartz countertops, GE stainless steel appliances, and a sleek glass backsplash. Enjoy laundry in unit plus a modern bathroom with glass walk-in shower. World class amenities include the rooftop social lounge with panoramic city views boasting a heated swimming pool, cabanas and movie theater. Work out in the sprawling fitness center with yoga and spin studio -- or let your inner-chef embrace the grills in the courtyard! This pet-friendly community has your paws covered with a self-service pet spa, and outdoor dog run! Moments to the new Hub on Causeway, anchored by a 65,000sqft grocery store, ArcLight Cinemas and dining experiences. Conveniently located by Financial District, Boston Common and MGH with 24 hour concierge. Garage parking available.
Courtesy: Compass 617Living
BROKER FEE PAID BY LANDLORD | PET FRIENDLY -- **VIDEO TOUR AVAILABLE** Welcome to the ultimate residential experience. This luxurious apartment offers floor to ceilings windows with hardwood flooring throughout. The stylish kitchen features quartz countertops, GE stainless steel appliances, and a sleek glass backsplash. Enjoy laundry in unit plus a modern bathroom with glass walk-in shower. World class amenities include the rooftop social lounge with panoramic city views boasting a heated swimming pool, cabanas and movie theater. Work out in the sprawling fitness center with yoga and spin studio -- or let your inner-chef embrace the grills in the courtyard! This pet-friendly community has your paws covered with a self-service pet spa, and outdoor dog run! Moments to the new Hub on Causeway, anchored by a 65,000sqft grocery store, ArcLight Cinemas and dining experiences. Conveniently located by Financial District, Boston Common and MGH with 24 hour concierge. Garage parking available.
Courtesy: Compass The Bushari Team Compass
Stunning and spacious one-bedroom home in Jeffries Point, East Boston. Built in 2018, this 904 SF condo features high ceilings, Ceasarstone waterfall counters, Samsung stainless steel appliances, in-unit Washer/Dryer, and a large private patio perfect for entertaining. Heat and hot water included. Bike storage available. Ideally located on the East Boston Greenway, with easy access to Logan Airport, Maverick Square T, numerous parks and dining options, as well as major transportation routes.
Courtesy: Revolution Realty, LLC Rev Realty Team
Renovated around 2017, this gorgeous rental in South Boston has a beautiful kitchen with granite counters and stainless steel appliances. Features include breakfast bar, hardwood floors, in-unit laundry, ceiling fans, modern bath, and more!
Courtesy: Revolution Realty, LLC Rev Realty Team
Exceptional value in South Boston! Spacious and bright 3-bedroom condo, featuring hardwood floors throughout and eat in kitchen with stainless steel appliances. Extra storage with new washer/dryer in the basement, for unit's exclusive use. Large private deck off kitchen and master bedroom. Steps to beach and all public transportation, including bus stop and Andrew T Station; routes 93 and 90 conveniently accessible.
Courtesy: Red Tree Real Estate Andrew Ruskowski
****VIDEO WALK THROUGH AVAILABLE**** New & Modern Kitchen w/ Island Kitchen w/ dishwasher and disposal. ****** Corner unit blasted with natural light & HIGH CEILINGS AND STUNNING VIEWS OF ALL OF CLEVELAND CIRCLE***** Corner View Condo overlooking Cleveland Circle and just seconds between the D, C and B line! Hardwood floors in the living room, hallway, and bedrooms! -New Windows, Modern Light Fixtures, Updated bathroom and Kitchen. BATHROOM IS HUGE WITH LARGE CLOSET AND UPDATED TILE AND FIXTURES POOL JACUZZI ELEVATOR PROFESSIONALLY MANAGED LAUNDRY IN BUILDING CLOSE TO C AND D LINE.
Courtesy: HMW Real Estate, LLC Yong Cai
Updated eat-in kitchen, hardwood floors throughout. Good size 4 bedrooms and 1 big full bath with extra 3 season sunroom. one off street parking. In unit private free washer/dryer provided. Big private sunroom and shared big yard. Gas heating . Convenient location with easy access to MA Pike, Boston landing commuter rail, Express Bus line (501,503,57) take you directly to BU, Copley Square and financial district.
Courtesy: Blackstone Williams Realty, LLC John Becker
Nice 2nd floor studio in quaint Pinckney Street! Big, beautiful windows overlooking Beacon Hill's hidden house. Updated kitchenette. Berber carpet. Laundry in building. Available 5/01/2020 or Sooner! Call for a Video.
Courtesy: Compass The McLaren Team
Updated SOWA LOFT available JUNE 1st – Specious one bedroom condo in the former Emerson Piano Factory features open floor plan, updated kitchen with stainless steal appliances and updated bathroom. Enjoy the industrial charm with original wooden beams ceiling, exposed brick walls and polished concrete floors throughout. Generous storage space, Central AC and in unit laundry complete this unit. Located just moments from Mass Pike and I93 and surrounded by many South End shops and restaurants including Whole Foods this condo is every commuter dream! Pet friendly building! Parking available for rent!
Courtesy: Seaport Realty Group Lynn Hollenbeck
Available 5/1/2020- HUGE 1BR with a fantastic E.Side location! ALL UTILITIES INCLUDED!!!! Awesome Open Layout make entertaining a breeze! Oversized bowfront LR and FULL eat in kitchen, KING size BR with large closet and Tiled full bath with stall shower. HW floors throughout, laundry hook-ups in basement & patio and yard area available for tenants use. NO PETS, NON SMOKERS ONLY. 1st, security and full fee due upon lease signing.
Courtesy: Keller Williams Realty Boston-Metro | Back Bay Paul Reardon
Bright & spacious 2 bed 2 bath second floor condo, steps from Andrew Square Red Line. Open floor plan with high ceilings & hardwood floors. Front facing Master bedroom fits a king bed & has en suite bathroom. 2nd bedroom has double closet and in-unit laundry. Eat-in kitchen appointed with Shaker style cabinets, subway tile backsplash, quartz stone counters, and stainless steel appliances including gas stove. Other features include a 2nd full bath, mudroom, and secure deeded storage in basement. A large wrap-around deck is private to the Unit. Shared common yard great for grilling. Quiet street adjacent to future Washington Village, walking distance to all the restaurants & shops South Boston has to offer, and near park and beach. A commuter's dream – minutes to subway and buses, easy access to Rte 93 and Seaport, and Logan Airport is a 10 min drive. Well behaved pets with references will be considered. Owner Occupied professional Association No undergraduates. No smoking or vaping
Courtesy: Keller Williams Realty Charlotte Liu
NO FEES! Concierge building in the highly sought after Symphony area at XII Stoneholm. Enjoy the luxury lifestyle of a doorman, roof top pool, gym, elevator, sun deck, all just steps from the Back Bay! This one bedroom loft style home has floor to ceiling windows, renovated kitchen, full loft space, built-in A/C and hardwood floors. Location nears Newbury Street, Boylston Street, Whole Food, Green and Orange T line and so much more! Available NOW or 5/1. Easy to show!!! **It can be either furnished or not**
Courtesy: William Raveis R.E. & Home Services Stephen McCarthy
Private Roof Deck Included with this immaculate top floor apartment anyone would like to call home. Hardwood floors, central AC, shared laundry in basement, stainless steel appliances and granite counters. Beautiful private roof deck with water views. Available 6/1 Be here in time for the summer! Convenient to the MBTA bus routes 7, 9, 10 and 11. Convenient to downtown, RT 93, RT 90 and Logan Airport. NO PETS AND NO SMOKING NO EXCEPTIONS.
Courtesy: City Realty Group CRM Rentals Team
FREE APRIL RENT! Spacious one bedroom unit on the ground level in a multi-family house located near Oak Square in Brighton. Unit has a combination of wall-to-wall carpeting, hardwood floors and vct floors. There is no laundry in the building but laundromat is nearby. HEAT AND HOT WATER INCLUDED IN THE RENT. Shared backyard. On-street parking with a residential permit. Garage parking available for an additional $250 per month. Easy access to the public transportation. Cat friendly for $50/month. Dogs are considered on a case by case basis for an additional $100/month. Security deposit negotiable. UNIT IS RENTED IN AS IS CONDITION. LEASE THROUGH 8/31/21.
Courtesy: Charlesgate Realty Group, llc Sarah Herbert
South End garden level one bedroom with high ceilings and updated kitchen and bath for June 1. Good closet space, gas cooking, open kitchen/living with room for dining as well. Heat and hot water included, common laundry in the building, cat considered. Great location near shopping, restaurants and all the South End has to offer.
