Blogging Off Part 2 (Post #8)

 

  Hello everyone, it is I, Tim Nieuwenhuis, here to tell you about the second half of our results, and sadly, sign off as there will be nothing left to blog about. But have no fear! Science continues forward, and I will be at Dickinson in the lab of Prof. Wohlbach pushing it along! Until then however, let us talk about the last things on our plate.

  Now as you all remember I was going to talk about histology and the results from that experiment, well instead we’re about to talk on how science does not always work out. In labs everywhere there are different constraints placed upon the people working there. There are resource restraints where you cannot acquire all the reagents for the experiment, technological restraints where you do not have access to the technology needed for the experiment, and the restraint that happened in our case, time restraints.

  When all the slides had been stained and prepped for viewing, we only had two days left before the end of the internship. With the amount of data and slides that needed to be looked at, compounded with only one person specializing in histology (a very brilliant fellow intern), we just did not have the time to view the slides. So I sadly have no concrete data to show you, however if you are just joining us, look at Post #6 to see the slides we were working on along with an explanation of how they were created.

  Now, if you recall at the beginning of this blog, I talked a lot about rats and mazes! Well guess what? It is time to harvest the fruits of those labors! If I could please direct your attention to Post #2 and Post #5 as they will catch you completely up to this point in these experiments. Currently I have only disclosed that the rats had learned as time went by, and so I will not show you the increase of completion and decrease of time. However after giving an explanation for terms used, I will show you all other graphs we made.

 

Total Arm Entries: How many times a rat entered an arm. This data was used to create percentages of other arm entering statistics along with testing learning.

 

% Total Errors: The amount of errors made divided by total arm entries. This data was used to test if the rats were learning the maze.

 

Adjacent Arm Entries: How many times a rat would go into an arm directly next to the one it is currently in. Often associated with response strategies.

 

Below you can see the data from our 10 days of testing these rats:

Rat1 Rat2 Rat3

 

  As you can see, there is no clear different trends between the groups, and any you might see are of no statistical significance. So it appears that antibiotics had no effect on the rat’s ability to learn, however we were not quite convinced if they were using the same strategies or not. So for one day we decided to remove the visual cues in the room. We expected this to possibly slow the speed at which some rats would go through the maze if they used the visual cues. The results from that experiment are below.

cue

  As you can see errors did not increase, on the contrary most groups decreased, but to no statistical significance. This means that most likely none of the rats were using spatial cues, and as a result, there was no noted difference in strategies used between the groups.  This means the only statistically significant discovery of all behavioral tests is that all rats learned.

  Now the last experiment I want to talk about is my side project, yes the one about feces! So to open up I would like to say I reran the experiment in more appropriate conditions, so the information from Post #4, while true, is a bit outdated. I reran the experiment almost exactly as last time, but I increased the amount of LB broth the samples were put in. This time, if you look at the picture below, you can see the results are different as every single condition had ampicillin resistant bacteria in it. The best way to see this is there is a lack of turbidity through the broth on all inoculated test tubes, all of which are on the right.
ratfeces

  As a result I decided to run a PCR on the bacteria that had grown, and sadly because we had very little time left, I was only able to run one. So I chose to run enterobacteria as it was known to bloom in the gut post antibiotics and the environment in the broth was suited for them. Out of the PCR came the picture below, and said picture was turned into a quantized graph.

Capture

  This picture means  that, indeed, at least some of the resistant bacteria was enterobacteria, and as a result my hypothesis was confirmed! If you look at Post #7 you can also see that enterobacteria also bloomed in the guts of the rats, pointing to the possibility that their resistance enabled them to, which is very exciting!
  Well thank you very much everyone for your support. Even though all of the experiments did not have the expected results, that is how science works. Just because you want a result to occur, never means it will actually occur. I would once again like to thank the Dickinson Internship program for sponsoring me and assisting me financially through the summer, without them this would not of been possible for me. And lastly I would like to thank everyone as BRInj, it was incredibly nice working with you all, your kindness and welcoming attitude created the nicest work atmosphere I could ask for. So goodbye everyone, see you at Dickinson College for my final year!

