In vivo and in vitro work

In vivo and in vitro work

One of the goals of our deployment is to collect skin, blubber, and skeletal muscle biopsies from young adult male Weddell seals. Allyson and Luis have extensive experience performing biopsies on marine mammals, including Weddell seals. Our two main priorities are to keep our team safe (even though Weddell seals are docile and slow-moving, they do have a pretty impressive “pointy end” that one better stays away from) and to make sure we cause as little discomfort to the animal as possible. Although this type of procedure is pretty straightforward, there always is a risk that an animal reacts poorly to the anesthesia (as is the case in any human undergoing a procedure). So preparation is key.

The day before our first procedure, we do a dry run in the lab where everyone is reminded of their responsibilities. Manu will learn how to coax the Weddell seal into a headbag (a way to prevent the animal from moving away while sedation and anesthesia meds are administered, a job for Luis who will also draw blood from the arterial line through which meds are administered) and will monitor the animal’s breathing (any sign of prolonged breath holding would trigger an emergency response that would include stimulating breathing and administering reversal meds). Allyson will collect the biopsy samples and will also get fecal/nasal/oral and skin swabs (which we will use to study the Weddell seal microbiome); Kaitlin will measure intra-ocular pressure and assist Allyson and Luis with collecting all the samples. Allyson and Kaitlin also prepare a cooler filled with all the sterile surgical tools and reagents we will need, while Luis calculates and prepares the appropriate dilutions of the meds.

On the day of our first procedure, we head to a familiar location: Tent Island (Picture 1: Weddell seal on the rough sea ice off Tent Island). We previously spotted biopsy candidates at Tent and we are sheltered from the prevailing easterly winds. After spotting our 400kg heavy “volunteer”, Allyson is the one to put the headbag on (a learning experience for Manu and Kaitlin), making sure the seal’s breathing is not impaired. Once the bag is on, Luis quickly administers an intra-muscular sedative, before putting in a venous line. As soon as our seal is “down”, the headbag is removed and a clean towel (we never use a towel twice to prevent transmitting parasites from one seal to another) is gently draped over the eyes of the seal to keep him as calm as possible. As Allyson, Kaitlin and Luis quickly work to get the samples we need, Manu consistently calls out the breathing rhythm of the seal so Luis can adjust the anesthesia as needed (enough to keep the seal calm but not so much that he stops breathing). The seal breathing varies between 4-16 breaths/min. In less than an hour, we get what we need. Not even 10 minutes after we back away from our seal, he seems to have recovered (well, he still looks a bit sleepy but he moves around like nothing happened, Picture 2: sleepy Weddell seal recovering from anesthesia). Mission accomplished. Well, almost: getting back to town and processing all the samples will take us another 4 hours. We’ll miss dinner (on a Sunday no less) but the galley serves pizza 24/7 so it’s pizza night!

Collecting tissue is only the first of many steps in our protocol. A major focus of our project is to grow and immortalize cell lines from those tissues. Cell lines are a valuable resources that will allow us and other researchers to study Weddell Seal biology “in vitro”, without us having to travel to Antarctica or disturb the animals. One source of tissue we are particularly interested in is the placenta, a highly vascularized tissue. Collecting placenta only requires minimal interaction with the Weddell seal and causes no harm (Picture 3: Allyson and Kaitlin collecting arteries from a fresh placenta at Hutton Cliffs; Picture 4: Kaitlin and Luis collecting arteries from a fresh placenta). In addition, the placental and umbilical vasculature is of specific interest to us, as its contractility needs to be carefully controlled, so that blood flow to the fetus remains unimpaired during long dives by the mom-to-be. The tricky part is to get to the placenta before it freezes (which, as you can imagine happens really quickly after the placenta is delivered on the ice) and before the Skua get to it. Skua are large and incredibly brash gulls that are currently migrating into McMurdo Sound as the weather starts to warm; Skua are even known to take live penguin chicks or peck at the eyes of abandoned seal pups. With the help of our friends from B009, we have been able to find and collect arteries from ten fresh placentas. We are trying to harvest and grow two types of cells: endothelial cells and smooth muscle cells. The endothelial cells form the “inner” tube of the artery, while the smooth muscle makes up the outer, contractile layer of the artery that allows the vessel to contract and relax to regulate blood flow. Kaitlin and Allyson practiced culturing these types of cells at the lab in Boston with tissues from mice, but we are anxious to see how our method translates to culturing Weddell seal tissues. After a few days of anxiously checking our culture flasks for cells, we finally can confirm that we have cells (Picture 5: Weddell Seal smooth muscle cells in a dish)! It’s only a few smooth muscle cells to start, but we are now a lot more confident in our ability to isolate and process these cells (Picture 6: Allyson, Kaitlin and our friend Dan with big smiles as we confirm that we cultured Weddell Seal smooth muscle cells). The endothelial cells are a bit trickier. It takes a few different isolation methods before we succeed. We’ve got a long way to go for these cells to be viable for long-term experiments at home, but we’re ecstatic that we’re off to a good start.

Science, both in the field and in the lab, is cool!

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Team B267

NMFS permit 19439; ACA permit 2016-005; Photos by Allyson Hindle and @manu_buys