Courtesy: Unity Realty Robert Cassano
Amazing opportunity to live in lovely and desirable Bay Village/Back Bay! This fantastic first floor unit is located in a duplex on a quiet, idyllic, tree lined street. Modern and bright kitchen with attractive back-splash and high end stainless steel appliances. Living area features unique tin ceiling and decorative fireplace. Well maintained hardwood floors. Sun-filled 2 bedroom/2 bath has convenient laundry in unit. 1000 square feet of space in an ultimate location in Boston close to all desirable amenities. Literally steps away from MBTA access, great restaurants, shopping, schools, etc. Don't miss your chance to live in the heart of Boston.
Courtesy: Gibson Sotheby's International Realty Madelyn Kanter
Beautiful and spacious 3 bedroom, 2 bath second floor unit located on a great street in Fort Hill right across the street from a community garden and a half mile to the Jackson square T stop. The unit boasts a light filled living room and recently renovated modern kitchen with granite countertops, stainless steel appliances, and new floating shelves (being installed). The large master bedroom includes a walk in closet, private deck, and new master bath - currently undergoing renovation. Two more bedrooms, each large enough for a queen sized bed, and a renovated full bath complete the unit. There is dedicated, free laundry for the unit in the basement. No pets and no undergrads per condo docs. Video tour upon request.
Courtesy: Kimball Borgo Real Estate Caroline Morson
Landlord will pay half the fee! Bright and sunny corner 4 bedroom apartment with Boston Harbor views. This spacious apartment with large size rooms throughout features a large living room and a large eat in kitchen with a separate pantry room. In-unit washer and dryer. Newly renovated kitchen, stainless steel appliances and newly renovated bathroom. Wood floors, traditional wood molding details await in this large unit in very vibrant and conveniently located Meridian street in East Boston. Ideally close to restaurants and shopping, highway access and Blue line T stop makes for an easy commute to work downtown or for a day of fun to the beach!
Courtesy: CONWAY - BRIGHTON David Morganstern
Flexible move-in date--now, May 1 or June 1. Fabulous recent construction in existing building! Separate side entrance, walk out ground level. Open floor plan. Kitchen with stainless steel appliances & granite countertops, custom cabinetry. Smaller second bedroom. Beautiful hardwood floors and recessed lighting. Central air conditioning. Washer/dryer in unit. Walk to Boston College and the Reservoir. Green Line is at your door.
Courtesy: Compass Terrance Moreau
Generously sized 3 bedroom apartment conveniently located on quiet, one-way street in Jamaica Plain - share the joys of being blocks away from Forest Hills T-Station, a short walk to the Arnold Arboretum and several nearby restaurants. This first- level unit features a welcoming entrance, hardwood floors throughout, elevated ceilings and ample, in-unit storage. A private deck is also located just off the fully-applianced, eat-in kitchen and all bedrooms are accommodating, spacious enough for king or queen beds + additional pieces. Private storage is located in the basement and unit-specific washer/dryer is also here - no need for quarters! 1 off-street parking spot included in rent and garage parking is available for rent. June1 move-in. Don't Miss Out!
Courtesy: Not Available Thomas Alexander
STUNNING new construction. Set on a 14,000 sq ft lot and with nearly 3,000sq ft of living space, this brand new luxury home on the Fairmont Hill/ Milton line has it all. From the over-sized 2 car garage to the sprawling open concept kitchen, this is your dream rental. The first floor features a mudroom, kitchen and dining room with walk out sliders to the patio and enormous yard, living room and office, plus a half bath. Stairs from the kitchen lead to the large unfinished basement for endless storage. Upstairs, you'll find large bedrooms with ample closet space, a dedicated laundry room and a bathroom with double sinks. Finally, the master suite is truly one of a kind, featuring a luxurious en suite bathroom and a walk in closet the size of an office. Central A/C throughout. DON"T MISS THIS RARE GEM!!
Courtesy: Boston Proper Real Estate Calla Gillies
Simply stunning penthouse duplex with PRIVATE ROOF DECK on lovely Gray Street. Modern chef's kitchen with high end appliances, gas range, farmhouse sink and granite countertops. Open layout with front facing living room and cozy decorative fireplace. Seperate dining alcove with built-in banquette seating. First floor features two decent sized bedrooms with custom built-ins and additional BONUS walk-in closet that could pair as an office nook. Laundry IN-UNIT and shiny hardwood floors throughout. Spectacular roof deck with amazing city views. Moments from Restaurant Row, dozens of cafes, SoWa market and access to everything the South End has to offer!
Courtesy: Boston Proper Real Estate Calla Gillies
FURNISHED spectacular studio in the heart of the city at Garrison Hall. Front facing on to Garrison Street this unit is filled with natural sunlight. Kitchen renovated with granite countertops and stainless appliances including a GAS range, dishwasher and convection microwave. Modern bath, ample closet space and hardwood floors throughout. Fantastic furnishings include functional murphy bed, linens and kitchen supplies. All in immaculate elevator building with beautiful marble lobby. Live-in super, professionally managed and laundry in the building. UNBEATABLE location next to Prudential Center, Copley Square, Star Market, Jaho Coffee Shop, Orange and Green Line and tons of shops and restaurants.
1–25 of 1,923 properties displayed
How may I help you?
Get property information, schedule a showing or find an agent
Boston ZIPs02111 Homes for Rent 02113 Homes for Rent 02114 Homes for Rent 02115 Homes for Rent 02117 Homes for Rent 02119 Homes for Rent 02121 Homes for Rent 02122 Homes for Rent 02123 Homes for Rent 02125 Homes for Rent 02126 Homes for Rent 02130 Homes for Rent 02131 Homes for Rent 02133 Homes for Rent 02134 Homes for Rent 02196 Homes for Rent 02203 Homes for Rent 02211 Homes for Rent 02222 Homes for Rent 02297 Homes for Rent


text:
Various Versions Released For Android
Precisely what is Android?
Apakah Android Itu, It is really an operating system utilized on smartphone's, lap tops, tablet pc's and so forth. Android mobile phone applications are a group of software that features os, key applications and middle-ware. By having an open and versatile Android development platform Android developers will have a way to build rich, innovative and interesting applications. Google took over the Android Inc in 2005 and thereafter released android code under the name of Apache License. Android developers write the applications in customized version of JAVA. This is listed as the top selling Smartphone application in 2010 by the C analysis whilst still being the most sought after android mobile apps.
Android Mobile apps incorporates loaded features which can be sure to take the android developers by storm. The features include:
Better connectivity using CDMA, Bluetooth, WiFi, NFC, WiMax, GSM, LTE etc Android push messaging service which is a part of Android Cloud to Device Messaging Framework also known as C2DM Supports numerous human languages Adapts to both traditional smartphones as well as VGA, 2D graphics, etc Better and greater storage medium Supports audio/video formats like JPEG, GIF, BMP, AMR, AAC, MPEG-4, WebM, MIDI, WAV, PNG etc Contains web browser base on Web-Kit, Chrome's V8 Java script engine which can be the best Is capable of multi tasking and multi-touch options Supports hardware like touch screens, accelerometers, barometer, magnetometers, pressure sensors, thermometers, 3D graphics and much more Also supports tethering that assists in the phone used as wireless Wi-Fi spot Recent Releases along with their added features
There have been various versions of android mobile applications that were released with the intention of fixing some particular bugs of previous version plus adding to the report on latest features. The various versions show the continuous android development that makes the component and its features customer friendly and versatile enough for easy and efficient usage. The following are the latest versions of android
2.3 Gingerbread
Better graphical user interface Enhanced copy/paste features Improved gaming experience Supports SIP Also supports Near Field Communication 3.0 Honey comb
New Gui features Adapts to larger screen devices Supports multi core processors Supports hardware for better graphic presentation 3.1 Honey Comb
Has enhanced input devices Books Applications Google Movies compatibility USB host mode that enables transfer of internet data from cameras and devices directly 3.2 Honey comb
Supports many android powered devices Provides Zoom to fill screen compatibility mode Provides better control for the developers in display functionality Supports transfer of media files directly from Facts Improved screen support API 4.0 Frozen goodies Sandwich
Facial recognition unlock facilities Controls and monitors network data usage Supports unified social network contacts Enhances the photography features E-mail searching authorized even offline NFC feature for information sharing Future Trend
The extensive uses and packed without standing features Android has had about a revolutionary change in the mobile industry. Android continues to be gaining the lion's share in the market growth as against its competitor RIM. The Gingerbread version of Android has been at the top position with more than 38.7 percent share depending on the reports in October. With the latest in the pipeline that is certainly Frozen goodies sandwich Bing is planning to cancel the front facing buttons and go in for the simple screen with headphones and power button.
