Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures

The overall goal of this procedure is to introduce neonatal rodents to intermittent bouts of mild and brief lowered oxygen levels. This is accomplished by first flushing plethysmography chambers with 21%room air oxygen for two minutes. In the second step, the input gas is quickly changed to a hypoxia mix of 10%oxygen for two minutes.

The cycle is then repeated 20 times, producing an acute intermittent hypoxia protocol. In the final step, neural stem and progenitor cells harvested from the treated animals are harvested for subsequent culture. Ultimately, microscopic measurement and immuno cyto chemistry of the resulting neuros sphere.

Cultures is used to demonstrate changes in the neuros sphere, population expansion and cell fate choice in response to the lowered oxygen level exposure respectively. System calibration is essential when performing this technique. Verification of the appropriate setup connections and flow rates of the instruments is critical to the successful gas delivery, intermittent hypoxia exposure and safety of the animal.

Before beginning the procedure, run a one eighth inch diameter piece of plastic tubing from the flow meter of compressed air tanks containing the 21%baseline gas and the 10%challenge or hypoxia gas to a flow meter. Then to a bias flow regulator, enabling flows of one liter per minute to each chamber. Run the same size tubing from the bias flow regulator to the plethysmography chambers and seal all of the unused connections to and from the bias flow unit and all unused openings to the chambers so that no gas flow escapes.

Then place the chambers into an incubator for e equilibration to 34 to 37 degrees Celsius to calibrate the cycle times. First, confirm that all of the tubing is properly connected between the gas tanks and the rest of the equipment. Then connect an ambient oxygen sensor to its corresponding handheld oxygen meter and attach the sensor to the plethysmography chamber lid.

Open both gas tanks and clamp the off cycle flow of gas with one pair of surgical long handled hemostats insulated with plastic tubing so that only one type of gas is delivered to the chambers at a time. Then record the time required for the chamber oxygen to reach the target level of 10%and to return to 21%After confirming that all of the tubes are properly connected, place the neonates in the plethysmography chamber and house the plethysmography chamber lids in the chamber base. Confirm a proper closure of the O-ring seal as well as the closure of any unused chamber connections to ensure the appropriate delivery of the gas mixes.

And then place the chambers holding the pups to be treated into the 37 degree Celsius incubator. Allow the chamber and pups to equilibrate to 37 degree Celsius at room air oxygenation. Then open both gas tanks and use one pair of hemostats to clamp the off cycle flow of gas as just demonstrated.

Using a handheld timer precisely time the cycles indicating the time for switching the hemostats between the tubes to alternate the flow of gas to the chambers. Record the completion of each cycle using a treatment log. Monitoring the incubator temperature at every cycle.

Change to ensure the animals are maintained at the designated range. Closely monitor the rodent pup activity throughout the protocol to ensure that the animals tolerate the cycles without visible distress. To isolate the neuros sphere forming cells, remove the pups from the chamber immediately after the intermittent hypoxia is completed, and harvest the sub ventricular zone tissue within 30 minutes.

Finally, add the tissue to the neuros sphere culture and conduct the standard passage and expansion for derivative neurospheres. Following intermittent hypoxia administration has just demonstrated subventricular zone derived neural stem and progenitor cell populations cultured as neurospheres for 14 days. Exhibit a nearly twofold increase in diameter, demonstrating an increased expansion within each forming sphere.

Further cells that are plated in permissive conditions, for example, in the absence of mitogenic factors yield significantly more beta three tubulin positive cells, indicating a more extensive neuronal differentiation compared to the cells harvested from normoxic treated control pups. Following this procedure, other methods such as stem cell harvest and culture can be performed in order to answer additional questions about how neural cells behave following intermittent hypoxia exposure.