High Compression Video Clips

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Chemokines are hormone-like proteins that help direct the movement of cells through tissues. Several chemokines are suspected to be particularly important in regulating the migration of leukocytes out of the bloodstream at sites of inflammation. We have studied the role of the interleukin-8 (IL-8) family of chemokines which has been implicated in neutrophil movement.

Intravital microscopy allows us to observe leukocytes moving through the iris vessels and infiltrating the surrounding iris tissue. In the first video clip, one can easily see movement through iris blood vessels of a mouse which had received an intravenous injection of a fluorescent stain, rhodamine. At this magnification, individual cells are difficult to discern. Notice that very few cells (seen as dark shadows) are apparent in the spaces between the vessels. Video 11)

Injection of endotoxin into the vitreous of a mouse eye induces an inflammatory response that includes emmigration of neutrophils from the microvasculature of the iris. Six hours after injection many infiltrating cells (dark spots between the vessels) can be seen. Video 12) At higher magnification, one can see several leukocytes sticking to the inner surface of the vessels. (Note: the smaller white particles are rhodamine-labeled platelets). In the first part of this clip, a leukocyte rolls into view in the upper right corner and then sticks. Video 13) In this experiment, the inflammation had already begun to subside at 24 hours after endotoxin injection. There are fewer infiltrating cells Video 14) and fewer sticking and rolling cells Video 15). In these videos, one can also see heterogeneity between vessels. Some vessel segments are heavily involved in leukocyte rolling, sticking, and diapedesis while others are spared.

What happens in mice missing a gene needed to respond to the IL-8 family of chemokines? To study the role of the IL-8 family of chemokines in this process, we also injected endotoxin into mice that have been genetically altered to not express the murine homolog of the human CXCR2 IL-8 receptor (mIL-8Rh). The baseline video Video 16) is similar to that for the controls. Video 11) However at six hours, these mIL-8Rh knockout mice show a striking difference from controls. There are very few infiltrating cells seen Video 17) even though the leukocytes are rolling and sticking to the vessels. Video 18) The recordings made at the 24-hour time point show that the number of infiltrating cells has increased since the six-hour time point Video 19), but is still less than the congenic controls. Video 14) A higher magnification view reveals that rolling and sticking still occurs in some vessel segments at 24 hours. Video 20)

These data suggest that the IL-8 family of chemokines is not critical for leukocyte rolling and sticking during endotoxin-induced uveitis, but is important for migration of the adherent leukocytes across the vessel wall into the surrounding tissue.

These data have been published: 
Becker MD.  O'Rourke LM.  Blackman WS.  Planck SR.  Rosenbaum JT., Reduced leukocyte migration, but normal rolling and arrest, in interleukin-8 receptor homologue knockout mice. Investigative Ophthalmology & Visual Science.  41(7):1812-7, June 2000.

Time-lapse photography
Once the infiltrating cells leave the blood vessels, their movement is too slow to be appreciated with normal video recording.  With the aid of time-lapse photography, a 90 minute recording can be seen in 10 to 20 seconds.  This 6-second video clip of a mouse iris ( Video 22) spans 30 minutes of real time beginning 6 hours after intravitreal injection of endotoxin to induce inflammation.  Video frames were captured every 20 seconds and are set to play back at 15 frames per second.  The movie has been processed by a NASA scientist with an image stabilization program designed to compensate for the movement of the eye due to the mouse's breathing and natural eye rolling.

The refractile dots are infiltrating leukocytes, primarily neutrophils.  Most of the leukocytes are in motion and follow a non-linear path.  Frame-by-frame measurements reveal that the mean speed of these cells is 8 microns (1 millionth of a meter) per minute.  The top speed of indivdual cells ranged from 20 to over 40 microns/minute. These data are consistant with the movement expected of neutrophils looking for bacteria at the site of an infection.
 
The time-lapse technology is a powerful tool that is now being coupled with different cell-labeling methods to study additional aspects of immune responses.  For example, we are looking at the distribution and movement of sentry cells that ingest foreign proteins and activate leukocytes.