Interested in being familiar with Tutorial dan Tips Trik Android, popular Android smartphones like the new Bionic, Iphone, Paket Internet Android, Smartphone Comparison plus much more?
Visit my web page to learn the answer to the question Just what Android and much more.


text:
19 Years
Since We Started
Happy Customers
Cities with Students
Teachers in Network
Gift Certificates Available. SHOP NOW!
...I am dedicated to the development of proficient instrumentalists. My teaching style is pretty transparent. It really depends on the needs of the student. I am dedicated to the development of proficient instrumentalists. ... Read More
...I'm goal-oriented and methodical, but I'm also passionate about what I do and I love nothing more than to impart that passion. I currently teach extensively throughout northern Virginia and hold the position of music teacher at the Ambleside School in Herndon. ... Read More
...Music has always been a driving force in my life and one of the greatest things I've been involved in. I hope to give my students the same opportunities I have had and make music an important part of their lives as well. ... Read More
...It is important and rewarding for both me and the student to have a passion for their music and learning. Because of this, it is important to me that each student sets their own goals.... Read More
...I have been playing the flute for 30 years! Back in school, I performed and competed actively. I love to play and love to teach. In another life, I would be a professional musician, but for now, I play in my spare time and try to encourage others by teaching.... Read More
...Students can expect to work from a variety of method books to study instrumental techniques, and music theory. For beginning students I prefer Hal Leonard's Essential Elements, but also work with any method book a student uses in their band class.... Read More
...With young students who are reading or starting to read, I usually use a combination of the Suzuki method and traditional French methods like Moyse, focusing on singing, memory, and tunefulness while allowing students to self-direct the learning of reading sheet music.... Read More
19 Years
Since We Started
Happy Customers
Cities with Students
Teachers in Network
We'll then reach out to the teachers for you.
Schedule the risk-free trial lesson directly with the teacher.
Continue with that teacher or try someone else.
Select all the days/times the student would be available to start lessons. Selecting "3pm - 7pm" means the student can start as early as 3pm or start as late as 7pm. It is important that you select as many days and the widest window of start times for each day as possible. That will help us make a match with one of our teachers.Ok
Are you sure that's your only availability? The more availability you easier it will be to arrange a teacher for you.


text:
Bravo, my dear! Seriously, when was the last time you finished a fic? hehe! It's been an absolute joy reading your fic - and yes, a steaming hot NC17 cookie is much appreciated (they're in a heart-shaped VIBRATING bed! We need some very detailed lovemaking for this bit!).
You're still one of the best, & I can't wait for the next MoM chapter
:D Just keeps getting better. Can't wait for Ginny's POV.
Oh terribleness, I had hoped I wasn't right. I hope it gets better quickly hear those things are awful. My friend had one and oof, I definitely would never wish one on anyone.
This story is nuts and I LOVE it!
Dumbledore patted her hand good-naturedly. "Yes, it is rather a nice arse, isn't it?" he whispered conspiratorially. "Mr. Weasley's tushie rather reminds me of Gilbert Grindelwald in his prime, actually."
You're more right than you know, thought Hermione.
Okay THAT made me laugh hard. Brilliant. Dumbledore saying 'tushie'. Ha ha.
FOUR CHAPTERS, Anise. I'm impressed. That has to be a record!!!
yay this is sooo fun lol
WOO HOO! I can't wait! As always, thanks for sharing =) Much Love.
This extremely short story has been so... uncomplicated. I'm not sure that I can believe that there is only one chapter left.
I fully expect that a chartreuse coloured Nick will show up along with Delerium and take us all off to platform 9 and 3/4 19 years later after the bat-bogey has come and the death train has left the station.
I guess I will just have to wait and see.
Thanks for all the great never-ending stories.
Yay for Ron and Hermione!
GEE! So he isn't gay! :)
The 'Top-Secret Gryffindor Male Homosexuality Subtext Gymnasium and Leather Bar' made me think of fight club for a strange reason...
Can't wait for the last chapter ;)
okay, ron's secret club has me laughing so loud my daughter got out of the shower to see what was wrong.
yay that ron isn't gay. i mean, yay for Hermione, boo for Justin and everyone else.
i loved this chapter. it was most excellent!
Lol! That was hilarious! This story is such fun! I can't wait 'til you post the next chapter!
And here I thought you'd posted it all. I continue to be crudely amused, and am slightly ashamed of myself >_>
Ron is paranoid delusional... and gay.
Undershorts of Evil! *throws confetti* :D
Congrats on finishing another fic! :)
While I appreciate the lack of Harry, I have to wonder if he'd provide another avenue for entertainment with HIS view on why Ginny's being cozy with Malfoy. Lol. Hermione, I think, is well written, despite that she doesn't know what 'minions' are. I think she'd be appalled (or at the very least surprised and sceptical) that Ron knows a word she doesn't.
So far, we've only had secondhand D/G but I love the dialogue and the blatant double entendres. The first years become likable characters for the brief moments we're with them, and Ron's temper fits in so perfectly with the runaway-plot crack!fic.
On a side note, maybe Ron should see Franz Kafka for more details on turning Malfoy into a cockroach a la The Metamorphosis. Haha.
Ohhhhh put poor Ronniekins out of his addled mind!
You are too frickin funny!
You're crazy, Anise. CRAZY!
Hmmmm what was I going to say? I'm tired :O Yeah so good fic. I like this one. I'm kinda preferring it to MOM, because i don't like dark, twisted Draco/Ginny sex :( Thats not gonna happen in this fic, is it??? HAPPINESS!!!! PLEASE!!!
You made me laugh aloud a LOT. So thanks for that. I was feeling down and all and you cheered me up. Get better sooon!
Did you have a kidney stone? I hear those are rather painful... nevertheless good to hear you are no longer in the hospital and I hope you are feeling better soon!
Ron kills me. He's so ridiculous and I love that all these little kids from other houses are pointing it out in their own funny ways. I think the Ravenclaw that was rather like a mini Hermione was the best though Cyanara was pretty hilarious too. Cyanara Slanderpool, funny name btw.
Haha this fic is hilarious! Especially the "pussy" one. Totally cracked me up.
Ron seems to be rather obsessed with this. Nevertheless, it was amusing. :)
When are we going to find out whether Ron is gay or not? I'm rather curious. o.o
My nick is pretty awesome if I should say so myself. ;)
I have a feeling the next chapter is going to be excellent.
lol poor ron! :) next please
I love this story! I can't help but laugh the entire time! And I cannot wait to see from Ginny's point of view!!! I'm about bouncing up and down in my seat right now! Ron is awesome, by the way!
That pussy one was so dirty.
I hope it's IT, not for Ron to see it, but when showing Ginny's POV!
Great fic!
best of luck in the hospital! ;)
I'm pretty sure Ron has both of the things that Sophia asked about! ha! i never realized how many sexual innuendos were out there or how funny it would be if you actually acted them all out!
i can't wait for THE ONE! ha! and ron's reaction.
also, i'm glad you're doing ok! sorry to get all fan-girlie on you, but your fics have always been a HUGE inspiration. whenever you include The Endless, I get chills :D Just had to let you know.
I just love all of your stories, Anise. Love them. I always start them, get up to wherever you've posted to, then get all flustered because most of your chapters on the long ones are all cliffies!
Then by the time you update, I have to go back and reread them! Which doesn't bother me one bit because when that happens, I usually find out something new. And I love how they're all interconnected some how! Your Keeping Christmas on the Death Train was the reason I signed up for this site XD. ((Which you need to FINISH!, so i can go reread it again! AND Nineteen years later!))
Anyway, I LOVE this story still. Its so funny! Either Ron is gay, or completely oblivious to EVERYTHING lol. I like the oblivious Ron, I think.
and....happy thoughts for Anise!!


text:
- Oct 16, 2015
- 335
- 0
- 28
- 22
I really doubt that Phineas & Ferb will be forgotten.Here's a few awesome current cartoons that will be forgotten,
Clone High - The show was amazing/funny with epic comedy/awesome hilarious characters, loved Principal Scudworth in his funny antics, a shame it didn't had another season.
