3 Mindful Steps To Better Decision-Making – Forbes Link @ Time / Country / Life / Lives / Health / Person / People / City / Lives / Cities / Longevity / Age / Economy / Actions / Internet Images @ TOP 10 BENEFITS OF USING ARTIFICIAL INTELLIGENCE FOR YOUR BUSINESS @ Google researchers have reportedly achieved “quantum supremacy” & Brain-computer interface: huge potential benefits and formidable challenges & The heterotopic heart transplantation in mice as a small animal model to study mechanical unloading – Establishment of the procedure, perioperative management and postoperative scoring

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  • – > Mestrado – Dissertation – Tabelas, Figuras e Gráficos – Tables, Figures and Graphics – ´´My´´ Dissertation @ #Innovation #energy #life #health #Countries #Time #Researches #Reference #Graphics #Ages #Age #Mice #People #Person #Mouse #Genetics #PersonalizedMedicine #Diagnosis #Prognosis #Treatment #Disease #UnknownDiseases #Future #VeryEfficientDrugs #VeryEfficientVaccines #VeryEfficientTherapeuticalSubstances #Tests #Laboratories #Investments #Details #HumanLongevity #DNA #Cell #Memory #Physiology #Nanomedicine #Nanotechnology #Biochemistry #NewMedicalDevices #GeneticEngineering #Internet #History #Science #World

Pathol Res Pract. 2012 Jul 15;208(7):377-81. doi: 10.1016/j.prp.2012.04.006. Epub 2012 Jun 8.

The influence of physical activity in the progression of experimental lung cancer in mice

Renato Batista Paceli 1Rodrigo Nunes CalCarlos Henrique Ferreira dos SantosJosé Antonio CordeiroCassiano Merussi NeivaKazuo Kawano NagaminePatrícia Maluf Cury


Impact_Fator-wise_Top100Science_Journals

GRUPO_AF1 – GROUP AFA1 – Aerobic Physical Activity – Atividade Física Aeróbia – ´´My´´ Dissertation – Faculty of Medicine of Sao Jose do Rio Preto

GRUPO AFAN 1 – GROUP AFAN1 – Anaerobic Physical Activity – Atividade Física Anaeróbia – ´´My´´ Dissertation – Faculty of Medicine of Sao Jose do Rio Preto

GRUPO_AF2 – GROUP AFA2 – Aerobic Physical Activity – Atividade Física Aeróbia – ´´My´´ Dissertation – Faculty of Medicine of Sao Jose do Rio Preto

GRUPO AFAN 2 – GROUP AFAN 2 – Anaerobic Physical Activity – Atividade Física Anaeróbia – ´´My´´ Dissertation – Faculty of Medicine of Sao Jose do Rio Preto

Slides – mestrado – ´´My´´ Dissertation – Faculty of Medicine of Sao Jose do Rio Preto

CARCINÓGENO DMBA EM MODELOS EXPERIMENTAIS

DMBA CARCINOGEN IN EXPERIMENTAL MODELS

Avaliação da influência da atividade física aeróbia e anaeróbia na progressão do câncer de pulmão experimental – Summary – Resumo – ´´My´´ Dissertation – Faculty of Medicine of Sao Jose do Rio Preto

https://pubmed.ncbi.nlm.nih.gov/22683274/

Abstract

Lung cancer is one of the most incident neoplasms in the world, representing the main cause of mortality for cancer. Many epidemiologic studies have suggested that physical activity may reduce the risk of lung cancer, other works evaluate the effectiveness of the use of the physical activity in the suppression, remission and reduction of the recurrence of tumors. The aim of this study was to evaluate the effects of aerobic and anaerobic physical activity in the development and the progression of lung cancer. Lung tumors were induced with a dose of 3mg of urethane/kg, in 67 male Balb – C type mice, divided in three groups: group 1_24 mice treated with urethane and without physical activity; group 2_25 mice with urethane and subjected to aerobic swimming free exercise; group 3_18 mice with urethane, subjected to anaerobic swimming exercise with gradual loading 5-20% of body weight. All the animals were sacrificed after 20 weeks, and lung lesions were analyzed. The median number of lesions (nodules and hyperplasia) was 3.0 for group 1, 2.0 for group 2 and 1.5-3 (p=0.052). When comparing only the presence or absence of lesion, there was a decrease in the number of lesions in group 3 as compared with group 1 (p=0.03) but not in relation to group 2. There were no metastases or other changes in other organs. The anaerobic physical activity, but not aerobic, diminishes the incidence of experimental lung tumors.

https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0214513

https://www.news-medical.net/news/20190911/Brain-computer-interface-huge-potential-benefits-and-formidable-challenges.aspx?showform=printpdf

https://www.technologyreview.com/f/614416/google-researchers-have-reportedly-achieved-quantum-supremacy/?fbclid=IwAR2Ksu1K5fP_EVWphR4rzzqm9Jn8VFsNrAlWBV11VjvtRVs1w4egMJJbuWw

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https://infotech.report/blogs/top-10-benefits-of-using-artificial-intelligence-for-your-business/11860?fbclid=IwAR2wPxCjsj6YtmJoqgLLB9EV5E1jtWZ3eNEVkwe8o8VJvUsNh8NivV3Ljq4national

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3 Mindful Steps To Better Decision-Making

Janice Marturano

Janice MarturanoContributor Careers

Learning to make good decisions is helped by mindful leadership training.
Some decisions are simple but some can change our life, and the lives of others.GETTY

Every day we are asked to make decisions. Some are of little consequence while others can literally change our lives and the lives of others. When those important questions arise, we can find it difficult to choose. We might feel paralyzed by an overload of input from others, or we might feel as though there is no clear “right.” So, are there ways a mindful leadership practice can help? Let’s look at 3 Steps to Better Decision-Making:  

Stop And Unplug 

In a time when we are constantly tempted to divide our attention, it is important to cultivate your ability to focus your mind on the question to be decided. Good decision-making needs us to quiet our busy mind and body so we can open to all the ways of knowing available to us. Removing the external distractions is a good way to start. Turn off the technology and find a quiet place to focus on the sensations of your breath for a few moments. When your mind becomes distracted, redirect it back to your breath. Feel yourself -mind and body-settling into the moment. I described more about this practice to strengthen your focus in my Forbes blog entitled “Are you Living on Autopilot?

Define The Question 

It may not be what you think. One way of defining the question is to begin by calling to mind the issue or situation, and asking a more general question first: “what is called for now?” In other words, step back from the specific question to one that is a little broader or more general. More than a few of the clients I work with have said that this reflection often lets them see that the reason an answer couldn’t be found was because they had the wrong question.

Don’t be in too much of a hurry to get to the precise answer to a narrower question. The smaller answer may be just that…small, rather than creative or breakthrough or compassionate. 

Reflect

Once you begin to feel your body and mind settle into the present moment and you have defined the question, it is time for the final step-reflection. This is not analysis, or even thinking. It is approaching the question with open curiosity. Allow there to be some spaciousness around the question so the answer or answers can arise, generated by your inner wisdom. No need to go searching, the answer will come to you. This decision-making reflection is also an opportunity for you to practice patience. Sometimes it may take a few dedicated reflections with your question to discover the answer so don’t try to push to a conclusion in your first reflection. You already have everything you need to make those important decisions and the more you practice with this approach, the more confidence you will gain in your capacity to choose.Follow me on Twitter or LinkedIn. Check out my website orsome of my other work here.