BRINJTim

Blogging Off Part 1 (Post #7)

  Hello everyone! Is it the holiday season, because I feel like everything is wrapping up *Buh-dum-tish*! Alright I’m done with the jokes, but in this post and the next, my goal is to present the results of the research done by my colleagues and I. It has been an incredible journey, but journies have destination and we are now at ours. Our research ended up being broken into three main sections: the microbiome, gut inflammation, and behavior. However, gut inflammation was also broken up as well, so they shall be covered in the two separate posts.

  Here’s the last quick recap of what we were researching! Recently studies have shown a connection between the bacteria living in one’s gut and their mental state. A recent paper has gone far enough to find a correlation between the microbiome and depression in humans. This was tested by examining fecal samples of those who have major depressive disorder and those who don’t. After comparing the microbiomes and seeing a difference in the flora the scientists then completed fecal transplants from the humans to rats and noticed similar mental conditions.

  Another issue that frequently occurs in the NICU (neonatal intensive care unit) is intestinal inflammation. This can become a serious issue for newborn babies as it can develop into life threatening diseases such as necrotizing enterocolitis (NEC). And so part of our experiments included looking into how intestinal health is affected by the bacteria that live there and if there is a correlation between certain bacterial families and inflammation.

  Now, the way we connect and test all of this is via antibiotics! As you know antibiotics are used to kill bacteria, but on of the issues with using them is that they kill indiscriminately (if the bacteria fall into their spectrum of effectiveness). That can cause problems for newly born children, particularly premature ones, who, even at the first sign or bacterial infection, are given antibiotics. Now this leads us to wonder, does this change in the microbiome due to antibiotics change the mental and physical state of the rats? If so, how are they actually affected by the resulting changes? Now all these questions and more will be answered, so ready yourself, for knowledge!

  To start off we should first discuss the models used for these experiments. We completed all experiments related to the main experiment in vivo with rats. For an explanation of the groups of rats and their naming please consult the table below.

ratgroups

The antibiotic chosen was ampicillin, as it is commonly used in the NICU and covers a wide spectrum of bacteria. The injection itself was done based off of the weight of the rats. For the mothers they received the ampicillin for the 3 days before the birth of the pups, and for the pups they received ampicillin for the first 10 days of life.

  The first topic of the actual experiments we’ll tackle is PCR. If you want to know how PCR works my fourth post discusses the ins and outs of the processes. So with how PCR works being known, we need to know what bacteria we’re looking for. First we tested for Lactobacillus, an anaerobic (does not use oxygen) gram negative bacteria, which has shown to have a positive effect on health. Enterobacteria, facultative anaerobes (can use oxygen, but doesn’t need to) gram negative bacteria that is known to be harmful in large amounts such as Escherichia coli. Clostridia Cluster XIVa, a gram positive anaerobic group known to produce butyrate, a fatty acid that helps reduce inflammation and protect the epithelial defense layers of the gut. And lastly Bacteroides, an anaerobic gram negative bacteria that is resistant to ampicillin and used as a positive control.

  Below are the graphs of the data received from the PCRs, made quantitative by measuring band strength. A star denotes statistical significance.

Lacto entero Clostr Bact

As you’ve just seen only enterobacteria seems to have statistically been flourishing in the gut of those who post nately took antibiotics, as expected. The lack of difference between bacteroides was also expected as we had them as our positive control. However, we found no data of significance on clostridia or lactobacillus, which was not what we expected. But, we would like to point out the trend in the mother having antibiotics and the slightly decreased lactobacillus. This is interesting because some lactobacillus is transferred from the mother to the rat pups via milk, so researching the effects of antibiotics on milk may still prove important.

  The second major topic is the result of the Western Blots, which we were using to test for different proteins that would present themselves in the case of inflammation. The first protein we looked for is Receptor for Advanced Glycation End Products also known as RAGE, a receptor upregulated during inflammation. The second protein was NF-B, a short term inflammatory marker. Below you can see the results quantized based off the band readings from the Western Blots.

rage NFKB

None of the data had statistical significance which points to antibiotics possibly having no effect on whether or not inflammation occurs. In the future we feel that looking at TLR4, Claudin 5, and Occludin, all a part of inflammatory response, will help further confirm the data from this experiment.