Phineas and Ferb - I'll so miss it, the show was amazing/funny with best scenes with Phineas/Ferb and friends and Perry/Dr. Doofenshmirtz.
Dan Vs. - Sucks it got cancelled, the underrated show was awesome/funny with great dark comedy and Dan/Chris's enjoyable crazy revenge adventures, and cool it had monsters and a dinosaur in it.
The 7D - The show was great/enjoyable with great comedy, loved the Glooms Hildy & Grim who stole the show though, I'll miss it.
Monsters Vs. Aliens: The Series - A shame it got cancelled, it was the best awesome enjoyable CGI movie-based comedy series ever.
Axe Cop - It was awesome/funny with great comedy, Axe Cop and his friends and the supervillains were awesome characters, I'll miss it.
Penn Zero: Part-Time Hero - The show was great/decent with great comedy/action, amazing detailed animation/character designs, loved the awesome enjoyable villains like Rippen, the Evil Chinchilla, Professor Evil Professor, and Count Destructo(his major role will come), I hope season 2 comes.
Pig Goat Banana Cricket - It was great/funny with sweet bizarre animation/character designs and great comedy.
Dan Vs. and Penn Zero are so good, but I agree.


text:
A disabled cat, which injured his hind legs and became disabled after a car accident is seen with his wheelchair made from toy truck, at an animal shelter in Edirne, Turkey on October 16, 2019. The cat found by university students in Edirne and started to move with the aim of the wheel chair made by shelter officials. (Photo by Gokhan Balci/Anadolu Agency via Getty Images)
Spanish Legion's masco goat parades during the Spanish National Day military parade in Madrid on October 12, 2019. (Photo by Oscar Del Pozo/AFP Photo)
A Greek magpie bird holds in its beak a nut, at Nafplio, Peloponnese, Greece, 17 October 2019. (Photo by Bougiotis Vangelis/EPA/EFE)
This handout picture taken on October 11, 2019 and released by International Animal Rescue shows an orangutan nicknamed Mama Sifa (R) and her cub Sifa being released into the forest in Ketapang, West Kalimantan. Sifa and her mother Mama Sifa were rescued from the recent massive forest fires in Indonesia that have caused a toxic haze to spread as far as Singapore and peninsular Malaysia seriously affecting endangered orangutans and their habitat. (Photo by International Animal Rescue/AFP Photo)
King penguins are seen at Volunteer Point, north of Stanley in the Falkland Islands (Malvinas), a British Overseas Territory in the South Atlantic Ocean, on October 6, 2019. The Falklands has an incredibly rich biodiversity including more than 25 species of whales and dolphins, but it is the guaranteed ability to get up close and personal with penguins that makes it such an enticing destination. There are five penguin species in the archipelago – King, Rockhopper, Gentoo, Magellanic and Macaroni. (Photo by Pablo Porciuncula Brune/AFP Photo)
King penguin chicks are seen at Volunteer Point, north of Stanley in the Falkland Islands (Malvinas), a British Overseas Territory in the South Atlantic Ocean, on October 6, 2019. (Photo by Pablo Porciuncula Brune/AFP Photo)
In this photo provide by the Berlin Zoo on Friday, October 18, 2019, a person holds one of the Zoo's Panda twins at the Zoo in Berlin. Zoo Berlin's panda twins, born on Aug. 31, 2019, open their eyes for the first time. (Photo by Zoo Berlin via AP Photo)
Western lowland gorilla Lou Lou and her daughter are seen at the zoo in Belo Horizonte, Brazil, on October 14, 2019. The baby gorilla was born on July 8, 2019, and is the fourth of the species -which appears on the International Union for Conservation of Nature (IUCN) red list as critically endangered- to be born in the zoo, the only zoo in South America to succeed in breeding the species. (Photo by Douglas Magno/AFP Photo)
A search dog patrols the Royal Gallery ahead of the State Opening of Parliament at the Palace of Westminster in London, Britain on October 14, 2019. (Photo by Leon Neal/Pool via Reuters)
This handout from the Krabi Pitakpracha Foundation taken on October 13, 2019 and received on October 15, 2019 shows members of the rescue foundation holding a four-metre long king cobra that was found in a sewer in Krabi. Rescuers pulled a fiesty four-metre long King Cobra from a sewer in southern Thailand after an hourlong operation, a rescue foundation said on October 15, calling it one of the largest ever captured there. (Photo by Krabi Pitakpracha Foundation/AFP Photo)
A flock of Adjutants storks sit on a garbage dump at Borgaon in Guwahati, Assam, India, 17 October 2019. Wildlife activists and environmentalists have protested in the past for shifting the garbage dump to another site since it is situated near the Deepor Beel Bird Sanctuary. (Photo by EPA/EFE/Stringer)
20 Oct 2019 00:03:00, post received 0 comments


text:
Top Documents of the Week
- Zoom: Troubleshoot Audio (Faculty & Students)
- Zoom: Record a Meeting/Lecture/Presentation (Faculty & Students)
- Apple Mail - Add a Shared Mailbox
- Zoom: Using Zoom on a Chromebook
- Zoom: Start a Meeting (Faculty & Students)
- Zoom: Breakout Rooms (Faculty/Hosts)
- Share from OneDrive (Faculty & Students): View Only
- Blackboard (Students) - Hyperlink Text in the Text Editor
Most Recently Updated Documents
- Microsoft Teams - Welcome to Teams Calling
- Microsoft Teams - Transfer call to off-campus phone number
- Microsoft Teams - I do not see the Calls app in Teams on mobile.
- Microsoft Teams - How do I move my Phone number to Teams?
- Zoom: Record a Presentation and Post it to Blackboard (Students)
- Zoom: Start a Meeting (Faculty & Students)
- Blackboard (Faculty) - Assignments: Clear, Ignore, and Allow Additional Attempt
- Blackboard (Students) - About LockDown Browser


text:
More Projects
Zero Point were looking for a marketing solution to represent their brand and services. Fullestop proposed a solution to Zero Point in form of a website. We upheld the development including the code, creatives & content management which accomplished project essentials.View Website Get a quote
Hello Baby Direct is an ecommerce platform for baby and nursery products based in St Albans, UKView Website Get a quote
Trinus Group wanted a fresh look for their existing website. The existing website had a lot of information to be presented to user.View Website Get a quote
The Goal was to display the content to tell people about Todd Bender's Navy SEAL Foundation.View Website Get a quote
Gourmetpet or Dr. Hahnz gave ecommerce a new concept of selling Pet Food of finest quality online. The website is both CMS & Ecommerce and hence provides detailed information about all the products and other Pet Care articles.View Website Get a quote


text:
Powerful, gripping and told in a stunning, authentic voice: this thriller is guaranteed to get people talking.
Sign up to our newsletter using your email.
By clicking subscribe, I acknowledge that I have read and agree to Penguin Books Australia's Terms of Use and Privacy Policy.
To help us recommend your next book, tell us what you enjoy reading.


text:
Married couples separated but living together I Am Wanting Swinger Couples
We can meet in advance, married couples separated but living together we can feel mrried with each other before meeting on the trail or where ever you want. Because if they do, I like it. So hit me up, lets brainstorm, seeking to start sooner than later If ya like sports that's a plus. I dont want to be street anymore. I am educated, experienced, an attractive man next door, 43 years old, fit athletic, disease free.
|Relationship Status:
|I Look For Men
|Chandler, AZ
|Relation Type:
|Girls Want Housewives Wanting Sex
Views: 6161
Cyril Byrne.Women Seeking Large Men
My marriage ended about four years ago. However, because of economic reasons, my ex and I were forced into what most would deem an intolerable situation: Our marriage was finished but we could not afford to spilt up with a mortgage, two kids married couples separated but living together our own small business to support.
Tucsonia pussy im so sorry, I moved into the spare room as we both tried desperately to keep ourselves sane and functioning.
Having two bathrooms helped.
Married couples separated but living together I Am Look Real Sex
The first couple of years were tough — panic attacks, fear, loathing myself, him, the situation, togetehr world all featured constantly. But secretly I was screaming for someone to help me. Except for a few very close friends, separatec one knew.
The shame of failure is a most powerful force. In the past few years I have had to married couples separated but living together myself many times why this is? Why is the shame so heavy? Why, when I talk married couples separated but living together my marriage ending, do people glaze over?