Janice Marturano

Janice Marturano

I am the Founder and Executive Director of the Institute for Mindful Leadership, a nonprofit organization dedicated to offering leaders an in-depth exploration of mindf… Read More

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NBA All-Star Kevin Durant And His Thirty Five Ventures Partner Rich Kleiman To Join Speaker Lineup At Forbes Under 30 Summit In Detroit, Presented By Rocket Mortgage By Quicken Loans

Forbes Press Releases

Forbes Press ReleasesForbes StaffLeadership

NBA All-Star Kevin Durant And His Thirty Five Ventures Partner Rich Kleiman
FORBES

DETROIT – August 22, 2019 – NBA All-Star Kevin Durant and Rich Kleiman, his cofounder and partner of Thirty Five Ventures, will join the speaker lineup for the 2019 Forbes Under 30 Summit, Oct. 27 – 30 in Detroit, presented by Rocket Mortgage by Quicken Loans.

The two-time NBA Champion, Finals MVP and ten-time All-Star, Durant founded Thirty Five Ventures in 2017 with sports/entertainment executive Rich Kleiman. The duo will share with other entrepreneurs and innovators their insights, lessons-learned and first-hand experiences on and off the court.

“Our Under 30 Summit is a once-in-a-lifetime opportunity for the next-generation of leaders to connect with visionaries like Kevin and Rich, who have established track records in starting companies, investing in emerging brands and in disrupting existing markets,” said Randall Lane, Chief Content Officer of Forbes. “Kevin and Rich join an all-star lineup that includes other founders such as Palmer LuckeySerena Williamsand Jon Oringer whose real-world lessons are templates for everyone embarking on something new or looking to up their game.”

Durant earlier this year landed in the No. 10 spot on Forbes’ 2019 Highest-Paid Athletes list, with $65.4 million in earnings.

Thirty Five Ventures is the umbrella for Durant’s and Kleiman’s business interests in sports, tech and culture and also includes Durant’s personal brand/marketing and on-court contracts; a diverse investment portfolio of companies such as Acorns, Coinbase, Overtime and Postmates; a media and creative development arm with current projects including the ESPN the sports business vertical “The Boardroom,” in partnership with ESPN and the scripted series “SWAGGER” with Imagine Entertainment and Apple.

Now in its sixth year, the Forbes signature Under 30 Summit, presented by Rocket Mortgage by Quicken Loans, brings together the world’s most successful entrepreneurs and young leaders representing more than 70 countries across various industries including tech, finance, science, public policy, media and more. Attendees will have the opportunity to network, collaborate, recruit and exchange ideas with likeminded game changers who are disrupting industries and challenging the status quo. The summit will kick off with our music festival featuring The Chainsmokers and 21 Savage, with special guest Normani.

To learn more, please visit here. To join the conversation on social, follow #Under30Summit.

Rocket Mortgage by Quicken Loans is the sponsor of the 2019 Forbes Under 30 Summit. Courtyard by Marriott is the official hotel and a presenting sponsor. The Macallan Single Malt Scotch Whisky is a presenting sponsor. Ally Financial and the official timepiece brand, IWC Schaffhausen, are partner sponsors. Tequila Herradura is a supporting sponsor.

About Forbes

The defining voice of entrepreneurial capitalism, Forbes champions success by celebrating those who have made it, and those who aspire to make it. Forbes convenes and curates the most-influential leaders and entrepreneurs who are driving change, transforming business and making a significant impact on the world. The Forbes brand today reaches more than 120 million people worldwide through its trusted journalism, signature LIVE events, custom marketing programs and 40 licensed local editions in 70 countries. Forbes Media’s brand extensions include real estate, education and financial services license agreements. For more information, visit:ForbesMedia.com.

About Quicken Loans / Rocket Mortgage

Detroit-based Quicken Loans Inc. is the nation’s largest home mortgage lender. The company closed nearly half a trillion dollars of mortgage volume across all 50 states from 2013 through 2018. In late 2015 Quicken Loans introduced Rocket Mortgage, the first fully digital mortgage experience. Today, 98% of all home loans originated by Quicken Loans utilize Rocket Mortgage Technology.

Quicken Loans moved its headquarters to downtown Detroit in 2010. Today, Quicken Loans and its Family of Companies employ more than 17,000 full-time team members in Detroit’s urban core. The company generates loan production from web centers located in Detroit, Cleveland and Phoenix. Quicken Loans also operates a centralized loan processing facility in Detroit, as well as its San Diego-based One Reverse Mortgage unit. Quicken Loans ranked highest in the country for customer satisfaction for primary mortgage origination by J.D. Power for the past nine consecutive years, 2010 – 2018, and also ranked highest in the country for customer satisfaction among all mortgage servicers the past six consecutive years, 2014 – 2019.

Quicken Loans was once again named to FORTUNE magazine’s “100 Best Companies to Work For” list in 2019 and has been included in the magazine’s top 1/3rd of companies named to the list for the past 16 consecutive years. In addition, Essence Magazine named Quicken Loans “#1 Place to Work in the Country for African Americans.”

Contacts

Forbes:

Matthew Hutchison mhutchison@forbes.com 

Christina Vega cvega@forbes.com

Laura Brusca lbrusca@forbes.comForbes MediaFORBESSend me a secure tip.

Forbes Press Releases

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Everything and anything you need to know about Forbes, the global media brand that celebrates entrepreneurial capitalism. In this section, you’ll find the latest Forbes… Read More

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The August Smart Lock Pro Is One Of The Best Smart Locks, And It’s On Sale Now

Forbes Finds

Christian de Looper ContributorForbes FindsContributor Group Forbes FindsI write about Tech for Forbes Finds.

Forbes Finds covers products we think you’ll love. Featured products are independently selected and linked to for your convenience. If you buy something using a link on this page, Forbes may receive a small share of that sale.

For Labor Day 2019, you should expect some pretty sweet smart home deals, but a week before Labor Day the pre-Labor Day deals are already rolling in. August is one of the companies that have been leading the charge on smart security, thanks to its Smart Lock series — and you can now get its best smart lock ever at a pretty impressive discount.

The August Smart Lock Pro is one of the best smart locks out there for a number of reasons. Notably, the device works with your existing deadbolt — so you won’t need to get new keys, or replace your entire deadbolt, to install it. That’s good news for those that rent or with big families who don’t. want to replace their keys, as those old keys will continue to work.

August Smart Lock Pro

August Smart Lock Pro AUGUST

This particular deal also includes the August Connect, which is an important piece of the pie for those building a smart home. The Connect essentially allows you to control your lock remotely, through Wi-Fi — instead of the Bluetooth connectivity you’ll get without the Connect. Bluetooth only works within around 35 feet. The remote control can make the lock a whole lot more helpful — it means that if you’re on holiday or work and need to let someone in, you can do so quickly and easily.

The August Connect also allows the lock to interact with other smart home devices, through ecosystems like Apple HomeKit, Google Assistant, and Amazon Alexa. That’s a handy feature — and with it you’ll be able to automate control based on other devices, and control the lock straight from the Apple Home app or using Google Assistant or Alexa.

August Smart Lock Pro + Connect AMAZON

The August Smart Lock Pro is well-designed and easy to install — and the app walks you through the installation process, so you should be able to install it yourself. It’s available for $162, which is a pretty massive discount on the normal price of $280 — so if you’re in the market for a new lock, it’s worth acting quick.

Shop Now: $162

Here are more pre-Labor Day deals for your smart home:

August Smart Lock 

 AMAZONAugust Smart Lock

The August Smart Lock Pro isn’t the only August lock on sale. The standard August Smart Lock is on sale too — though it does not come with the August Connect. Because of that, it’s a good option for those that really only want to control their home from nearby, or those that don’t need their lock to integrate with other smart home ecosystems. The standard Smart Lock is available for a super affordable $77 — which is almost half off the original price of $150.