  Thank you very much for reading my blog, as the next post will be my final one as I talk about the results of the histological experiments, the behavioral experiments, and my personal experiment. See you soon!

A Slice of Life (Blog Post #6)

            Hello everyone! It is I, Tim Oldfield Nieuwenhuis, and I’m here to tell you about everyone’s favorite thing, SCIENCE!  This is a particular field of science that, while I have been exploring at my internship, I have failed to touch upon up until this point. Now you’re probably wondering “Wow Tim, you’ve already done so many cool hip things in the lab, what else could you of possibly done?” and the answer to that is “PATHOLOGICAL HISTOLOGY”! And don’t copy and paste that into google, because I’m about to tell you what that is right now!

            Now you might hear the word pathology and pathological tossed around willy nilly, so I will explain it first. In its most barebones definition, pathology is the study of disease, and that can be from its causes to effects. So with such a broad topic of “disease” there are many more specific fields of studies denoted by pathology. Botanical pathology is the study of disease in plants, meanwhile pathological bacteria are bacteria that cause disease. Now with that being cleared up, you’re probably wondering what the second half of that phrase is, and let’s get right to it.

            Histology is the study of the anatomy on the microscopic level, and if you took high school biology you probably know a little bit about it. So as we all know anatomy is the study of the physicality of the body, so when you learned anatomy you probably learned the names of all the bones and muscles. However, in your schooling you probably learned at one point what cells comprised the muscles, intestines, or the different layers of the skin. The microscopic world of your anatomy is histology, and via microscopic imaging we can take a look at these structures to further our knowledge on them.

            Now the reason this is important to us in our research is, if you remember, we were looking at issues that could occur in the gut due to microbiome imbalances.  One such issue is inflammation, and luckily, through histology, we are able to look at tissue to see if it had actually happened. Now, to look at tissue there are three main portions of the process. The first is creating the slide from the tissue, the second is staining the slide to make the different markers of inflammation visible, and lastly is looking at the slide itself. We’ll start off with the slide creation as it is the first and one of the most complex steps.

            To create a slide, at least in the method we used in our lab, you start off with the extracted tissue and a paraffin machine. The point of a paraffin machine is to imbed the tissue into a block of wax. The way this is done is by placing the tissue into a small metal tray, and then pouring hot wax into the tray thus surrounding the tissue. Once the tray is filled with wax it is moved to a cooling station where the wax solidifies around the tissue. Once cooled, the block of tissue is sliced in a paraffin microtone, a machine which can slice the tissue as thin as ten micrometers in width. After we slice the wax blocks to the size needed, we place them on positively charged slides and prepare them for staining.

            For staining there different methods available depending on what your goal is when viewing the slide. In this post I will cover two different staining methods, each with their own purpose. The first is Hematoxylin and Eosin staining, also known as H&E Staining. This staining allows us to look at the physicality of the tissue itself. Hematoxylin will dye the negatively charged components of the tissue blue, such as the nucleotides and basophils. Eosin however, dyes the positively charged components pink and orange such as amino acids, cytoplasmic filaments, and extracellular fibers. These color changes allow us to see closely where and what kind of damage has been done.

            The other staining we will talk about is proliferating cell nuclear antigen (PCNA) staining. PCNA staining is a method that uses antibodies and presented proteins to help determine if cells are proliferating, somewhat like a Western Blot. Using DAB, which reacts with the antibodies to make a brown secretion, we can visibly see the cells which we believe are proliferating. The reason this is important is because, during inflammation, proliferation of epithelial cells is decreased. Comparing between different conditions allows us to quantify the physical aspect of inflammation, thus creating comparable data.

