March 22, 2023

Quantifying reactive astrogliosis during prolonged forced abstinence from oxycodone self-administration.

Thank you for the introduction good morning everyone my name is jordan allen i am a senior here at north central college graduating in the next few weeks that is if i can make it that long and i will be presenting on my thesis on quantifying reactive astrogliosis during prolonged forced abstinence from oxycodone self-administration i know that’s a mouthful and it

Sounds like an awful title but hopefully by the end of this presentation you will understand that it is the most comprehensive summary of this research that i could put into a title let’s get started so before we jump into the specific research i want to provide a broad sense of why we conducted this research in the first place most of you know by now that there’s

An opioid epidemic taking place and this is largely due to the over prescription of opioids in recent years oxycodone is a semi-synthetic opioid and opioids in general are just a class of pain relieving drugs and opioids are a huge problem right now because they’re causing millions of individuals to lose their lives to an addiction some frightening statistics

Here approximately 27 million people worldwide are currently suffering from an opioid use disorder and roughly 100 000 opioid overdoses occur each year i also want to make sure before we get started that everyone understands what drug addiction is it’s defined as the brain disease characterized by compulsive drug seeking despite negative consequences and what’s

Important to realize here is that drug addiction is a brain disease it may start off as a choice to take drugs but once an addiction develops it becomes a necessity having spoken to hundreds of injection drug users myself most of them crave a life without drugs but just cannot seem to abstain from them no matter what they do so among the primary drivers of this

Opioid epidemic is a lasting vulnerability to relapse which is the largest issue with treating drug addiction and part of this lies in the fact that there are some pretty nasty withdrawal symptoms from opioids such as insomnia anxiety cold sweats nausea much more in the top right we can see the timeline of these symptoms which peak after 72 hours and subside after

About a month interestingly though people are vulnerable to relapse even after their withdrawal symptoms disappear and this may be due to a phenomenon known as the incubation of drug craving which is defined as the time dependent intensification of drug craving during withdrawal and uh the incubation of drug craving has been seen in humans with nicotine addictions

And in rats with a variety of drugs and we can see this demonstrated by the figure in the bottom right uh where we can see that the rats pressed a lever that gave them infusions of cocaine more and more the longer they were in withdrawal so most research on drug addiction up to this point has focused on the neural adaptations or the structural and chemical

Changes that take place in neurons however researchers are beginning to understand the critical role of glial cells which are the support cells for neurons astrocytes shown here are the star-shaped glial cell and they have projections to neurons and blood vessels they’re the most abundant type of glial cell in fact they outnumber neurons and they seem to be

Critically involved in maintaining substance use disorders they mainly function to modulate neurotransmission maintain glutamate homeostasis and provide neurons with nutrients after an injury or some other traumatic insult on the brain astrocytes can undergo a process called reactive astrogliosis which is a description of active astrocytes that is characterized

By astrocyte proliferation and hypertrophy which is the enlargement of astrocytes and they are also characterized by the secretion of cytokines which are inflammatory substances that are involved in the immune system and they are pro-inflammatory meaning that they triggered neuroinflammation so there are two main types of reactive astrocytes the a1 astrocytes

Which are inflammatory and neurotoxic and a2 astrocytes which are beneficial the a1 astrocytes lose almost all of the normal astrocyte functions and such as their ability to promote neuronal survival outgrowth and synaptogenesis the creation of synapses and importantly they induce cell death which explains why they’re implicated in neurodegenerative diseases

Such as alzheimer’s for this presentation what you should remember is that a1 astrocytes are marked with c3 which is a complement cascade protein involved in immune responses a2 astrocytes on the other hand up regulate many neurotrophic factors that promote neuronal survival and serve to protect neurons gfap or glial fibulary acidic protein is an intermediate

Filament protein and it’s primarily expressed in astrocytes and it’s responsible for providing the structural support to astrocytes and assisting with shape changes and movements of the astrocytes and c3 again is our marker of a1 astrocytes previous research has shown increases in g-fap after exposure to opioids and what i want you to remember is that an increase

In g-fat is indicative of astrogliosis and so using this information from previous research we hypothesized that as a compensatory response to no longer having oxycodone we would actually see decreased gfa and c3 levels on withdrawal day one and an even larger decrease in gfa on nc3 on withdrawal day 15 as the traumatic insult of the exposure of oxycodone is

Further removed so in order to assess the craving of oxycodone we first had to train the rats to self-administer oxycodone so to do this we put the rats in operant chambers for six hours a day for 10 days and while they’re in this chamber the rats can either press the active lever which would result in an infusion of oxycodone paired with a light or they could

Press the inactive lever which did nothing and then following this self-administration training the rats remained in their cages for 1 or 15 days while they underwent withdrawal otherwise known as forced abstinence because the rats are being forced to abstain from the drug okay so what do we find so looking at this figure on the top left we can see on the x-axis