Shop Now: $77

Google Home – Smart Speaker & Google Assistant

 AMAZONGoogle Home – Smart Speaker & Google Assistant

Looking for a great smart speaker to control your smart lock with? The Google Home is on sale too — and it’s available for $79 from Amazon. The speaker comes with Google Assistant, and considering the fact that it normally comes at $129, it’s well-worth considering, especially for those that use Android phones and other Google devices.

Shop Now

Christian de Looper

Christian de Looper

Born and raised in Canberra, Australia, I lived in France and Minnesota before eventually landing in sunny California. I’ve written for a range of online publications, i… Read More

Forbes Finds

Forbes Finds

Forbes Finds is a shopping service for our readers. Forbes searches premium retailers to find the new products — from clothes to gadgets — and the latest deals. Forbes F… Read More

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Andrew Luck Walking Away From Football And $500 Million Should Be A Wake-Up Call For Your Own Career

Jack Kelly

Jack KellySenior Contributor CareersI write real and actionable interviewing, career and salary advice.

Indianapolis Colts quarterback Andrew Luck (AP Photo/Eric Christian Smith)

In a stunning postgame press conference this weekend, 30-year-old Indianapolis Colts quarterback Andrew Luck officially announced his early retirement from the NFL.  

Luck, formerly one of the highest-paid NFL players, was “mentally worn down” and constant injuries have deteriorated his love for the game. He suffered torn cartilage in two ribs, a torn abdomen, a lacerated kidney, a concussion and a torn labrum. This pre-season, Luck missed parts of training camp due to an ankle injury.

Jim Irsay, the owner of the Colts football team, claimed that Luck earned almost $100 million over his seven-year career and could be walking away from about $500 million.   

In what seemed like an impromptu news conference, Luck said, “I haven’t been able to live the life I want to live. It’s taken the joy out of this game…the only way forward for me is to remove myself from football. He emotionally added, “This is not an easy decision. It’s the hardest decision of my life. But it is the right decision for me.”

This is a cautionary tale for all of us. In our parents’ and grandparents’ generations, they found jobs, trades and professions and stuck with them for their entire working lives. You put in your time, were loyal to your company and after 30 years, you retired with a gold watch and a decent pension.  

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We don’t have that luxury any longer. This is a new era of dramatic change. Automation, artificial intelligence, globalization, nearshoring and offshoring jobs, the gig economy and other factors strip away any sense of permanence in a job or career. There are no guarantees that the job you hold now will even exist in 10 or 20 years. The company you work for may be taken over, their products rendered obsolete or the jobs have moved to other countries. There is no loyalty to their employees—on the part of the companies—any longer. If you are of value today, they love you. If you don’t produce to their satisfaction or management can find someone cheaper who will work longer hours, they’ll let you go in a heartbeat without any regrets. 

It’s a free-agency economy for everyone in the corporate world. You alone are in control of your own destiny. No boss, manager or mentor will save you. You can’t solely rely on a company for a safe and secure future. This sounds cold and harsh, but it’s not. There is a zen calmness when you come to terms with the fact that you’re the quarterback of your own life. It is all up to you—fail or succeed. 

You need to prepare every day for being called into the human resources office and given a pink slip. However, you can also prepare every day to lead your best, most rewarding life on your own terms. Think of what you really want to do and achieve in your career and go for it. Don’t let anything hold you back. Give it your all. Some people deride Millennials, but there is validity in their ethos to move on if they’re not appreciated. 

It must have been tough on Luck to walk away from something he worked so hard to achieve—and the money. Yes, I know you will say that he has millions in the bank, so nobody is going to cry for him. However, that doesn’t mean that he isn’t a human being and subject to the same emotions as us. I don’t know if he has a back-up plan, but most people who aren’t millionaires require one. You need to always think about what’s next. Have a few plans in the works, so that if you cannot continue with your current job, are sick and tired of your career or are fired, you don’t have to scramble and start from a dead stop. 

A big part of Luck’s decision had to do with his mental well-being. He acknowledged that his mindset had changed and he no longer possessed the same drive, passion and enthusiasm for the game as he once had—due the constant injuries. It’s the same for all of us. We don’t like to discuss it, but sometimes we’re just mentally and emotionally exhausted in our careers. It wears us down, ruins personal relationships and makes us unhappy. Instead of ignoring this, you should find a job that is better suited for you—mentally and emotionally—or at least take a break to get yourself together, decompress and heal.

We may not be subject to the painful blows that a quarterback receives, but we do get our fair share of punishment in the corporate world—lack of recognition, failure to receive a much-earned promotion or raise, lack of mobility up the corporate ladder, discrimination and prejudices, getting laid off or just stuck in your career. Like Luck, you will need to know when to walk away and start something new and different. Have the confidence to follow your convictions and do what’s right for yourself, even if the fans in the cheap seats boo you.

Jack Kelly

Jack Kelly

I am a CEO, founder, and executive recruiter at one of the oldest and largest global search firms in my area of expertise, and have personally placed thousands of profes… Read More

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Landis, A Stanford Real Estate Startup, Raises $15M To Put Americans On The Path To Homeownership

Frederick Daso

Frederick DasoContributor Under 30I write about college students and recent graduates founding startups.

Frederick Daso

Frederick Daso

I write extensively on college students’ triumphs and failures in their journeys in entrepreneurship. I graduated from MIT with my Bachelor’s and Master’s in Aerospace E… Read More

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MIT Technology Review

Computing2h

Google researchers have reportedly achieved “quantum supremacy”

The news: According to a report in the Financial Times, a team of researchers from Google led by John Martinis have demonstrated quantum supremacy for the first time. This is the point at which a quantum computer is shown to be capable of performing a task that’s beyond the reach of even the most powerful conventional supercomputer. The claim appeared in a paper that was posted on a NASA website, but the publication was then taken down. Google did not respond to a request for comment from MIT Technology Review.

Why NASA? Google struck an agreement earlier this year to use supercomputers available to NASA as benchmarks for its supremacy experiments. According to the Financial Times report, the paper said that Google’s quantum processor was able to perform a calculation in three minutes and 20 seconds that would take today’s most advanced supercomputer, known as Summit, around 10,000 years. In the paper, the researchers said that, to their knowledge, the experiment “marks the first computation that can only be performed on a quantum processor.”

Quantum speed up: Quantum machines are so powerful because they harness quantum bits, or qubits. Unlike classical bits, which are either a 1 or a 0, qubits can be in a kind of combination of both at the same time. Thanks to other quantum phenomena, which are described in our explainer here, quantum computers can crunch large amounts of data in parallel that conventional machines have to work through sequentially. Scientists have been working for years to demonstrate that the machines can definitively outperform conventional ones.

How significant is this milestone? Very. In a discussion of quantum computing at MIT Technology Review’s EmTech conference in Cambridge, Massachusetts this week before news of Google’s paper came out, Will Oliver, an MIT professor and quantum specialist, likened the computing milestone to the first flight of the Kitty Hawk in aviation. He said it would give added impetus to research in the field, which should help quantum machines achieve their promise more quickly. Their immense processing power could ultimately help researchers and companies discover new drugs and materials, create more efficient supply chains, and turbocharge AI.

But, but: It’s not clear what task Google’s quantum machine was working on, but it’s likely to be a very narrow one. In an emailed comment to MIT Technology Review, Dario Gil of IBM, which is also working on quantum computers, says an experiment that was probably designed around a very narrow quantum sampling problem doesn’t mean the machines will rule the roost. “In fact quantum computers will never reign ‘supreme’ over classical ones,” says Gil, “but will work in concert with them, since each have their specific strengths.” For many problems, classical computers will remain the best tool to use.