Or, more often, become instantly distant? They back away suddenly to an errand they had forgotten needed their urgent attention.
Or worse, they get preachy or angry at me, as if I am some kind of leper who still has the audacity to think they can continue to be part of society. I do not have an infectious condition. This bitter divorce storyline has become the standard for all of us because it is perfect at providing high-octane drama for TV, film and books.
The basic narrative is this married couples separated but living together a marriage breaks down, both parties hate each. They fight and bicker and look for any opportunity to stab one another in the back or get one-ups on the. They go to court for a long drawn-out battle where the only winners are the lawyers. The kids get screwed up because of the whole process. Finally the exes go their separate ways but neither one ever, ever forgives or forgets the betrayal or hurt of the.
It started out with flailing for solutions.Wife Is Slut
Married couples separated but living together I had the strength, I would frantically crunch numbers and look at job sites hoping something would add up housewives wants sex tonight IA Washburn 50706 an escape route. Nothing ever did and perhaps that was because most of my energy was taken up with pretending everything was okay while being a working partner in the business married couples separated but living together raising two kids.
I zombie-walked through days, then paced my room all night. There were some days when I actually hid in a cupboard. Then, one day in a shopping centre, I ran into another woman who was going through a break-up. As I listened to her pain and anger, I felt myself togethher into a hole of helplessness.
Her anger was palpable and understandable and my heart ached for her.
But listening to her struggles, I realised that I could make a decision. I could decide how I was going to be.Eorld Sex
I could decide who I was going to be. And with that one realisation everything changed. From that point on, I tried spearated base all my other decisions from this basic premise — who did I want to be? It was extremely hard at. I recognised that my power was in who I decided to be, not proving who was right or wrong.When A Man Teases You What Does It Mean
After years of feeling helpless and trapped, I had a sense of power back and I began to heal. I could decide who I wanted to be — not for separtaed good of anyone else but me.
I could make my life better no matter the circumstances.
Once I tia cyrus escort I could make decisions to better my own life everything mmarried because I took back control and, with this, my head started to become clearer. All the things that hurt me or made me angry about the marriage were no longer relevant because we yes, he and I had decided to not be married.
I had to change my expectations of him and he of me. I stopped apologising for myself, started to see who I was and started to let go of my old idea of who he was or married couples separated but living together be.Women Seeking Hot Sex Grenola
We had a new relationship now where the issues of being husband and wife were no longer relevant. We were now a partnership raising thai girl singapore children and keeping a business afloat.
I, of course, must acknowledge that my ex had his own healing to do and he worked hard at becoming a great dad and rebuilding our married couples separated but living together into something new. This can be hugely liberating. All separzted of us still live in the same house and neither he nor Liiving see any reason to change this even if we. I have no desire for our children to see less of their father.
For us, ironically, it has now become the easier option to stay. Honestly, I married couples separated but living together not know if we would have reached this point if we had not been ladies want sex Claiborne into this situation but man, am I glad now that we. But it is hard to explain. We are not a couple but we are not room-mates. We are also not just friends because we have two kids together and a history.
There are still plenty of hiccups and arguments and working out stuff but we are both sure that this is the best solution, for us.
Of friends, let me say. Mine are the best. No, really, the best. Those special few who patiently held my hand or kicked my arse. I just wanted to put it out there that the story of a marriage separation can be different.
Married couples separated but living together
They are never easy, they are never not sad but they can help you develop into something new. A new type of friendship. It is not the most exciting story, but I can tell you everyone comes out okay in the end. The author of Beloved and Jazz won both a Nobel and Pulitzer prize for her fiction.
Married couples separated but living together I Am Look Men
Asian folding screens Down togetherr whose success with First Derivatives was widely recognised. French businessman spent 18 years on the foundation that oversees the Irish College in Paris.
We use cookies to personalise content, target and report on ads, to provide social media features and to analyse our traffic. For more information see our Cookie Policy.
I Am Search For A Man
Separated but living in married couples separated but living together same house with our kids: Ashe Conrad-Jones. Conrad Jones My. More from The Irish Times Beauty. Sponsored Heading west: Married couples separated but living together the Dyson V11 through its paces. Coast to coast: Subscriber Only.
How to holiday cheaply in France: Everything you need to know. Our son stole from us to fund his cocaine habit. Heading west: Toni Morrison obituary: Writer who trailblazed the African American experience The author of Beloved and Jazz won both a Nobel and Pulitzer prize for her fiction.
Brian Conlon obituary: Jean Bijasson obituary: Pillar of the Irish community in Paris French businessman spent 18 years on the foundation that oversees the Irish College in Paris.
Something for the weekend. A Friday roundup of the latest exclusive offers and competitions, and a sneak preview of what's coming up over the weekend livig The Irish Times. Sign In. Don't have an account? Forgot Password? Not an Irish Times subscriber?


text:
- 3 beds
- 2 baths
- $95,000 price
- 21652775 mls id
- Square Feet: 1,560 sqft
- Year Built: 1914
- Lot Size: 0.095 acres
- Neighborhood: Marion - Center Ne
- Status: Pending
Completely remodeled All Electric 3 bedroom, 2 bath 2 story. Spacious great room opens to large, light filled dining room. Kitchen has all new white cabinets, white subway tile, drop in electric range in center island and dishwasher. Master Bedroom on the Main floor. Downstairs full bath with ceramic tile floor and shower. Upstairs is two bedrooms and a full bath. All new doors, windows, plumbing, flooring, kitchen, baths, hot water heater, carpet, paint and back privacy fence. New front deck adds to curb appeal.
Related Listings
4 Pictures
A true diamond in the rough, this home has real potential. Perfect for an investor looking for a new project, or a DIY-er. New roof and ducts in 2017, furnace in 2007. Situated in an area seeing rapid revitalization, character abounds with high ceilings and original hardwoods, built ins and...
3207 Broadway Street Indianapolis, IN
3 beds, 2 baths, 2,199 sqft, $94,999
Listing Agent: David Landau
Courtesy of: Carpenter, REALTORS®
14 Pictures
Updated 3 bedroom home in Tuxedo Park that's waiting on someone's special touches to call home. Buyer gets to pick ALL interior selections such as cabinets, flooring, paint, tile, countertops and lighting with some updating on the roof. This home is priced with all STANDARD FEATURES included in...
810 North Colorado Avenue Indianapolis, IN
3 beds, 1 baths, 2,688 sqft, $97,500
Listing Agent: George F. Hemphill
Courtesy of: Makeover Realty
1344 North Olney Street Indianapolis, IN
3 beds, 1 baths, 1,096 sqft, $98,900
Listing Agent: Michael Ruth II
Courtesy of: Keller Williams Indpls Metro N
Courtesy of: The Madsen Group, LLC
Office Phone: +13172844400
Listing Agent: Pamela Madsen
Updated: 30th March, 2020 12:24 PM.
2021 Hillside Avenue Indianapolis, IN


text:
Your institution must subscribe to JoVE's Biology section to access this content.
Fill out the form below to receive a free trial or learn more about access:
Restenosis following cardiovascular procedures (bypass surgery, angioplasty, or stenting) is a significant problem reducing the durability of these procedures. An ideal therapy would inhibit smooth muscle cell (VSMC) proliferation while promoting regeneration of the endothelium. We describe a model for simultaneous assessment of VSMC proliferation and endothelial function in vivo.
Cite this ArticleCopy Citation | Download Citations | Reprints and Permissions
Yu, D., Makkar, G., Sarkar, R., Strickland, D. K., Monahan, T. S. Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function. J. Vis. Exp. (124), e55201, doi:10.3791/55201 (2017).
