Here we present a tail-tip blood sampling protocol for frequent sample collection in unrestrained mice. This method is useful for assessing patterns of pulsatile luteinizing hormone secretion and could be adapted for analysis of other circulating factors.
The overall goal of frequent tail-tip blood sampling is to obtain whole blood samples for the measurement of pulsatile luteinizing hormone secretion referred to as LH in awake non-anesthestized and normal behaving mice. This method can help answer key questions in the field of reproducive neuroendochrinology by providing a reliable protocol to assess luteinizing hormone pulsatile secretion in mice. The main advantage to this technique is that frequent handling and habituation will ensure robust and reliable luteinizing hormone pulse measurements following acute or chronic experimental treatments.
For five weeks prior to the blood collection, handle the mice on a daily basis from Monday to Friday. In the final two weeks, handle the mice on Saturday and Sunday as well. Perform the mouse handling regime in a bio-safety cabinet.
Remove the first mouse from its cage and place it on a suitable surface within the cabinet. Gently restrain the mouse on this surface by holding the base of its tail. Then with the other hand firmly stroke the ventral surface of the tail from the base to the tip.
Do this a total of six times and then return the mouse to its cage. This constitutes one handling set. Now wait six minutes and perform another handling set.
Continuing this pattern with inter set intervals, perform four handling sets per handling session. In total this provides twenty-four tail strokes per session. If experimental treatments during the sampling period will include any additional handling methods like scruffing or an intraperitoneal injection then also acclimatize the mice to such handling activity during the handling sessions.
Two weeks before the planned blood collection day, house the mice in pairs to minimize their disruption during the sampling period. For the blood collections prepare 0.6 milliliter micro centrifuge tubes for blood storage. Label the tubes and for three microliter blood collections aliquot fifty-seven microliters of ACES buffer into each tube.
Once prepared store the tubes at four degrees Celsius. At the bio-safety cabinet have the following items ready:pipette tips, a waste bin, a timer, an experiemental log, an ice bucket containing the blood collection tubes, sharp, steril scissors, a permanent marker, and small gauze. Forty-five minutes before starting the blood collection, remove the first mouse from its cage and place it on a suitable surface.
Then mark the tail to quickly identify this mouse from its cage partner. Now use scissors to remove one to two millimeters of the tip of the tail. After clipping that tail carefully stroke the tail until a droplet of blood forms at the tip.
Then collect three microliters of blood and discard both the blood and the tip into the waste bin. Next ensure that the blood flow has stopped. If needed apply gentle pressure with sterile gauze.
Then return the mouse to its cage for six minutes. Now on six minute intervals over forty-five minutes, stroke the tail firmly to take additional blood samples from the mouse. If a blood droplet does not form, dab the tail with a gauze pad soaked in saline to help clear the blood clot.
During the pre-sampling period, discard all the blood aliquots. After forty-five minutes start the sampling period. When taking samples transfer three microliters of blood into a collection tube mix the tube by inversion and put it back on ice.
Now proceed with the sampling for as long as necessary. Once over either euthanize the mice or ensure that their tail blood flow has completely stopped and then provide the mice with a clean cage. Next transfer blood samples to a storage box and store the samples at minus twenty degrees Celsius until they can be analyzed.
Luteinizing Hormone or LH was measured in frequent blood samples to determine the response to an acute psychosocial stress challenge. Adult female C57 black six mice were ovariectomized and blood samples were collected for ninety minutes to establish a pretreatment baseline of LH pulsatile secretion. For the stress treatment during the ninety minute sampling experimental animals were put into rodent restraint devices and moved into a new cage while controls remained in their home cages and were not restrained.
The analysis showed that control animals had clear unambiguous LH pulses throughout the entire sampling period. In contrast the psychosocial stress of the restraint rapidly and unambiguously suppressed pulsatile LH secretion in the experimental group. When preparing to use this technique, it's important to remember to minimize environmental disturbances such as noise or interruptions by other investigators which could stress the mice and interrupt pulsatile LH secretion.
Since its development by Dr.Stein and others in the Chen laboratory this technique has begun to pave the way for researchers in the field of neuroendocrinology to explore LH pulse regulation in a species where complex molecular and genetic tools are practical and available.