            I will talk about the results of these experiments incredibly soon, but until then, I’ll show you some pictures of what we’ve talked about. I would also like to thank the internship grant given to me by Dickinson College. Without their funding of my expenses, the reality of this internship for me would of maybe not come to fruition. So a big shout out to them, and hope to see you soon!

illeum(an H&E stain)

DAB (PCNA stain with DAB)

 

I Am Still Just a Rat in a Maze (Post #5)

        Hello everyone! It is I, Tim Nieuwenhuis, and instead of using a regular pun for a title, I’ve incorporated a pun/song reference… thing. Anyhow, today I’m here to talk about the rats and how they’ve been doing in this whole crazy maze experience. While we are still analyzing the data, there have been some anecdotal occurrences with the rats that, in my opinion, are interesting, and I hope are interesting to you too!

        So to recap, we tested the rats in something known as a radial arm maze, for more information on the test see post #2. Using this maze we planned on examining the rats’ ability to learn and also what strategies they used to complete the task of eating all the fruit loops at the end of the arms. All of the data from the experiment by this point has been collected from: 20 rats, 13 days, 8 fruit loops (per day), 4 groups, 3 scientists, and 1 maze… however, we’re running statistical analyses on what was recorded so we don’t have any solid claims yet, besides a few select ones.

        Such as the rats are learning! Falling within the range of statistical significance, which means by analyzing the data on the quantitatively recorded occurrences in the maze, it was determined the rats learned! The way this was determined was by both a decrease in total time in the maze along with total errors. Now this is not unexpected, we wouldn’t be running experiments on rats if we thought they were unable to learn, but the kicker is, all groups learned the same amount. What this means is that learning, in this specific experiment, seemed to have very little change between all variable groups and the controls. While we weren’t sure what to expect, because these are juvenile rats, and most experiments of this sort are completed on adult rats, it is still novel data.

        The rest of the data I’ll save for a later post when the entire project comes together, however for now I’ll explain an interesting finding. To refresh our memory, one of the things we looked at were adjacent arm entries, this was done to see if the rats were using spatial memory or a reaction strategy. If a rat looked around at the positional cues that we left up and used those to determine where it wanted to go next, the rat would be using a spatial strategy. If a rat walks directly out of the arm, realizes every time it takes a sharp left that there is food, and continues to take sharp lefts, they are using a reaction strategy. As a result one would think that more adjacent arm entries meant that rats are using a response strategy, which is correct. However, we also noticed a different kind of response strategy shown below.maze strategy

        If you start at the red arrow at the bottom and follow the paths (or ROYGBIV), you’ll see you can follow the same strategy the entire maze without an error or adjacent entry, and several rats seemed to have been following this strategy. Now off these anecdotal observations no conclusions can be made, but stay tuned, because we have a way to test what strategy is being used, and it’s coming up!

Collecting Feces From Other Species (Post #4)

Guess who’s back everyone!

You’re probably right with whatever your guess is because I’m still kinda sure that I’m the only one who can edit this blog. Someone please tell me if anyone else is doing so, because that would be an issue.

Anyways in this blog post I would like to talk about and expand upon my experiments regarding my testing of the rat stool samples, while also explaining the way that some of the processes I’m using work. To catch up anyone who hasn’t read the previous posts, in the lab at BRInj we’re working on how taking antibiotics effects juvenile rats in a plethora of ways, and how it can relate back to human health. On of these areas of focus is the gut microbiome, which is the environment of an animal’s intestinal tract. We’re trying to see what the effects of antibiotics have on the bacteria that live in the gut by comparing 4 different conditions of rats, some with antibiotics, some without (look at blog post #2 for exact conditions).

Now one of the major worries on the global stage right now is antibiotic resistance, which for those who don’t know, means that bacteria that we treat with certain medicine are becoming resistant to said medicine. While I don’t want to spend a lot of time on the topic itself, it is an important and threatening issue and I heavily suggest reading up on the topic yourself if you’re not knowledgeable in it. However, this antibiotic resistance is something I wanted to focus on, because I wanted to see if there is a correlation between those bacteria who antibiotic resistance, and what bacteria bloom after usage of antibiotics. However, if you’ve been keeping up you know all of this.