And another but: Quantum computers are still a long way from being ready for mainstream use. The machines are notoriously prone to errors, because even the slightest change in temperature or tiny vibration can destroy the delicate state of qubits. Researchers are working on machines that will be easier to build, manage, and scale, and some computers are now available via the computing cloud. But it could still be many years before quantum computers that can tackle a wide range of problems are widely available.ShareLinkAuthor

Martin GilesImageGoogle | Erik Lukero

Quantum supremacy

Google researchers have reportedly achieved “quantum supremacy”01.Google thinks it’s close to “quantum supremacy.” Here’s what that really means.May 201802.Google has enlisted NASA to help it prove quantum supremacy within monthsNovember 2018

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Brain-computer interface: huge potential benefits and formidable challenges

Dr. Liji Thomas, MD

By Dr. Liji Thomas, MDSep 11 2019

A new Royal Society report called “iHuman: blurring lines between mind and machine” is for the first time systematically exploring whether it is “right” or not to use neural interfaces – machines implanted in or worn over the body to pick up or stimulate nervous activity in the brain or other parts of the nervous system. It also sets out recommendations to ensure the ethical risks are understood, and to set up a transparent, public-driven but flexible regulatory framework which will allow the UK to lead innovative technology in this field.

Neural interfaces, brain-computer interfaces and other devices that blur the lines between mind and machine have extraordinary potential. Image Credit: Iaremenko Sergii / Shutterstock

Neural interfaces, brain-computer interfaces and other devices that blur the lines between mind and machine have extraordinary potential. Image Credit: Iaremenko Sergii / Shutterstock

The thrust of the report is on making sure the government understands the ethics of neural interface technology, its large benefits in hitherto hopeless conditions, and the need to guarantee that, as co-chair Tim Constandinou says, “these emerging technologies are implemented safely and for the benefit of humanity.” The reason for this detailed report is that, willy-nilly, the technology is going to be put in place, if not safely, then in an unregulated and dangerous manner. As Constandinou continues to say, “In 10 years’ time this is probably going to touch millions of people.”

Neural implants already being used

At least 400 000 cochlear implants have already been put in place in deaf children, to bypass the auditory apparatus and allow the person to hear sounds from outside simply by converting them into electrical signals that directly reach the brain. Deep brain stimulation in conditions like Parkinson’s disease is also an established modality of treatment, suppressing tremors and stiffness by targeted stimulation of dopaminergic neurons in specific areas of the brain. Even more excitingly, there are artificial arms and legs that move in response to the patient’s thought.

By 2040, says the report, conditions like Alzheimer’s disease will probably be treated using a BCI.

Elon Musk is already working on Neuralink, a BCI initiative that will allow paralyzed people to use computers to communicate using their thoughts alone. This could improve the quality of life for people with locked-in syndrome, for instance, where the brain is normal but is totally cut off from the rest of the body. However, Musk has plans that are far in advance of helping people to just replace something they have lost. He foresees that artificial intelligence (AI) could advance so rapidly and so much that it forces humans to become subsidiary, something like a house pet. Installing an AI layer would be a good way to stay in step with AI instead, he says, and the “neural lace” interface his company is producing is an initiative designed to do just that. He plans to begin clinical trials of these neural threads next year.

Facebook is also actively working on a 100-words-per-minute brain-typing interface that will replace manual typing one day.

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However, scientists in this pioneering field make it clear that we have not even scratched the surface of the potential applications of brain-computer interfaces (BCI), which could not only help solve medical issues like dementia, epilepsy, untreatable depression, and obesity, but could help people communicate without sound and even without words. We could share sensory experiences with others far away, as by sending “neural postcards”, letting others visually experience their trip or “taste” the food they are eating, by sharing the brain’s neural activity

The report speaks of previously unimaginable levels of thought sharing: “People could become telepathic to some degree, able to converse not only without speaking but without words – through access to each other’s thoughts at a conceptual level. This could enable unprecedented collaboration with colleagues and deeper conversations with friends.”

The danger

At the same time, the dangers of commercializing this field are obvious, not only in the area of leveraging BCIs to read others’ thoughts even when the subject is not willing, but if Big Tech companies manage to obtain monopolistic access to human thoughts and ideas. This could lead to the sacrifice of more useful but less profitable avenues of neural interface technology on the altar of financial gain. According to co-chair Christofer Toumazou, “The applications for neural interfaces are as unimaginable today as the smartphone was a few decades ago…However, if developments are dictated by a handful of companies then less commercial applications could be side-lined.”

The report also says, “Access to people’s thoughts, moods and motivations could lead to abuse of human rights.” Co-author Susan Chan says, “As our experience with social media has shown, we do need to think ahead to guard against possible harmful uses. If recent experience has shown us anything, it’s that individual consent and opting in or out is not enough to protect either individuals or society more widely.”

However, deeper issues are also raised about how such implant-driven changes in a person’s thoughts and decisions affect the humanness of the person. Will the person one deals with still be human or a mix of computer and human once such a process is set in motion?

The recommendations

The Royal Society report recommends:

  • The government should launch a national investigation to understand the ethical issues behind this technology, including what data is permissible to collect and safety measures during data storage
  • The creation of a UK Neural Interface Ecosystem to promote greater sharing of technology and increased output of new and useful ideas from both sides, academic and industry
  • Governmental regulatory frameworks should look at the best methods of ensuring innovation while checking the tendency of big technology companies to take over the field. One suggestion is a “sandbox” approach where new medical devices are tested out in a controlled environment to ensure they are safe and effective
  • Encouraging public dialogue and using these opinions to modify the applications, thus ensuring public good is served, while also protecting the rights of individuals to opt out of the neural thought sharing network

At present, the British public strongly support the development of BCI to help patients to recover essential or very useful functions lost due to brain injury or illness, but not so much for healthy people to boost their memory, attention spans or physical skills. Thus, a regulated healthy development that includes due safeguards is aimed at, to improve the lives of millions of people without costing humans their very identity.Sources:

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Dr. Liji Thomas

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Dr. Liji Thomas

Dr. Liji Thomas is an OB-GYN, who graduated from the Government Medical College, University of Calicut, Kerala, in 2001. Liji practiced as a full-time consultant in obstetrics/gynecology in a private hospital for a few years following her graduation. She has counseled hundreds of patients facing issues from pregnancy-related problems and infertility, and has been in charge of over 2,000 deliveries, striving always to achieve a normal delivery rather than operative.

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PLOS ONE

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PEER-REVIEWED

RESEARCH ARTICLE

The heterotopic heart transplantation in mice as a small animal model to study mechanical unloading – Establishment of the procedure, perioperative management and postoperative scoring

PLOS

Abstract

Background

Unloading of failing hearts by left ventricular assist devices induces an extensive cardiac remodeling which may lead to a reversal of the initial phenotype–or to its deterioration. The mechanisms underlying these processes are unclear.

Hypothesis

Heterotopic heart transplantion (hHTX) is an accepted model for the study of mechanical unloading in rodents. The wide variety of genetically modified strains in mice provides an unique opportunity to examine remodeling pathways. However, the procedure is technically demanding and has not been extensively used in this area. To support investigators adopting this method, we present our experience establishing the abdominal hHTX in mice and describe refinements to the technique.

Methods

In this model, the transplanted heart is vascularised but implanted in series, and therefore does not contribute to systemic circulation and results in a complete mechanical unloading of the donor heart. Training followed a systematic program using a combination of literature, video tutorials, cadaveric training, direct observation and training in live animals.

Results

Successful transplantation was defined as a recipient surviving > 24 hours with a palpable, beating apex in the transplanted heart and was achieved after 20 transplants in live animals. A success rate of 90% was reached after 60 transplants. Operative time was shown to decrease in correlation with increasing number of procedures from 200 minutes to 45 minutes after 60 operations. Cold/warm ischemia time improved from 45/100 to 10/20 minutes. Key factors for success and trouble shootings were identified.

