Blebs are herniations of the cell membrane. They are commonly associated with both necrotic and apoptotic cell death, although they are often seen during growth and morphological changes of many cell types. In hair cells, blebs typically appear in response to various insults, such as acoustic trauma, ischemia, application of extracellular ATP, and application of ototoxic drugs, as well as during development in the bustling mouse. Although blebs can apear in the subnuclear region of the cell, they typically form on the apical surface. Such blebs grow from the region above the basal body of the kinocilium (which sits in a notch in the cuticular plate). The images below show a schematic drawing of a typical hair cell on the left, and a similar hair cell with a bleb on the right.
(Click on any image to see a larger version)
Blebs have been seen in a wide variety of hair cell organs in a wide variety of species. This poster discusses in particular blebs in the cochlea of the alligator lizard. The image on the left (from Tilney et al, 1982) shows an SEM of the tectorial region of an alligator lizard cochlea, in which nearly every hair cell contains blebs (the spheres in front of the hair bundles). This cochlea was exposed to acoustic trauma (110 dB SPL for 24 hours) which typically induced a temporary threshold shift (TTS) in these lizards; that is, the lizards recovered normal hearing thresholds after exposure. When the authors of that study looked in ears that had recovered from TTS, the blebs were gone; presumably the cells had recovered from blebbing (a similar recovery has been seen in mammalian cochleae). Blebbing is also seen in hair cells immediately following acoustic trauma that induces permanent threshold shifts (PTS); the image on the right shows an SEM of a guinea pig cochlea exposed to impulse trauma with a peak pressure of 160 dB SPL, from a study by Hamernik et al (1984). Blebs are visible as spheres next to most inner hair cells near the bottom of the image. Taken together, these results imply that hair cells can recover normal function after blebbing, but do not necessarily do so.
Blebbing is also commonly seen in in vitro studies of hair cells. However, in such studies blebbing is commonly assumed to be synonymous with cell death. Although this assumption may be valid in practice, hair cells in vitro are often isolated and/or bathed in a single medium, which may restrict their ability to recover. In addition, as will be shown below, blebs can be very difficult to see under many imaging conditions; this fact often results in blebs being overlooked or ignored until they grow to be very large, at which point the cells may indeed be dead or dying.
In this study we examined the effect of bathing media on the ability of hair cells to recover from blebbing. To simulate the in vivo environment as closely as possible, we isolated intact cochleae from southern alligator lizards (Gerrhonotus multicarinatus and clamped them between two fluid spaces which could be perfused separately. Blebbing was typically induced by the surical isolation procedure. By perfusing different media on each side of the cochlea and observing their effect on bleb size, we identified several factors that influence the growth of blebs and the subsequent recovery from blebbing.
The image below, taken using brightfield illumination microscopy, shows the tectorial region of the alligator lizard cochlea immediately following surgical dissection. The cochlea is seen from the top; regions with small circles (such as the one pointed to by the black arrowhead) are hair bundles; the circles within these regions are individual stereocilia. Although this preparation has some obvious blebs (e.g., black arrow), many others are much harder to see (white arrows). Consequently, images of our preparation had to be examined one bundle at a time, explicitly searching for blebs, in order to assess the extent of blebbing. Several other research groups have provided us with anecdotal evidence to suggest that blebs are practically invisible unless one is looking for them.
Although blebs can be hard to see in some conditions, they are painfully obvious in others. For example, if we maintain the cochlea in a typical in vitro environment, in which the cells are bathed with artificial perilymph (AP) on all sides, blebs grow over time to become very large. The left-hand image below shows a region of one cochlea immediately following dissection. Blebs are visible to the left of each hair bundle in this image. The right-hand image shows the same cochlea after an additional hour of being bathed in artificial perilymph on all sides. In this image the blebs fill the entire field of view, so the hair bundles are no longer visible. The poster shows a plot of bleb diameter vs. time in AP for several blebs; bleb diameter typically saturates at 20 - 30 micrometers after 10 - 30 minutes of growth. This growth of blebs was seen consistently in more than 80 cochleae.
Our initial hypothesis was that maintaining the cochlea in an environment that more closely resembled the in vivo condition would aid in the recovery of hair cells from blebbing. To this end we bathed several cohcleae with AP on the basolateral surface and artificial endolymph (AE) on the apical surface (we refer to this bathing condition as AE/AP). Because the surgery was performed in a single fluid (AP), blebs were present immediately following dissection. However, in about half the cochleae in which we bathed AE apically, these blebs shrank over time and eventually disappeared entirely. The images below show part of a cochlea immediately after dissection (left-hand image) and the same cochlea two hours later (right-hand image). Blebs are present immediately following dissection, but are not visible after two hours in the AE/AP medium. The poster shows a plot of bleb size vs. time as blebs shrank in this bathing condition. Note that the growth is relatively slow, compared to osmotic swelling (which typically takes no more than a few minutes to saturate).
Although the AE/AP bathing condition allowed blebs to shrink in half the cochleae we examined, blebs continued to grow in the other cochleae. However, as shown in a plot on the poster, the growth occurred more slowly in AE/AP than when AP was bathed on all surfaces. Because of this result, and because blebs were typically already present immediately following dissection, we reasoned that the state of the cochlea during dissection was important in determining whether hair cells could recover from blebbing. Since only a single bathing medium can be used during dissection, we examined whether changing that bathing medium had an effect on blebbing.
We discovered several changes to the dissection medium that affected blebbing. Bleb growth was stopped when Na+ was replaced by NMDG+, a large, relatively impermeant cation. The same result was achieved by replacing Cl- by gluconate-, a poorly permeant anion. Subsequently perfusing AP led to bleb growth. These results suggest that bleb growth occurs by concomitant entry of Na+ (or, in some cases, possibly K+) and Cl-.
Bleb growth was also stopped by adding 1 mM Gd+3 to AP. The left-hand image below shows a cochlea dissected in AP with Gd+3. Note that no blebs are visible in this image. Removing Gd+3 from the apical surface led to bleb growth. The center image shows the same cochlea after being bathed with Gd+3-AP basally and AE apically for 22 minutes. Blebs are visible to the right of each hair bundle. This bleb growth was reversible; subsequently bathing Gd+3-AP apically caused blebs to shrink and disappear, as shown in the right-hand image. These results suggest that the entry of ions through Gd+3-sensitive channels in the apical surface are sufficient to allow bleb growth.
Gd+3 blocks several channels, including the hair cell transduction channels, stretch-activated channels, and many voltage-dependent channels. However, blocking transduction channels with gentamicin had no effect on blebbing. In addition, a 10-minute exposure to EGTA-buffered, Ca+2-free AP following dissection in NMDG did not prevent subsequent bleb growth in AP. These results suggest that the transduction channel is not the primary site for ion entry in blebbing.
The presence of Ca+2 may be important during an early stage of blebbing, however. Dissecting cochleae in EGTA-buffered, Ca+2-free medium prevented bleb formation, even when cochleae were subsequently bathed in AP. These results suggest that blebbing has a Ca+2-dependent initiation stage (such as Ca+2 entry through transduction channels) followed by a Ca+2-independent growth stage (such as Na+ entry through stretch-activated channels).PDF version of the poster.