Translate text to:
Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell (VSMC) proliferation. Common murine models study small vessels such as the carotid and femoral arteries. Herein we describe an in vivo system in which both VSMC proliferation and endothelial barrier function can be simultaneously assessed in a large vessel. We studied the infrarenal aortic response to injury in C57BL/6 mice. The aorta was injured from the left renal vein to the aortic bifurcation by 30 transmural crushes of 5-seconds duration with a cotton-tipped applicator. Morphological changes were assessed with conventional histology. Aorta wall thickness was measured from the luminal surface to the adventitia. EdU integration and counter staining with DAPI and alpha-actin was used to demonstrate VSMC proliferation. Activation of ERK1/2, a known moderator of intimal hyperplasia formation, was determined by Western Blot analysis. The effect of inflammation was determined by immunohistochemistry for B-cells, T-cells, and macrophages. En face sections of endothelium were visualized with scanning electron microscopy (SEM). Endothelial barrier function was determined with Evans Blue staining. Transmural injury resulted in aortic wall thickening. This injury induced VSMC proliferation, most prominently at 3 days after injury, and early activation of ERK1/2 and decreased p27kip1 expression. Injury did not result in increased B-cells, T-cells, or macrophages infiltration in the vessel wall. Injury caused partial endothelial cell denudation and loss of cell-cell contact. Injury resulted in a significant loss of endothelial barrier function, which returned to baseline after seven days. The murine transmural blunt aortic injury model provides an efficient system to simultaneously study both VSMC proliferation and endothelial barrier function in a large vessel.
Restenosis following cardiovascular procedures (bypass surgery, angioplasty, or stenting) is a significant problem reducing the durability of these procedures. All revascularization procedures are plagued by restenosis. Present strategies to prevent restenosis (drug-eluting stents and drug-coated balloons) inhibit both vascular smooth muscle cell (VSMC) and endothelial cell proliferation (EC). Consequently, these interventions prevent VSMC mediated restenosis, but also prevent the regeneration of the endothelium. Without an intact endothelium, patients are required to be on potent antiplatelet agents to decrease the risk of in situ thrombosis at the risk of bleeding complications. An ideal therapy would inhibit VSMC proliferation while promoting regeneration of the endothelium. Thus, there is a need to simultaneously study VSMC proliferation and endothelial barrier function in vivo.
Presently, there are several mouse models of restenosis 1. These models include carotid ligation and femoral artery wire injury 2. Aortic models include stent placement 3, balloon injury 4, and aortic allograft 5. All of the present models are limited. Carotid ligation generates a flow-mediated neointimal lesion and does not have endothelial injury. Additionally, both carotid and femoral arteries have many fold fewer cell layers than human vessels, limiting their translational value. The mouse aorta which is approximately 1.3 mm in diameter, is the only vessel that approximates a clinically relevant (coronary) human artery (3).
Despite the translational potential of murine aortic models of disease, current models have limitations. These models require advanced microsurgical skills and specialized equipment such as angioplasty balloons and stents. Herein, we present a novel, reproducible technique to simultaneously induce VSMC proliferation and disrupt endothelial barrier function.
Subscription Required. Please recommend JoVE to your librarian.
Ethics Statement: The protocols for animal handling were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Maryland (protocol number 0416009) and conducted according to AAALAC-International standards.
1. Surgical Procedure
- Anesthetic Technique
- Sterilize all instruments used in survival surgery with steam sterilization at 121 °C for 30 min.
- Induce anesthesia via an induction tank with 100% O2 and 2.5% isoflurane delivered via precision vaporizer. Post-induction, discontinue the isoflurane and flush the chamber with O2. Maintain anesthesia with 1.5-2% isoflurane via face mask and 1 L/min O2 by inhalation.
- Attach both the induction chamber and the face mask to a charcoal scavenger for waste gas adsorption to protect personnel. Ensure an adequate anesthetic plane by demonstrating that there is no response to noxious stimuli (toe pinch).
- Create an operative field consisting of a surgical tray with isothermal pad to provide thermal support during surgery. An additional isothermal pad will provide thermal support to animals in their recovery cage.
- Animal Preparation
- Carry out the following investigation on 10-12 weeks old male C57BL/6 mice.
- Remove the hair on the ventral abdominal surface of the animal from the sternum to the inguinal region with a depilatory agent or an electrical clipper with a number 40 blade.
- In case of depilatory agent, apply this compound to the surgical area for 2-3 min and then remove it with cotton. We use a commercially available compound of calcium hydroxide and sodium hydroxide.
- Prep the area over the shoulder with 70% alcohol and subcutaneously inject carprofen (5 mg/kg) with a 25 gauge or smaller bore needle. This treatment will provide postoperative analgesia for the animal.
- Transfer the animal to the surgical field and position in dorsal recumbency.
- Prepare the surgical site by scrubbing 8-12% providone-iodine with a clean cotton applicator or cotton gauze. Then rinse the skin twice with 70% alcohol.
- Place ocular lubricant in both eyes to reduce the incidence of corneal desiccation. Cover the surgical site with a sterile drape.
- Operative Technique
- Make a median abdominal laparotomy incision approximately 2-2.5 cm in length with a scalpel beginning immediately caudal the xiphoid process and extending towards the pelvis.
- Mobilize the small bowel and duodenum and reflect laterally to the right. Roll up a strip of sterile cotton gauzed and soaked with sterile saline for injection to allow packing of the viscera to improve exposure.
- With the small intestine mobilized to the right side of the abdomen, expose the retroperitoneum and expose the abdominal aorta from the left renal vein to the aortic bifurcation (Figure 1).
- With a sterile cotton-tipped applicator, deliver 30 consecutive crushes, each five seconds in duration.
- Remove the packing and allow the viscera to return to their native position.
- Close fascia with a running 4-0 absorbable monofilament suture (polydioxanone). Skin is closed with a running 6-0 non-absorbable monofilament suture (nylon).
- Recovery and Post-Procedure Care
- After the procedure, place the animal in a recovery cage with clean bedding on an isothermal pad to continue thermal support until the animal is able to ambulate normally. Do not leave the animal unattended until it demonstrates the ability to maintain sternal recumbency.
- Monitor the animal every hour for the first 4 h after surgery. Once the animal is ambulating normally return it to the assigned husbandry room. The animal will not be housed in the company of another animal until it has fully recovered.
- Monitor the animal twice daily for the first 72 h after surgery and at least 3 times a week thereafter. Monitoring includes weighing the animal three times a week.
- Administer carpofen (5 mg/kg) subcutaneously twice daily for the first 72 h after surgery.
2. Procurement of Tissue
- Method of Euthanasia
- Euthanize the animals at predetermined time points.
- Induce anesthesia via an induction tank with 100% O2 and 2.5% isoflurane delivered via precision vaporizer.
- To study the integrity of the endothelium, Administer Evans Blue dye to the animal.
NOTE: Evans Blue is a negatively charged azo dye with a high binding affinity for albumin and can only stain blood vessels in the absence of an intact endothelium6.
- For endothelial integrity studies, at the time of euthanasia, perfuse the animals with 5 mL of 0.3 % Evans Blue dye followed by 5 mL PBS at physiological pressure for 5 min. The animal is maintained in a surgical plane of anesthesia during the perfusion procedure.
- To study the integrity of the endothelium, Administer Evans Blue dye to the animal.
- After achieving a deep anesthetic plane, open the chest with a sternotomy. Make a laceration in the right atrium to allow drainage of blood from the animal and access the left ventricle is accessed with a 21 G needle and inject phosphate buffered saline (PBS) until the effluent from the right atrium is clear.
- After perfusion with PBS, enter the abdomen through a midline incision.
- Once again, mobilize the small intestine to the right side of the abdomen exposing the infrarenal aorta, sharply dissect the aorta from adjacent tissues and excise it from the left renal vein to the aortic bifurcation.
- Store the excised aorta in a 4% paraformaldehyde solution until tissue processing occurs.
- For endothelial integrity studies, open the aorta longitudinally and pin it to a wax sheet exposing the entirety of the luminal surface6. Carry out a qualitative assessment of endothelial integrity by the degree of staining with Evans Blue dye6.
- For other histologic assays, section the aorta transversely and embed it in optimal cutting temperature (OCT) compounds as dictated by the histological method to be used 7.
Subscription Required. Please recommend JoVE to your librarian.
Representative Results
Transverse sections aorta embedded in OCT were sectioned, and stained with hematoxylin and eosin then counter stained with Verhoeff-Van Gieson (VVG) stain to identify the internal and external elastic lamina 7. Crush injury induced aortic wall thickening compared to the aortas of animals treated with a sham procedure (laparotomy and small bowel mobilization alone). Wall thickness, as assessed by the distance from adventitia to the lumen, was greatest three days after injury (42.2 ± 1.7 µm vs. 22.1 ± 1.1 µm for laparotomy alone) (Figure 2A-B). Injury resulted in the cells having a more rounded appearance and an irregular contour of the luminal surface (Figure 2C).