Conclusion

Abdominal hHTX in the mouse may enable future examination of specific pathways in unloading induced myocardial remodeling. Establishment of the technique, however, is challenging. Structured training programs utilising a variety of training methods can help to expedite the process. Postoperative management, including daily scoring increases animal wellbeing and helps to predict survival.

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Citation: Westhofen S, Jelinek M, Dreher L, Biermann D, Martin J, Vitzhum H, et al. (2019) The heterotopic heart transplantation in mice as a small animal model to study mechanical unloading – Establishment of the procedure, perioperative management and postoperative scoring. PLoS ONE 14(4): e0214513. https://doi.org/10.1371/journal.pone.0214513

Editor: Frank JMF Dor, Imperial College Healthcare NHS Trust, UNITED KINGDOM

Received: September 2, 2018; Accepted: March 14, 2019; Published: April 12, 2019

Copyright: © 2019 Westhofen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: This study was financed by DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Implantation of left ventricular assist devices (LVADs) is regularly used in patients with end-stage heart failure, either as a bridge to transplantation, as a bridge to recovery, or as a destination therapy.[1] LVADs increase cardiac output and strongly reduce the cardiac workload. In some patients, this mechanical unloading may induce a reverse remodeling with beneficial consequences on ventricular geometry, myocardial structure, contractility and pump function.[29] On the other hand, mechanical support has also been reported to provoke myocardial atrophy and fibrosis, and to impair cardiac electrophysiology and calcium handling.[1016] The mechanisms underlying the beneficial and the detrimental effects of LVADs on cardiac physiology are poorly understood.

The fully unloaded heterotopic heart transplantation (hHTX) in rodents is an internationally accepted animal model which is mainly used to investigate transplantation biology.[1730] In this experimental model, the heart of a donor animal is heterotopically transplanted in a recipient animal. Due to the configuration of the anastomoses, the graft beats with markedly reduced left ventricular filling while coronary perfusion is preserved. Thus, the hHTX is a suitable model to study unloading associated remodeling. The first heterotopic abdominal heart transplantation was published using rats by Abbott et al. in 1964.[31] Following its modification by Ono et al. a couple of years later, it has been widely adopted as a rodent model.[17183240] Notably, most studies using the hHTX addressing unloading induced cardiac remodeling have been performed in rats. Compared to mice, genetic modifications in rats are limited which reduces the possibilities of testing mechanistic hypotheses. The hHTX in mice, however, is technically highly demanding with a low error tolerance. Establishing the technique is, therefore, time-consuming and costly which may prevent laboratories from adopting the hHTX.

To support investigators learning the hHTX in mice, we here present our experiences establishing this method following several years performing the hHTX in rats. We provide details of the operation procedure and of the perioperative management. Furthermore, we present several aspects of trouble shooting and a scoring protocol for increased postoperative animal care.

Materials and methods

The transplantation technique has previously been reported and leads to complete mechanical unloading of the donor heart (Fig 1A).[171841] Briefly, the heart of a donor animal is explanted, preserved in cooled saline solution, and transplanted in the abdomen of a recipient animal. The donor ascending aorta is anastomosed to the recipient infrarenal aorta, and the donor pulmonary artery is anastomosed to the recipient inferior vena cava (IVC, Fig 1B).

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Fig 1.

Schematic illustration of the heterotopic abdominal heart transplantation (a) and of the anastomoses (b). The donor ascending aorta (Ao asc) was anastomosed to the recipient infrarenal aorta (Ao), and the donor pulmonary artery (PA) was anastomosed to the recipient inferior vena cava (IVC). Due to the absence of atrial filling, and in the presence of competent aortic and pulmonary valves, the left and the right ventricle (LV, RV) are mechanically unloaded.

https://doi.org/10.1371/journal.pone.0214513.g001

To establish the hHTX in mice, an educational program was devised with discrete learning objectives utilizing a range of teaching methods [15182842]. Using a targeted approach, the transplant establishment was broken down into a series of steps with the aim of improving efficiency in the acquisition of the technical skills required to perform the operation.

Step 1: Current literature and video tutorials were reviewed, and key factors from these technical descriptions that influenced outcome were identified.

Step 2: The hHTX was observed and performed in rats to develop microsurgical skills. This step was performed in our own institution that has extensive experience in rat hHTX.

Step 3: Initial technical skills were developed in mice on cadaveric specimens with the donor and recipient steps of the operation performed using a single animal. When the donor procedure was completed in less than 20 minutes and the recipient procedure performed in less than 120 minutes the surgeon would progress to step 5.

Step 4: A mentorship program was undertaken with two other institutions that had successfully established the procedure in mice.

Step 5: The procedure was performed in live animals when the operation was performed in cadaveric specimens in less than 140 minutes. For the live procedure two animals were used–a donor and a recipient animal.

Animals

Male and female FVB mice with a mean age of 7–12 weeks weighing 16-35g were acquired from our animal facility. Mice were maintained in specific pathogen-free animal facilities with ad libitum access to food and water at the Institute of Cellular and Integrative Physiology, University Medical Center Hamburg. The animal study was reviewed and approved by the local authority for animal protection (Behörde für Gesundheit und Verbraucherschutz Hamburg, Approval No. A8a/785 and G13/098). All experiments were performed in accordance with the German legislation on the protection of animals.

Preparations of donor and recipient animals

Thirty minutes before the induction of anesthesia, 0.1 mg/kg of buprenorphine and 5.0 mg/kg of carprofen were injected intraperitoneally (i.p.). The inhalative anesthesia (isoflurane, 3% during the induction and 1.5% during the maintenance of the anesthesia) was delivered via a nose cone from an anesthesia device system. Animals were placed in a supine position and the body temperature was maintained at 37°C with the use of a heating pad (TR-200, Fine Science Tools, Heidelberg, Germany). The operative procedure was performed using a dissection microscope (WILD Heerbrugg 355110, Leica) with 6–25 -fold optical magnification.

Donor operation

After a midline abdominal incision was made, as much blood as possible (0.5–0.8 ml) was aspirated with a 1-ml syringe and a 30-gauge needle from the IVC to reduce cardiac preload. Then, 0.5 ml of ice-cold heparin solution (100 U ml-1) was injected into the IVC using a 30-gauge needle. Following a short delay, the abdominal aorta was punctured in order to prevent volume overloading of the heart. After 1 minute for the systemic heparinization, the abdominal incision was extended towards the thoracic inlet by cutting along both sides of the thoracic spine. The mobilized anterior chest wall was reflected superiorly. The thymus was resected (Fig 2A) to aid access to the aortic arch. The aortic arch was dissected and 0.5 ml of ice-cold heparin solution was slowly injected using a 30-gauge needle. Then the superior and inferior venae cavae were ligated using 8–0 silk sutures. Afterwards, the ascending aorta was transected below the brachiocephalic artery, and the main pulmonary artery was transected proximal to its bifurcation (Fig 2B and 2C). Connective tissue between the ascending aorta and the pulmonary artery was carefully dissected. Finally, the pulmonary veins and the azygos vein were ligated as a group with a single 8–0 silk suture (Fig 2D). Carefully, the graft was bluntly dissected from the remaining connective tissue. Until transplantation, the heart was preserved in ice-cold saline solution.