So far, to test this theory, I’ve completed several steps.  I took one dropping from each condition, which, while not giving enough of a sample size for strong conclusions, could still give interesting preliminary data. Then with the fecal matter I homogenized it, which I did in PBS buffer, a water based solution that somewhat matches conditions in certain areas of the human body. After that, I pipetted a small amount into separate LB broth containing tubes. For those who don’t know LB broth is used to grow certain bacteria aerobically, which means with oxygen, so not all possible bacteria had the chance to grow. As a control I also placed bedding from each rat cage into LB broth containing tubes as well.

After letting the bacteria grow in an incubated shaker for one day, I noticed only major growth in the tubes with fecal matter and so I proceeded in the experiment with them. Presently there is now growth in the bedding tubes which will be tested later. However, with the fecal samples, I created four different conditions in LB broth with ampicillin concentration, the type of antibiotic the rats were treated with. After inoculating the antibiotic tubes and waiting one day, all groups except for three had shown growth, and that’s where I’m currently at with the research.

The next step I need to complete is running a polymerase chain reaction, also known as PCR. This technique can be used to determine if certain DNA is in a sample of something. By using what is known as a primer, you can enhance a section of DNA so it becomes more visible when you run it on a gel, which separates DNA by size. This is useful because, while most life shares common sections of DNA, there are also vastly different ones. For example if you had a primer that enhances the section of DNA for human hemoglobin (the protein that helps carry oxygen), the primer would not have a section of DNA to enhance on a crab. So following this logic, we can also find out which bacteria exist in my ampicillin resistant tubes by enhancing their DNA using primers for specific families of bacteria. Until the experiment is done, I will not be sure what bacteria is resistant, but if it is the same type as the one we find blooms the most in the ampicillin treated rats’ guts it would be interesting to the narrative.

pcr

(Above is a simplified visual representation of PCR from Wikipedia to visually aid with the process)

Once again thank you all for reading and see you soon!

Milking the Situation (Post #3)

  Hello everyone! Once again it is I, the one and only blogger of this page! That’s right, Tim Nieuwenhuis! For this post, I would like to talk about ideas my lab group and I have come up with for possible experiments to help our research on necrotizing enterocolitis (NEC). As I mentioned, we as interns, are allowed to come up with methods to approach what we’re researching, and as a result, each person has been coming up with different approaches to the topic. I have spearheaded two major possible veins of research, and in my explanation of them, I’ll also explain why I think they might be beneficial to the study.

  To recap, NEC is a disease that affects the intestines of primarily premature infants. The normal cause of it is antibiotic use which causes a microbiome imbalance in newborns. The purpose of this antibiotic use is for infections that would normally be incredible dangerous to a newborn. However, the imbalance in the microbiome results in a decrease of helpful bifidobacteria and increases harmful proteobacteria. A disclaimer I would like to make is that everyone’s microbiome is different and only having one or the other of any type of bacteria is not good. There is a healthy balance for all phylums of gut based bacteria, and none, if controlled, are harmful. I just don’t want to besmirch the names of any of my microscopic friends.

  Now with this knowledge in mind, and my proclivity towards microbiology, I came up with two ideas. The first was to milk the mothers of the pups. Yes, I know, “rat milking”, but trust me this is not a Simpsons’s episode gag. You see, a mother’s milk is an extremely important factor in the health of premature babies, as giving milk over formula greatly decreases the chance of NEC occurring. There are many reasons this is true, as mother’s milk has various different compounds to help nourish the child. However, our experiments deal with antibiotics which lead me to wonder, how different is the bacterial load in the milk of control rats compared to that of those that took antibiotics? Using PCR, I hoped to be able to find and compare bacterial concentrations of the mother rats and relate it back to the microbiome of the pups, because there may be a connection between the bacteria in mother’s milk and how a baby’s microbiome is affected.

  This idea however, did not come to fruition, for many understandable reasons. The initial reason is BRInj uses IACUC (Institutional Animal Care and Use Committee) to ensure minimal stress levels and safety for the animals used in the facility. The milking of the mothers would very well have created more stress for them, and so the AICUC would’ve needed to be revised and reviewed. As a result, we thought to try to collect milk after sacrifice, but none was obtained, which ended the possibility of the experiment. I would like to say however, that the means of milking rats doesn’t appear to be that hard, as there are papers out there with pretty simple DIY methods or creating milking apparati. So if you ever want to pursue this idea, by all means go ahead and throw me on as an “et al”.