Increased wall thickening of the injured aorta is at least in part mediated by increased proliferation of medial vascular smooth muscle cells. To prove that wall thickening is due to increased proliferation as opposed to cell hypertrophy, we used an EdU proliferation assay. In this assay, the thymidine analogue 5-ethynyl-2'-deoxyuridine (EdU) is administered intravenously to the mouse 24 h prior to euthanasia. EdU is incorporated into DNA during synthesis. EdU is detected by a click reaction (copper-catalyzed azide-alkyne cycloaddition using green-fluorescent dye) 8. Sham mice exposed only to laparotomy had no observed fluorescence in any layer of the aorta. Conversley, mice subjected to aortic injury demonstrated fluorescence in both the media and intima of the aorta (Figure 3).
Aortic wall thickening after crush injury activates cell signaling pathways implicated in restenosis in humans. The mitogen-activated protein kinase (MAP Kinase) ERK1/2 is one of the principle mediators of intimal hyperplasia formation In response to stimulation from a mitogen, ERK1/2 is phosphorylated and activated 9,10. Data from our laboratory have also demonstrated that the cyclin-dependant kinase inhibitor p27kip1 is decreased in response to mitogenic stimuli in the vessel wall12. Mice were euthanized at 1, 3, 7 days, and 1 month after aortic crush injury. The infrarenal aorta was isolated and protein expression of ERK1/2, phospho-ERK1/2 (the activated form of ERK1/2), and the cyclin dependent kinase inhibitor p27kip1 were determined by Western blot analysis (Figure 4A). No change was observed in total ERK1/2 protein expression over time. However, three days after injury, phospho-ERK1/2 (Pi-ERK1/2) expression was over 200% greater than baseline Pi-ERK1/2 expression. p27kip1 expression decreased at early time points and reached a nadir of approximately 20% of baseline seven days after injury. Both Pi-ERK1/2 and p27kip1 expression approximated baseline levels one month after injury (Figure 4B). Thus the observed thickening of the aortic wall is at least in part due to increased medial proliferation.
The aortic injury model allows simultaneous assessment of both medial smooth muscle proliferation and endothelial barrier function. Scanning electron microscopy (SEM) was used to characterize the effect of the crush injury on the morphology of the endothelium. The luminal surface of the aorta is lined by a confluent, endothelium that prevents adhesion of blood-born cells and molecules (Figure 5A, Scale bar = 100 µm). In contrast, crush injury results in disruption of endothelial cells and partial denudation of the endothelium (Figure 5B, Scale bar = 100 µm). As seen at higher magnification, disruption of the endothelium results in adhesion of particles consistent in size and shape with platelets. Adhesion of these particles occurs throughout the entire zone of injury, and is not limited to areas of gross denudation observed at lower magnification (Figure 5C, Scale bar = 50 µm). The morphology of the adherent particle is best observed at higher magnification. These particles are consistent with platelets and fibrin (Figure 5D, Scale bar = 10 µm).
In addition to endothelial morphology, the barrier function of the endothelium can be assessed with this model of vascular injury. The azo dye Evans Blue cannot permeate the normal, intact endothelium of the murine aorta. Damage to the barrier function of the endothelium allows staining of the basement membrane with Evans Blue. Mice treated to a laparotomy alone (sham) maintained the barrier function of the endothelium and these aortas were white. Crush injury resulted in a diffuse loss of endothelial barrier function and these aortas stained dark blue. The intensity of staining with Evans Blue was decreased three days after crush injury, and full barrier function was restored on week after injury (Figure 6A). Endothelial barrier function can be further quantitated by weighing the samples and homogenizing in 10-fold volume of 50% trichloroacetic acid solution. Diluting the supernatant in 4-fold ethanol allows for determination of fluorescence intensity (excitation 620 nm and emission 680 nm) as described by Aoki et al.11. The amount of Evans Blue eluted from the infrarenal aorta was over three-fold higher in aortas immediately after injury compared to aortas subjected only to laparotomy (Injured - Day 0). The aorta continued to stain with Evans Blue at one and three days after injury, but less intensely than at the time of injury, suggesting a gradual recovery of endothelial barrier function. One week after injury the injured aorta had equivalent staining as an aorta treated with laparotomy alone (Figure 6B).
Figure 1: Injury is Created by Bluntly Crushing the Aorta from the Left Renal Vein to the Aortic Bifurcation. Figure adapted with permission from Yu et al., PLoS One 2015 12. Please click here to view a larger version of this figure.
Figure 2: Crush Injury Induces Aortic Wall Thickening. (A) Crush injury induced morphological changes in the aorta. Representative 5 µm cross sections were stained with VVG at 0, 1, 3 and 7 days after injury. Scale bars = 100 µm. (B) Wall thickness was greatest 3 days after injury, 42.2 ± 1.7 µm, and was significantly greater than the aorta of sham animals exposed to laparotomy alone, 22.1 ± 1.1 µm. Statistical significance was determined with the Student's two-tail t-test. * Denotes p <0.05, n = 3. (C) Crush injury induced a rounded appearance of medial VSMCs (stained in green for α-smooth muscle actin) and changing cell orientation. Nuclei were counterstained with DAPI (Blue). Scale bars = 10 µm. Data are presented as means and standard deviations. Please click here to view a larger version of this figure.
Figure 3: Crush Injury Induces Cell Proliferation in the Aortic Wall. Mice were exposed to either laparotomy alone (sham) or laparotomy with aortic crush injury. Thymidine analogue 5-ethynyl-2'-deoxyuridine (EdU) was administered 24 h prior to euthanasia. EdU is incorporated into DNA during synthesis. EdU is detected by a click reaction (copper-catalyzed azide-alkyne cycloaddition using green-fluorescent dye). There was no detectable EdU (green) in the aortic wall of the sham animals. In the animals subjected to crush injury, EdU stained prominently in the media of the aorta and to a somewhat lesser degree in the intima. Vascular smooth muscle cells were identified by staining with α-smooth muscle actin antibody (red). This finding suggests that the observed wall thickening observed in Figure 2 is due to an increase in cell proliferation. Scale bars = 10 µm. Please click here to view a larger version of this figure.
Figure 4: The Mitogen-activated Protein Kinase (MAP Kinase) Pathway is Activated in Response to Crush Injury. (A) The MAP Kinase ERK1/2 is phosphorylated when activated and is associated with intimal hyperplasia and restenosis. Mice were euthanized at 1, 3, 7 days, and 1 month after aortic crush injury. Western blot analysis was used to assess protein expression of total ERK1/2, phospho-ERK1/2, and the cyclin dependent kinase inhibitor p27kip1. Protein expression was normalized to a sham animal treated with laparotomy alone. (B) No change was observed in total ERK1/2 protein expression over time. However, three days after injury, phospho-ERK1/2 (Pi-ERK1/2) expression was over 200% greater than baseline Pi-ERK1/2 expression. p27kip1 expression decreased at early time points and reached a nadir of approximately 20% of baseline seven days after injury. Both Pi-ERK1/2 and p27kip1 expression approximated baseline levels one month after injury. Please click here to view a larger version of this figure.
Figure 5. Crush Injury Disrupts Aortic Endothelial Cells. (A) Scanning electron microscopy (SEM) was used to characterize the effect of the crush injury on the morphology of the endothelium. The luminal surface of the normal aorta is lined by a confluent, endothelium that prevents adhesion of blood born cells and molecules. (B) Crush injury results in the disruption of endothelial cells and partial denudation of the endothelium. (C) As seen at higher magnification, disruption of the endothelium results in adhesion of particles consistent in size and shape with platelets. Adhesion of these particles occurs throughout the entire zone of injury, and is not limited to areas of gross denudation observed at lower magnification. (D) At higher magnification, these particles are consistent with platelets and fibrin. Please click here to view a larger version of this figure.
Figure 6: The Blunt Aortic Injury Allows Quantification of Endothelial Barrier Function. (A) Damage to the barrier function of the endothelium allows staining of the basement membrane with Evans Blue dye. Mice treated to a laparotomy alone (sham) maintained the barrier function of the endothelium and these aortas were white. Crush injury resulted in a diffuse loss of endothelial barrier function and these aortas stained dark blue. The intensity of staining with Evans Blue was decreased three days after crush injury, and full barrier function was restored on week after injury. (B) Binding of Evans Blue to the infrarenal aorta was quantitated by eluting Evans Blue from the specimen and quantification by fluorescence intensity. Data are presented as means and standard deviations. The most Evans Blue was eluted from the aorta immediately after crush injury. The at the time of injury, the aorta bound over three-fold times more Evans Blue than the aorta subjected to laparotomy alone. As the aorta was allowed to recover, there was less binding at one and three days after injury. One week after injury, the aorta bound the same amount of Evans Blue as the aortas subjected to laparotomy alone. Statistical significance was determined with the Student's two-tail t-test. * denotes p <0.05, n = 3, NS denotes not significant. Please click here to view a larger version of this figure.