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Fig 2.

a-d: Photographic illustrations of the donor operation. Fig 2A shows the donor heart in-situ with the thymus turned upwards. Fig 2B is a close-up of the heart and its superior vessels: the superior vena cava (SVC), ascending aorta (Ao asc). The SVC is being ligated, the Ao asc is transected just below the brachiocephalic artery, and the pulmonary artery (Trc pul) is transected just below its bifurcation. Fig 2C shows the inferior vena cava (IVC) before its ligation. Fig 2D shows the azygos vein, which is also being ligated, together with the pulmonary veins.

https://doi.org/10.1371/journal.pone.0214513.g002

Recipient operation

The abdomen of the recipient animal was opened with a midline incision from the pubis to the xiphoid. Using a retractor, the abdominal cavity was exposed. The intestines were carefully moved cranially and to the sides without removing them from the abdomen. The liver and the intestines were covered with a gauze drenched in warm saline. The reproductive organs and the bladder were also carefully moved to the sides and covered with a gauze drenched in warm saline (Fig 3A). Infrarenally, the abdominal aorta and the IVC were carefully dissected, and a group of centrally positioned lumbal vessels were ligated with a 8–0 silk suture. Using two clips (Yasargil Clip, Aesculap, Inc.–a.B. Braun company, Center Valley, PA, USA), the blood flow through the abdominal aorta and the IVC was interrupted. The first clip was placed proximally of the iliac bifurcation and the second one just distally of the renal vessels (Fig 3B). Meticulous ligation of all lumbar vessels was performed between the two clips. Using a 30-gauge needle, an aortotomy was made at the proximal end of the clamped aorta, and a venotomy was performed at the distal end of the clamped IVC (Fig 3C and 3D). Both vessels were flushed with ice-cold saline solution until no residual blood was left in the clipped section. The aortotomy was extended distally and the venotomy proximally in a longitudinal orientation, using fine scissors.

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Fig 3. Photographic illustration of the recipient situs.

Fig 3A shows the infrarenal abdominal aorta (Ao) and the inferior vena cava (IVC). In the left upper corner, the right kidney can be identified. At the lower border the reproductive organs can be found, dorsally of the reproductive organs is the iliac bifurcation. Fig 3B shows the Ao and the IVC after clamping. Fig 3C shows the Ao and the IVC after aortotomy and venotomy, and flushing of the vessels. Fig 3D shows a schematic illustration of the aortotomy (infra-renal abdominal aorta) and venotomy (subrenal IVC) after clamping the vessels.

https://doi.org/10.1371/journal.pone.0214513.g003

Heterotopic heart transplantation

The donor heart was taken out of the cold preservation solution and placed on the right side of the abdominal IVC. The ascending aorta and the pulmonary artery of the graft were orientated perpendicular to the IVC and abdominal aorta of the recipient animal. The ascending aorta was positioned ventral to the pulmonary artery (Fig 4). The donor heart was then covered with gauze drenched in ice-cold saline. The heart was regularly cooled (every 3–5 mins) by topical application of ice-cold saline solution whilst the anastomosis was performed. First, the arterial anastomosis was performed, starting with an anchor stitch placed proximally, then distally, with a 10–0 suture (Fig 5A). After knotting the distal anchor stitch, a continuously running suture of 4–6 stitches on the left side towards the proximal anchor stitch was performed (counter-clockwise direction, from outside-to-inside and inside-to-outside), and the suture was tied to the proximal anchor stitch (Figs 5B and 7A). The heart was then carefully flipped over to the left side of the abdomen, and the right side of the arterial anastomosis was finished with 4–6 stitches, and the suture was tied to the distal anchor knot (Fig 5C). For the venous anastomosis, again two anchor stitches were positioned, first proximally, then distally (Fig 6A and 6B). After the distal anchor stitch was performed, the suture was pulled through to the outside of the vessel, between the pulmonary artery and the IVC, so the continuous suture could then be started on the right side of the anastomosis, stitching from outside-to-inside and inside-to-outside (Fig 6C). Using this approach, the heart did not have to be flipped over a second time, improving economy of movement and reducing the strain on the vessels. After tying the suture to the proximal anchor knot, the left side of the anastomosis was finished (Fig 6C and 6D). For the venous anastomosis, 5–7 stitches were made on each side. The suture was not tightened firmly and a loop-suture was left in the middle of the first anastomotic side, to prevent congestion.

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Fig 4. The recipient situs with clamped abdominal vessels.

The abdominal aorta (Ao) and the inferior vena cava (IVC) were clamped and prepared for the anastomoses. The donor heart is already situated next to the vessels with the ascending aorta (Ao asc) and the truncus pulmonalis (Trc pul) in place.

https://doi.org/10.1371/journal.pone.0214513.g004

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Fig 5.

a-c: Schematic illustration of the arterial anastomosis on the abdominal aorta (Ao), while the broken line indicates the inferior vena cava (IVC). Fig 5A shows the proximal and distal anchor stitch. Fig 5B shows the continuously running suture on the left side from the distal anchor stitch towards the proximal anchor stitch. Fig 5C shows the right side of the arterial anastomosis after the heart was carefully flipped over to the left side of the abdomen.

https://doi.org/10.1371/journal.pone.0214513.g005

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Fig 6.

a-d: Schematic illustration of the venous anastomosis on the inferior vena cava (IVC), while the broken line indicates the abdominal aorta (Ao). For the venous anastomosis, also proximal and distal anchor stiches were performed (Fig 6A and 6B). Fig 6C shows how after the distal anchor stitch was performed, the suture was pulled through outwardly between the pulmonary artery and the inferior vena cava (IVC), so the continuous suture could then be started on the right side of the anastomosis, stitching from outside-to-inside and inside-to-outside (5–7 stiches). Fig 6D shows the left side of the anastomosis.

https://doi.org/10.1371/journal.pone.0214513.g006

After finishing both anastomoses, warm saline was poured over the heart, and small stripes of a hemostatic agent (Tabotamp, Ethicon, Inc., Somerville, NJ, USA) were draped around the anastomoses. The distal clip was removed first allowing slow retrograde filling of the heart (Fig 7B and 7C) before the proximal clip was released. If necessary, additional hemostasis using cotton swabs with light pressure was performed. Typically, the heart would beat spontaneously after a short period of fibrillation. The initial bradycardic heart rate recovered after about 1–2 hours. This process was supported by warming the abdomen with topical application of warmed saline. The intestines were repositioned avoiding torsion, and the laparotomy closed (subcutaneous suture and skin suture) with a continuous 5–0 nylon suture.

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Fig 7.

a-c: Photographic illustration of the heterotopic transplantation. Fig 7A shows the finished left side of the arterial anastomosis, afterwards the heart is being flipped over for the right side of the arterial anastomosis. Fig 7B and 7C show the transplanted heart after finishing all anastomoses. Fig 7B shows the transplanted heart before de-clamping. Fig 8C shows the heart which is directly perfused following removal of the clamps.

https://doi.org/10.1371/journal.pone.0214513.g007

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Fig 8. Scoring sheet used for daily postoperative evaluation of the recipient animal.

Five categories were used to score the animals postoperatively. For each category points were assigned, that were summed up in a total score to evaluate if further action was required.

https://doi.org/10.1371/journal.pone.0214513.g008

Postoperative care and scoring

After the operation, the recipient mouse was placed in a warming cage with access to food and water ad libitum. Postoperative care and observation of the operated animals were performed after 1, 3, 6, 8 and 12 hours. Afterwards, animals were scored on a daily basis, including five parameters: body weight, general health condition, spontaneous behavior, breathing and wound healing (Fig 8). For each criterium 0 to 20 points could be assigned and the total score was calculated each day to decide on whether further action was required. A score of 0 implicated no stress and no further action was necessary. At a score of 5–9 (low stress) further observation and possibly supportive interventions (e.g. warmth supply) were indicated. At a score of 10–19 (intermediate stress), a start of medical support (analgesia, antibiotics) had to be discussed. If this score persisted longer than 72 hours, it was defined as a severe stress level. At a total score of ≥ 20 (severe stress), the animal welfare appointee had to be informed and intensified veterinary care had to be started. Furthermore, euthanasia had to be evaluated. Pain medication was administered daily (every 8 hours during the first 3 postoperative days) for the first 8 postoperative days using buprenorphine (0.1 mg/kg KG s.c.) and metamizole (300 mg/kg KG p.o.) Furthermore, the transplanted graft was assessed daily via palpation (presence or absence of regular contractions).