That we have the day of self-administration training and on the y-axis we have the number of lever presses either active or inactive so in the oxycodone routes the top left figure as the self-administration training progressed the number of active lever presses increased which is shown in red while the number of inactive lever presses the press lever that did

Nothing remain the same and this is shown in the white part so the rats in the oxycodone condition learned that they would receive an infusion of oxycodone with an active lever press so they significantly preferred that lever over the inactive lever as training progressed rats in the saline condition which just received a saline infusion which is not rewarding

Did not learn to distinguish between the levers and did not develop a preference towards either liver then we wanted to assess the incubation of drug craving or the intensification of drug seeking over withdrawal and to do this we recorded the number of active lever presses in a 30 minute drug seeking test and rest that preferred the active lever or press the

Active lever more we’re said to have an intensified drug craving for oxycodone and so going through the various elements of this graph we have the various withdrawal days in the saline and oxycodone conditions on the x-axis and on the y-axis we have active lever presses so between the oxycodone and saline animals we found that at both time points oxycodone animals

Press the active lever significantly more than the saline animals now just looking at the oxycodone refs we can see that the active lever presses uh shown in red were significantly higher on withdrawal day 15 and then they were on withdrawal day one which is the gray bar and so this indicates that the craving for oxycodone intensified or incubated over the for

15 days of forced abstinence and since we love neuroscience we had to look at their brains during this incubation of drug craving and so on the left we can see immunofluorescence images of the medial prefrontal cortex and the nucleus accumbens on withdrawal day one and which are all day 15 in the oxycodone rats and so the red markings that we see show us gfap

Which as i mentioned earlier indicate astrocytes generally so the red markings show us astrocytes and from these images it looks like astrocytes are more abundant in both brain regions on withdrawal day 15 compared to withdrawal day one of the oxycodone rats but when we quantified these g-fat markings based on their overall area which we can see in the figures

On the right we found that this pattern of slightly increased gfap levels on withdrawal day 15 compared to withdrawal day one was not significant okay so what about a1 astrocytes the cool inflammatory a1 astrocytes so the top row here is showing one spot in the medial prefrontal cortex and the bottom row is showing one spot in the nucleus accumbens across that

Row and looking from left to right in each row we have the first one being gfap which is again to measure astrocytes generally the second one the middle bar shows the c3 which was to measure a1 astrocytes uh so everything you see here is essentially an a1 astrocyte and a merged image all the way on the right to show where gfap and c3 overlap or co-localize so

The important finding here is that there were markings when we stained for c3 which we can see in the middle and the white dots and there is some degree of co-localization in both the mpfc and the nucleus accumbens which was expected since a1 astrocytes are transformed astrocytes and so this increase in c3 tells us that a1 astrocytes the inflammatory astrocytes

That cause cell death are present in both the medial prefrontal cortex and the nucleus accumbens during withdrawal or during the incubation of oxycodone craving so again the major findings one we were able to successfully induce the incubation of oxycodone craving meaning that the craving for oxycodone did intensify during withdrawal and there was a consistent

But non-significant pattern of increased activity or increased astrocyte levels on withdrawal day one compared to withdrawal date which are like 15 sorry compared to withdrawal day one in the oxycodone animals which as i mentioned earlier is indicative of astrogliosis so if these trends were significant they would tell us that there are more astrocytes in both

The medial prefrontal cortex and the nucleus accumbens the longer the rats were in withdrawal and so the last important finding is that a1 astrocytes are present in both the medial prefrontal cortex and the nucleus accumbens of oxycodone rats on withdrawal day 15. and the reason that such is such an important finding is that um it indicates that there is cell

Death occurring during the incubation of oxycodone craving or the withdrawal of oxycodone how exactly a1 astrocytes contribute to the incubation of drug craving we still don’t really know since we only were able to examine the brains of rats on withdrawal day 15 and for this reason we didn’t have a withdrawal day one time point to compare withdrawal day 15 and

Withdrawal day one so we can’t see how the levels of a1 astrocytes are changing over time um so we can’t make any conclusions based on like what they are exactly doing but this research just shows that they are present so future research should focus on this more and so better understanding a1 astrocytes can have profound impacts on the field of neuroscience

A1 astrocytes seem to be involved in neurodegenerative diseases such as alzheimer’s and traumatic brain injury so understanding their function in response to drug exposure will help us understand their roles and many other conditions and hopefully with an improved understanding of the neurobiological mechanisms of drug addiction we can help to prevent the loss

Of lives due to drug addictions thank you jordan i presume you can hear the uproarious applause

Transcribed from video
A1 Astrocytes in Oxycodone Addiction By Jordan Nowlin