Subscription Required. Please recommend JoVE to your librarian.
We have characterized the effects of a murine aortic injury model that results in medial hyperplasia and endothelial barrier dysfunction. Partial EC detachment along the aorta intima accompanied the loss of cell-cell contact and enhancement of cell protrusions. Correspondingly, endothelial barrier function was significantly impaired, which stimulated the mitogen-sensitive signaling pathways, leading to proliferation of VSMCs and thickening of the vessel wall. The strengths of this model is that it is technically easier to learn and execute than other aortic models of disease and allows for simultaneous assessment of both the proliferative response to injury and endothelial function. The critical step in this protocol is the actual crush injury. Variability in the extent of the crush injury could result in varying degrees of injury and thus variable amounts smooth muscle cell proliferation. One step that reduces this variability in our laboratory is to have one scientist, blinded to the treatment or experimental condition, perform all of the crush injuries.
A limitation of this model is its generalizability to clinically relevant human disease processes. Despite the early robust proliferative response of VSMCs in this model, it does not model the late events of restenosis, i.e. no neointima developed. Decreased inflammatory mediators such as cytokines might regulate this process. This finding could be due to the rapid recovery of the endothelium from a partial injury. Similar to a previously reported vascular crush injury model in rabbits 13, infiltration of immune cells was minor. In the present investigation we evaluated profile of inflammatory cytokines the sham animals and aortic injury animals with the profiler array. However, there was no significant difference in the cytokine profile that would explain the difference in the proliferative response between sham and injured animals (data not shown). The injury described in the present report is temporally limited. Whereas other models of aortic injury and intimal hyperplasia such as the aortic angioplasty model completely denudes the endothelium, the present model only partially denudes the aorta. Complete denudation of the endothelium requires significantly longer to recover and these animals experience the effects of injury for a longer period of time.
This model produces a rapid response of VSMCs and ECs to injury allowing quantification of morphological and biochemical changes in one week. It is more efficient than other models that take 14-28 days before the effect of intervention is evident. This model uses the murine aorta which is the only murine vessel that approximates a clinically relevant human vessel. The significant advantage of this technique compared to other aortic models is that this model is relatively easy to learn, and does not require expensive or difficult to obtain equipment 2,3,4,5. In conclusion, the murine aortic crush injury model provides a technically straightforward, inexpensive, efficient in vivo platform to simultaneously evaluate the response of VSMCs and ECs to vascular injury.
Subscription Required. Please recommend JoVE to your librarian.
This work was funded by the Department of Veterans Affairs Career Development Award (1IK2BX001553-01) (TSM) and the Vascular Cures E. J. Wylie Scholarship (TSM).
We thank Hsia Ru-ching PhD, from the Electron Microscopy Core Facility of University of Maryland School of Medicine, for her technical support in processing the scanning electronic microscopy samples.
|Catalog Number
|Ocular lubricant
|Pharmaceutical agents
|Pharmaceutical agents
|Pharmaceutical agents
|Precision vaporizer
|Summit Medical
|Surgical supplies
|Charcoal scavenger
|Bickford Inc.
|Surgical supplies
|Isothermal pad
|Harvard Apparatus
|Surgical supplies
|Sterile cotton-tipped applicator
|Fisher Scientific
|Surgical supplies
|4-0 absorbable monofilament suture
|Ethicon, Inc
|Surgical supplies
|5-0 non-absorbable monofilament suture
|Surgical supplies
|21-gauge x 1 inch needle
|BD Biosciences
|Surgical supplies
|25-gauge x 1 inch needle
|BD Biosciences
|Surgical supplies
|Dry sterilizer
|Surgical supplies
|Fine scissors
|Fine Science Tools
|Surgical instruments
|Adson forceps
|Fine Science Tools
|Surgical instruments
|Dumont #5 fine forceps
|Fine Science Tools
|Surgical instruments
|Vannas Spring Scissors 3 mm cutting edge
|Fine Science Tools
|Surgical instruments
|Needle driver
|Fine Science Tools
|Surgical instruments
|Scalpel handle #4
|Fine Science Tools
|Surgical instruments
|Scalpel blades #10
|Fine Science Tools
|Surgical instruments
|Reagents for tissue processing
|Evans Blue
|Reagents for tissue processing
|Reagents for tissue processing
|Modeling wax
|Reagents for tissue processing
|OCT compound
|Tissue-Tek Sakura
|Reagents for tissue processing
|Mayer's hematoxylin solution
|Reagents for immunohistological analysis
|Eosin Y solution alcoholic
|Reagents for immunohistological analysis
|Elastin stain kit
|Reagents for immunohistological analysis
|Click-it Edu Alexa-488 Imaging Kit
|Reagents for immunohistological analysis
|Anti-Erk1/2 antibody
|Cell Signaling Technology
|Reagents for immunohistological analysis
|Anti-phospho-Erk1/2 antibody
|Cell Signaling Technology
|Reagents for immunohistological analysis
|Anti-p27kip1 antibody
|Cell Signaling Technology
|Reagents for immunohistological analysis
|Trichloroacetic acid
|Reagents for immunohistological analysis
- Carmeliet, P. Mechanisms of angiogenesis and arteriogenesis. Nat Med. 6, (4), 389-395 (2000).
- Carmeliet, P., Moons, L., Collen, D. Mouse models of angiogenesis, arterial stenosis, atherosclerosis and hemostasis. Cardiovasc Res. 39, (1), 8-33 (1998).
- Baker, A. B., et al. Heparanase Alters Arterial Structure, Mechanics, and Repair Following Endovascular Stenting in Mice. Circ Res. 104, (3), 380-387 (2009).
- Petrov, L., Laurila, H., Hayry, P., Vamvakopoulos, J. E. A mouse model of aortic angioplasty for genomic studies of neointimal hyperplasia. J Vasc Res. 42, (4), 292-300 (2005).
- Li, J., et al. Vascular smooth muscle cells of recipient origin mediate intimal expansion after aortic allotransplantation in mice. Am J Path. 158, (6), 1943-1947 (2001).
- Radu, M., Chernoff, J. An in vivo assay to test blood vessel permeability. J Vis Exp. (73), e50062 (2013).
- Turbett, G. R., Sellner, L. N. The use of optimal cutting temperature compound can inhibit amplification by polymerase chain reaction. Diagn Mol Pathol. 6, (5), 298-303 (1997).
- Puchtler, H., Waldrop, F. S. On the mechanism of Verhoeff's elastica stain: a convenient stain for myelin sheaths. Histochem. 62, (3), 233-247 (1979).
- Salic, A., Mitchison, T. J. A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc Natl Acad Sci U S A. 105, (7), 2415-2420 (2008).
- Nelson, P. R., Yamamura, S., Mureebe, L., Itoh, H., Kent, K. C. Smooth muscle cell migration and proliferation are mediated by distinct phases of activation of the intracellular messenger mitogen-activated protein kinase. J Vasc Surg. 27, (1), 117-125 (1998).
- Rzucidlo, E. M. Signaling pathways regulating vascular smooth muscle cell differentiation. Vascular. 17, Suppl 1. S15-S20 (2009).
- Aoki, T., Sumii, T., Mori, T., Wang, X., Lo, E. H. Blood-brain barrier disruption and matrix metalloproteinase-9 expression during reperfusion injury: mechanical versus embolic focal ischemia in spontaneously hypertensive rats. Stroke. 33, (11), 2711-2717 (2002).
- Yu, D., et al. MARCKS Signaling Differentially Regulates Vascular Smooth Muscle and Endothelial Cell Proliferation through a KIS-, p27kip1- Dependent Mechanism. PLoS One. 10, (11), e0141397 (2015).
- Banai, S., et al. Rabbit ear model of injury-induced arterial smooth-muscle cell-proliferation - kinetics, reproducibility, and implications. Circ Res. 69, (3), 748-756 (1991).
0 Comments
Post a Question / Comment / Request
You must be signed in to post a comment. Please sign in or create an account.