Statistics

Continuous variables are presented as mean ± standard deviation, or median and quartiles, and categorical variables are presented as percentage. Comparisons of continuous variables that were normally distributed were performed with the Students t-test, comparison of continuous variables that were not normally distributed were performed with the Mann-Whitney-U-Test. Statistical software used was Prism 7.0c

Results

Learning curve

In a first step, theoretical knowledge was acquired, using literature research and video education, and first practical training was then performed in cadaveric procedures. Before practical training using live specimens was commenced, in a mentorship program with other institutions that had successfully established the method, technical advice and skills were acquired. Our experience on hHTX in alive mice is based on a total of 286 procedures, all performed by one surgeon, between December 2015 and July 2017. Twenty cadaveric procedures were performed to reach technical confidence. The first successful transplantation with a well-beating donor-heart and survival of the recipient ≥48 hours was achieved after 20 live procedures. Total operation time decreased in correlation with increasing number of procedures from 250 minutes to 45 minutes after 60 live transplantations (Fig 9A). Cold ischemia time improved in the early training phase from 45 to 8 minutes, and warm ischemia time from 120 to 25 minutes (Fig 9B and 9C). Operation time and ischemia times stabilized after ~60 procedures. A first success rate of 90% was reached after 60 live transplantations (Fig 10). Notably, even though the operation time which can be interpreted as a gross marker for the complication rates was stable, survival rates of the following operations had a strong variation (Fig 10). In the early training phase (<60 procedures) different mice characteristics (e.g. age, weight) contributed to fluctuating success rates. Heterogeneity in individual responses to unloading was also observed, especially in the early learning phase, leading to varying degrees of heart weight reduction post-transplantation (data not shown). This suggests secondary processes, i.e. valvular leakage, in some transplants and underlines the complexity of this procedure. Procedural semiquantitative scores for intraoperative bleeding complications, time until rebeating, contractility, rhythm within the first minutes after reperfusion, congestion and anastomosis problems, and any other technical complication were collected. Key factors for success and trouble shootings establishing this operation were identified.

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Fig 9.

a-c: Graphical illustration of the learning curve: Operation times. Total operation time (a), warm ischemia time (b), and cold ischemia time (c) with time in minutes on the y-axis and number of operations on the x-axis (#OP).

https://doi.org/10.1371/journal.pone.0214513.g009

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Fig 10. Graphical illustration of the learning curve: Success rate.

Success was defined by a well-beating donor-heart after transplantation and survival of the recipient ≥48 hours.

https://doi.org/10.1371/journal.pone.0214513.g010

Postoperative scoring

121 (42.3%) of the 286 operated recipient mice did not survive the planed observation period of ≥ 48 hours. Of these, 87 (72.0%) died during the first 12–24 hours after the operation, and could, therefore, not be included in the postoperative scoring routine which started 24 hours following operation. When animals showed signs of stress during postoperative observation and scoring was positive (≥20 points), supportive interventions like application of warmth, fluid injection (warm saline solution, i.p., 0.5ml), administration of pain medication or antibiotics (baytril) were performed. In the majority of cases, supportive interventions did not lead to an improved survival and therefore success. As expected, scoring analysis revealed that a lower total postoperative score correlated with better survival (P<0.001, Fig 11). Notably, 80% of the surviving animals had a score of 0. In a subgroup analysis of the five scoring parameters, only the scores relating to the spontaneous behavior and to the general health condition showed a significant negative correlation with survival (P<0.001; Fig 12). Weight loss did not predict survival, as e.g. 20% of the surviving animals had a score of 10 due to weight loss. We saw no case of breathing disorder, impaired wound healing or paraplegia.

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Fig 11. Graphical illustration showing the correlation between postoperative total scoring points and survival.

A comparison of the green curve (survival yes) and the red curve (survival no) shows that more animals (y-axis) with a lower score (x-axis) survived.

https://doi.org/10.1371/journal.pone.0214513.g011

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Fig 12.

a-c: Graphical illustration showing the correlation between postoperative scoring parameters and survival. Displayed are in detail body weight (a), general health condition (b) and spontaneous behavior (c), with the mean score on the y-axis and survival on the x-axis.

https://doi.org/10.1371/journal.pone.0214513.g012

Gender analysis

Operations of male and female mice were performed alternatingly over time (Fig 13). 162 (56.6%) of the 286 operations were performed in male mice vs. 124 (43.4%) in female mice. 81 (50.0%) of the male-mouse-transplantations were successful vs. 81 (65.3%) of the female-mouse-transplantations (P = 0.002). With regards to postoperative scoring, there was no statistically significant difference between the genders (P = 0.765). Male animals displayed a more stable heart weight reduction following unloading (data not shown).

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Fig 13. Graphical illustration of gender distribution over time.

The number of operations over time are displayed on the x-axis, with gender (male/female) on the y-axis. Each datapoint represents one operation.

https://doi.org/10.1371/journal.pone.0214513.g013

Procedural scores

Overall, intraoperative bleeding problems at the anastomotic side occurred in 43.3% during the first 60 live transplantations, mostly due to technical inaccuracy since the exact placement and spacing of stitches is essential for operative success. It was possible to reduce intraoperative bleeding problems to 24.7% during the following 226 operations (P<0.01). Retrograde bleeding due to insufficient ligation of lumbar vessels was observed in 26.6% during the first 60 live transplantations, and in 20.8% during the following 226 operations. This was usually noticed directly after incision of the recipient vessels, and therefore could be corrected before the blood loss was too severe. Early congestion of the graft was observed in 23.3% during the first 60 live transplantations, and in 15.5% during the following 226 operations. Late thrombosis of the graft in 11.7% during the first 60 live transplantations, and in 7.5% during the following 226 operations, mostly due to a too tight venous anastomosis. When anastomoses were technically accurate, grafts started to beat immediately after reperfusion. Usually, a short period of fibrillation was observed during the reperfusion phase. Recovery of normal contractility correlated with the quality of the anastomoses and short ischemia times. Hindlimb paralysis was only observed in one very early operation trying to use an electro-cauter for interrupting the blood flow from the lumbar vessels. The use of an electro-cauter had not been problematic in rats in our experience but was discontinued in mice.

Discussion

The hHTX in mice can be used to study cardiac remodeling in mechanically unloaded hearts and to address underlying mechanisms. Since its first description by Corry et al. in 1973, the model has been in widespread use and various modifications have been published.[3440] This underlines the technical complexity but also the value of this model. Different modifications of operative steps, pitfalls and troubleshooting have already been published, however the establishment of the technique remains challenging.[1718] To encourage working groups to use this animal model, and to reduce initial failures, we presented here our own experiences with emphasis on a structured educational outline, on technical aspects & trouble shooting and on the postoperative management.

Educational program

Our educational program consisted of five consecutive steps: (1), starting with a review of the literature and video education, a theoretical understanding was established. (2), watching and performing the hHTX in rats allowed to get a first practical experience. However, due to the considerable differences in size, the translation to mice was limited. In our view, this step was of limited value. (3), cadaveric procedures were performed to gain technical confidence. This step is of great value, since it reduces harm in alive animals. Switch to live procedures should only be performed when the sequence of the procedure and the sutures are well established. (4), experience from other working groups who already established the technique in mice was shared during laboratory excursions of several day’s duration. Here, the procedure could be discussed to avoid major pitfalls and to get a better understanding of the practical challenges in the mouse and how to handle these. This was extremely helpful after own experience was gained with cadaveric procedures. (5), live procedures were performed which were initially very time-consuming but improved quickly with respect to operation times and technical handling. To establish hHTX in mice a well-planned educational program including all these abovenamed steps, and working through them in the mentioned order can help improve the learning process and maximize efficacy and economic premises, as well as reproducibility.