  My second big idea however,  does seem to be a bit more feasible and relates directly to the research of the microbiome. Generally, when we see a bloom of proteobacteria in the gut after antibiotics, it seems to be one specific species that takes over (normally in the enteroebacteria family), and hearing that interested me greatly. The reason being, if the entirety of the gut bloomed with proteobacteria that would mean that the phylum itself is what’s doing so well, however if only one species blooms something else might be going on. What I want to test is possible antibiotic resistance to the most commonly used antibiotic, ampicillin.

  My current plan is to extract bacteria from the rat feces that we’ve collected and try to culture them.  We will have an aerobic and an anaerobic ampicillin broth to grow the bacteria in to test for any resistance. If bacteria appear to grow in the broths, I will then run a PCR on them, using enterobacteria primers that we’ll be using for feces analysis to see if the bacteria are the assumed pathogenic species. If they are, then we have a blooming bacterial group that is resistant to ampicillin, which could be the reason why it does so well in the gut of those who have had antibiotics.

  This plan will only happen if, after fecal analysis, we see a bloom in proteobacteria, but if there is, I think it will be an interesting path to follow. Anyways that’s all for this week, stick around to be kept in the loop and see you soon!

rat milk

Ratical Experiments (Post #2)

Well hello again everyone! It’s me, Tim Nieuwenhuis, the only person who I think can edit this page! I’m here to tell you about my second week at BRInj and I’m going to stop telling you I’m going to tell you something, and instead just tell it!

As I mentioned in my last post, I believed this week we were going to attempt some PCR (Polymerase chain reactions), but because the use of said technique isn’t applicable to us quite yet, we’re holding off on it. However this week we further narrowed down what we, the interns, want to research. Using the laboratory’s rats we plan on testing the rats’ ability to learn and use different forms of memory in a radial arm maze. We will also test for the composition of their gut microbiome and what proteins their intestines may express due to the antibiotics some were given to help simulate the disease Nec. We will also explore mental health issues caused by an imbalanced microbiome.

The four types of rat conditions created are as follows:

1) Those whose mother received antibiotics before the birth of the pups, and the pups receiving more antibiotics during early life.

2) One where only the mother was given antibiotics.

3) Another group where only the pups were given antibiotics.

4) A group where no antibiotics were given.

Now you’re probably wondering, “What is a radial arm maze?”, and if your not wondering that,  I’m going to tell you what it is anyways. First behold this general picture of a top down view of one!

radial

A radial arm maze is an eight arm maze that generally has arms which are baited with food to test the ability of the rat to remember where food is located in the maze, and where they have already been. Now this maze comes in different variations, some even have doors that open and shut on the center area to disorientate the rats and test their memory further. There are also different ways to set up the maze, such as partially and fully baiting it, which allows one to test different types of memory. However for our experiment we are not using any doors and are also fully baiting the maze, which means each at the end arm will have food in a cup.

So now you’re probably wondering “What information does this give us?”, and if your not, I’m still going to tell you. Given the time constraints placed on the internship we decided to do a fully baited radial arm maze, instead of a partially baited one. While a partially baited maze would have given us interesting information, it is just not the best idea with the limitations we have. Using a fully baited maze , we can look at how rats learn and use their memory, which comes in many different flavors that can overlap.

Here is a brief definition of the types of memory that we are observing:

Working Memory: The short term bank of memory used to deal with a current situation. The rats need to know where they have already gotten food, and make sure they don’t return to an arm now without food.

Spacial Memory: The ability to use spacial cues to navigate oneself to the desired goal. The rats use shapes placed in the rooms to orientate themselves to travel to arms they haven’t been in yet.

And of course long term memory, which would be, do the rats know how to navigate the maze? Testing the rats’ ability to remember and execute the maze will allow us to look into how well they’re learning, and to see if antibiotics are a detriment.

As the experiments go I’ll keep you all updated, so see you next week!