The overall goal of the following experiment is to study the reactive alterations of peripheral neurons to different pancreatic microenvironments. This is achieved by first generating whole tissue lysates from normal human pancreas and diseased pancreatic tissue. As a second step, DRG cells are isolated from newborn rats, which are subsequently treated with the isolated tissue lysates.
Next, after incubating for 48 hours, the cells are fixed for subsequent double immunofluorescence staining. The results show the differences in the neurite density branching pattern of neurons and the glial density of neurons cultured in different tissue extracts. This method can help facilitate studies in the neuro gastroenterology field, such as the role of molecular mediators, of neuroplastic alterations in different GI diseases.
Though this method can provide insight into the different neurotrophic attributes of pancreatic diseases, it can also be applied to other systems such as cancers or inflammatory states of the entire GI tract. We first had the idea to use that method when we have started our collaborations with research researchers in the field of neuropathic diseases in the pediatric system like, like the Ong disease, and applied that for our purposes To homogenize the human pancreatic tissue. Transfer five by five by five millimeter cubes of pancreatic tissue directly from negative 80 degrees Celsius to liquid nitrogen.
Then place the frozen blocks first into homogenization tubes that can contain a metal dissociation ball, and then into the tissue homogenizer homogenize without allowing the tissue to DeFreeze immediately after dissociation. Resus suspend the solid powder like homogenate in 300 to 500 microliters of ice cold 0.1 XPBS. Do not use any lysis buffer since this may.
Lice the neurons then centrifuge the homogenous at 21, 130 times G at four degrees Celsius for at least 15 minutes. Collect the clear supernat, which contains the tissue extract to collect cell line supernat from human pancreatic cell lines. Culture the cells to at least 70 to 80%confluence in normal growth.Medium.
When they are ready, wash the cells at least three times with cell culture, grade PBS and culture them in serum free medium for up to 48 hours. Next, measure the protein concentration of the tissue homogenate or cell line supernatants via the Bradford protein assay. Then aliquot the tissue extracts and the cell line supernatants so that the final concentration of the extract or supernatant in the neuronal medium is 100 micrograms per milliliter.
And store them at negative 80 degrees Celsius. Using a decapitated P two to P 12 newborn rat, cut the skin and expose the vertebral column. Then perform an anterior laminectomy to expose the spinal cord.
Next, expose and remove the dorsal root ganglia or d RRGs. Place the DRG in ice cold MEM supplied with Gentamycin and metronidazole. Continue to collect DRGs, beginning with the coddle DRGs and moving up to the upper cervical DRGs.
When finished, 52 DRGs are collected enough for a 24 well plate. Next, use micro scissors to separate the DRGs from their roots and nerves. Then cut away any remaining peripheral and central projections.
Following collection of the DRGs. Incubate them in HBSS supplied with collagenase type two at 37 degrees Celsius for 20 to 30 minutes. Following incubation, tritrate the DRGs through syringes with decreasing diameter.
Note that excessive tri can destroy neurons but is less destructive of glia. Once the medium containing the DRGs has become cloudy, centrifuge the suspension at 93.9 times G for five minutes. Then discard the medium and resus.
Suspend the cells in neuro basal medium to prepare the cells for culture. Use a hemo cytometer to estimate the total number of neurons and glia. Then seed the cells on 13 millimeter pre-coated cover slips.
In a 24 well plate the coating used depends on the experimental aims. Next, top the wells with neuro basal medium and allow the cells to attach to the wells overnight. The next day defrost the tissue extract supplemented media or the cell line supernatant supplemented media.
Next, aspirate the seeding medium from the DRGs and perform an optional, very gentle wash with PBS. Then slowly pipette the tissue, extract supplemented media or the cell line supernatant supplemented media onto the cells to yield 100 micrograms per milliliter. Now let the cells grow for 48 hours at 37 degrees Celsius in the tissue extract or cell line supernatant supplied media after 48 hours.
Aspirate the media and fix in 4%paraform aldehyde for immuno staining. For double immunofluorescent staining, use neuron specific and glia specific markers for morph geometry. Use an inverted light microscope equipped with a CCD camera in combination with automated software to measure neurite density using 200 x magnification.
Measure the neurite density of neuronal cultures by overlaying a 50 by 50 micron grid and counting the fiber density per square measured in the intersecting fibers. Repeat this analysis for four different regions of densest growth on four to five representative photo micrographs on each cover slip. Measure the neurite outgrowth mean number of branches per neuron, mean branch length and perianal size from 30 randomly selected solitary neurons from each cover slip by marking the neurites and peric DRG neurons cultured with a tissue extract from a normal pancreas are shown here.
In contrast, DRG neurons cultured with chronic pancreatitis or pancreatic cancer tissue extracts. Exhibit a greater neurite density shown. Here are myenteric plexus neurons cultured with various human pancreatic tissue extracts.
Myenteric plexus neurons grown in chronic pancreatitis or pancreatic cancer extracts build longer neurons and exhibit a more complex branching pattern than those grown in extracts from a normal pancreas. When DRG neurons were cultured with the supernatant from pancreatic cancer cells, an increase in neuro density was also observed similar to what was observed with tissue extracts. This increase, however, was reversible when selected neurotrophic factors such as our tein or nerve growth factor were depleted or blocked.
Ggl cells also respond differently to different pancreatic tissue extracts, extracts from chronic pancreatitis or pancreatic cancer, tissue enhanced glial growth and glial cell counts in DRG derived glial cultures. Although the effect was more pronounced with pancreatic cancer, then chronic pancreatitis extracts Once mastered. This technique can be done in four to five hours if it is performed properly After its development.
This method paved the way for researchers in the field of neuro gastroenterology to explore mechanisms of neuroplasticity in different GI diseases, including inflammatory and malignant GI disorders. After watching this video, you should have a good insight how to isolate peripheral neurons to study pancreatic neuro neuroplasticity in pancreatic and how to implement that system and other neural alterations and other neuroplasticity assays in GI disorders.