Technical aspects and trouble shooting

Sharing expertise is an important factor when starting the establishment of the hHTX in mice since the operation itself is technically demanding and small details can be pivotal for increasing success. During training, we were able to learn from other working groups that already established the operation. Therefore, some major pitfalls could be avoided. Nonetheless, we were able to identify several issues that affect the success of the operation and new key factors that further simplify the operation and increase success rates.

  • We studied procedural success using mice of different age groups (7–18 weeks) and found no statistically significant difference regarding survival. The technical complexity, however, was lowest for mice aged 8–10 weeks. Older mice had increasing amounts of fatty and fragile tissue while in younger mice the vessel diameter was too small. We did not observe significant differences regarding thrombosis rates regarding mouse age as suggested by Martins.[43]
  • We observed a better survival for female mice but more reliable and stable results regarding weight reduction of the unloaded hearts in male mice.
  • Technically, the male recipients were more difficult because of the gonadal vessels that frequently need to be dissected off the aorta/IVC.
  • The properties of the clips interrupting the blood flow during the anastomoses are essential. After trying different options, we used aneurysm clips (Yasargil Aneurysm Clip System, Aesculap, Inc., Center Valley, PA, USA), since they apply the right amount of pressure, and are very slim. This way retrograde bleeding as well as injury of the vessel wall could be avoided, and the length of vessels for the anastomoses can be maximumised.
  • The first and last stitches should be placed close to the stay sutures as the ends of the anastomoses are most vulnerable to bleeding or congestion.
  • The venous anastomosis was done with very loose stitches, and a loop suture was applied in the middle of the first side of the anastomosis to prevent congestion.
  • To keep the procedure as simple and fast as possible, and to minimize manipulation of the heart, the second side of the venous anastomosis was performed without flipping the heart over again. The suture was pulled through outwardly between the PA and the IVC after the distal anchor stitch was performed. The continuous suture was then started on the right side of the anastomosis, stitching from outside-to-inside and inside-to-outside.
  • Intestines should not be removed from the abdominal cavity to avoid torsion, mesenterial ischemia, excessive loss of fluids and loss of body temperature.
  • In the beginning, we used cardioplegic solution, to flush the graft at explantation, and as a storage solution during cold ischemia. With these hearts we saw significantly slower onset of spontaneous beating, sometimes the hearts did not start to beat at all, despite technically sound anastomoses. Remnant cardioplegic solution could lead to prolonged and incomplete recovery of the transplanted heart. Accordingly, we used ice-cold saline solution for storage and flushing of the graft.
  • When flushing the heart initially during the explantation procedure, the syringe had to be free of air bubbles. Once air bubbles are trapped in the heart it is difficult to prevent air embolism later on.
  • Furthermore, flushing of the heart by puncturing the aortic arch before harvesting the donor heart must be done with great care to avoid injury to the aortic valve which results in aortic regurgitation and filling of the left ventricle, thereby preventing mechanical unloading of the graft.
  • To increase the overview for the preparation of the ascending aorta and pulmonary artery, the thymus of the donor animal was dissected before harvesting the heart. This could easily be performed by tearing the left and right part apart.
  • The pulmonary artery and the IVC were handled very carefully with minimal manipulation to avoiding tearing of the vessel injury which is usually irreparable.
  • Intraoperative technical accuracy was in our experience the most relevant factor for the postoperative survival and success. Despite close monitoring and postoperative supportive strategies, intraoperative complications were in most cases irreversible.

In our experience, a training period of two months with constant operations, and a total number of 50–60 operations during this period seems to be appropriate to achieve stable operation times and a good operation routine. Major technical challenges are the ligation of the lumbar vessels in the recipient animal and the creation of the anastomoses. The venous anastomosis in particular requires a sensitive handling and precise placement of sutures.

We believe that the technical success during the transplantation is essential for the postoperative survival and success of the model. Gender analysis revealed a better postoperative survival in female mice, but a tendency to more stable unloading results in male mice. Given the more stable heart weight reduction, male mice could be preferred for unloading studies, despite the poorer survival rates.

Perioperative management

A structured postoperative management and scoring analysis was introduced during the establishment of the procedure in close cooperation with the in-house animal care facility. The scoring revealed the complexity of this operation and gave important feedback to the surgeon. Primarily, the scoring points relating to general health condition and spontaneous animal behavior corresponded well with the survival during the observation period. Change in body weight did not predict the outcome as this would develop too slowly. Also, breathing or wound healing were not associated with outcome. Interestingly, the outcome of the procedure could not be positively affected by the postoperative supportive strategies. The success of the procedure was determined intraoperatively which supports the importance of operative precision. However, the postoperative scoring had applications to limiting the suffering of animals. As the supportive interventions could not affect the survival of the animals, veterinary care and also euthanasia should be evaluated early when an intermediate stress level (as defined in the scoring) is reached.

Conclusions

hHTX in mice is an adequate model to study the remodeling of mechanically unloaded hearts. Establishing abdominal hHTX in the mouse, however, is laborious, time-consuming and costly, but can be improved considerably by sharing expertise, a structured programme and avoiding the identified pitfalls presented above.

Supporting information

pone.0214513.s001.xlsx

ABCD
1Operation times
2No of operationTotal operation time (min)Warm ischemia time (min)Cold ischemia time (min)
3123012045
4224511040
5325011040
6422011045
7523011040
8624011040
9721012045
10821011040
11921511030
121022011030
131120511040
141221011530
151321511035
161420012040
171520511035
181621011030
191720011030
201820512040
211920011030
222018010015
232120010515
242217010015
252317010015
262417010015
272517510015
282617010015
292718511020
302816510515
312916010015
323016010015
333116010015
343216510020
35331409015
36341559515
373516010020
38361309515
39371309515
40381309515
41391409015
42401308015
43411208015
44421309015
4543907510
46441008010
47451108010
484690808
49471157515
50481057010

S1 Fig 9figshare1 / 5 Download

Operation time, warm ischemia time, and cold ischemia time for each operation in chronological order are displayed.

(XLSX)

S1 Table. Learning curve for total operation time (min), warm ischemia time (min), and cold ischemia time (min).

Operation time, warm ischemia time, and cold ischemia time for each operation in chronological order are displayed.

https://doi.org/10.1371/journal.pone.0214513.s001

(XLSX)

S2 Table. Learning curve for success rate.

Success was defined by a well-beating donor-heart after transplantation and survival of the recipient ≥48 hours.

https://doi.org/10.1371/journal.pone.0214513.s002

(XLSX)

S3 Table. Correlation between postoperative total scoring points and survival.

A mean value for 10 consecutive operations was calculated.

https://doi.org/10.1371/journal.pone.0214513.s003

(XLSX)

S4 Table. Correlation between postoperative scoring points for body weight, general health condition, and spontaneous behaviour and survival.

Postoperative scoring routine started 24 hours after the operation. Therefore only animals that survived > 24 hours were included in the scoring routine.

https://doi.org/10.1371/journal.pone.0214513.s004

(XLSX)

S5 Table. Gender distribution over time for each operation.

For each consecutive operation the gender is listed.

https://doi.org/10.1371/journal.pone.0214513.s005

(XLSX)

Acknowledgments

This study was financed by DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck. For the support during the technical training, we acknowledge the Department of Surgery, Addenbrookes Hospital, University of Cambridge, especially Dr. Kourosh Saeb-Parsy and Dr. Jack Martin and the Department of Internal Medicine, University Hospital Regensburg, especially Dr. Gabriele Schiechl.

References

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