March 2012
Volume 53, Issue 3
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Retina  |   March 2012
Recovery of Axonal Transport after Partial Optic Nerve Damage Is Associated with Secondary Retinal Ganglion Cell Death In Vivo
Author Affiliations & Notes
  • Sylvia Prilloff
    From the Institute of Medical Psychology, Otto-von-Guericke University of Magdeburg, Magdeburg, Germany.
  • Petra Henrich-Noack
    From the Institute of Medical Psychology, Otto-von-Guericke University of Magdeburg, Magdeburg, Germany.
  • Bernhard A. Sabel
    From the Institute of Medical Psychology, Otto-von-Guericke University of Magdeburg, Magdeburg, Germany.
  • Corresponding author: Bernhard A. Sabel, Institute of Medical Psychology, Otto-von-Guericke University of Magdeburg, Leipziger Strasse 44, 39120 Magdeburg, Germany; bernhard.sabel@med.ovgu.de
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 1460-1466. doi:10.1167/iovs.11-8306
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      Sylvia Prilloff, Petra Henrich-Noack, Bernhard A. Sabel; Recovery of Axonal Transport after Partial Optic Nerve Damage Is Associated with Secondary Retinal Ganglion Cell Death In Vivo. Invest. Ophthalmol. Vis. Sci. 2012;53(3):1460-1466. doi: 10.1167/iovs.11-8306.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: Traumatic injury of the optic nerve leads to retrograde cell death of retinal ganglion cells (RGCs) but usually a certain percentage of neurons survive. It has been suggested that recovery of axonal transport is beneficial for survival. The present study was therefore performed to provide a synopsis of the temporal pattern of axonal transport decline/recovery and the viability of RGCs after optic nerve crush (ONC).

Methods.: Fluorescent dyes were injected into the superior colliculus to retrogradely label RGCs. Axonal transport kinetics into the RGCs was visualized with in vivo confocal neuroimaging (ICON) in uninjured rats and in rats which had mild or moderate ONC. Red fluorescent beads were injected on day 2 post-ONC and green beads on day 7.

Results.: At 2 to 4 days post-ONC significant axonal transport was detected, but within 1 week the transport of the fluorescent beads was decreased. Interestingly, during post-ONC week 3 the axon transport slowly recovered. However, despite this recovery, retrograde cell death rate continued and was even increased in a “second wave” of cell death in those neurons that displayed axon transport recovery.

Conclusions.: After damage many surviving RGCs lose their axon transport, but after approximately 3 weeks, this transport recovers again, a sign of intrinsic axon repair. Contrary to the prediction, axon transport recovery is not associated with better cell survival but rather with a second wave of cell death. Thus, the accelerated cell death associated with recovery of axon transport suggests the existence of a late retrograde cell death signal.

To observe trauma-induced dynamic changes in retrograde axonal transport in parallel to survival of neurons is of great interest because it may provide valuable information about the relationship between axonal transport recovery and neuronal viability. In the present study we used a double-labeling approach in the adult optic nerve crush model to address the following questions: (1) To which extent and when is axonal transport interrupted after mild or moderate lesions; (2) Is there recovery of axonal transport and, if so, what is its temporal pattern; and (3) Is axon transport recovery associated with increased or decreased retinal ganglion cell (RGC) survival? 
These questions have already been addressed before. 1 3 However, with the imaging method used here we were able for the first time to provide an in vivo synopsis of axonal transport dynamics and neuronal survival over time on a cellular level with the method of “in vivo confocal neuroimaging” (ICON). ICON was developed in our laboratory 4 and it is based on principles used in the standard repertoire of neurobiology: retrograde axonal fluorescent latex microspheres can be used to elucidate the organization of neural connectivities and circuits 5 7 and they can be used to label cells in tissue cultures. 8,9 Previous double-labeling studies have been used to determine the target areas of specific neuronal axons and axon collaterals. 10 12 With ICON, however, we are able to visualize individual neurons noninvasively in vivo for several weeks. This can reveal new aspects about the relation of different posttraumatic pathophysiological events 13 such as axon transport and cell survival. 
Until now the prevailing view has been that the early and extensive disturbance of axonal transport after nerve injury 3 is detrimental for neuron survival because of, for example, interrupted growth factor supply. 14 In line with this theory retrograde trafficking of injury signals was reported to upregulate “regeneration associated genes” in the cell body. 15 In contrast to this “helper function” of axon transport, it is also conceivable that cell survival of a disconnected cell does, in fact, not depend on any retrograde axonal signaling because so far there is no direct in vivo evidence for it. Rather, current knowledge builds on experiments that studied retrograde cell death after nerve injury in separate groups of animals which were all euthanized before tissue analysis. 1,2 Our study, in contrast, evaluates live tissue where the cell fate can be followed over time. With ICON we were able to image one and the same population of neurons again and again which is a much more sensitive method than the observation of dead tissue to detect the dynamics of RGC. Using this in vivo set-up we now show that impaired axon transport after optic nerve trauma recovers in the first 2 to 3 weeks, but cells with recovered axon transport do not show better survival rates but are rather more likely to die in a second wave of cell death. 
Material and Methods
Animals
Twenty-four male hooded rats (HsdOla:LH; two per cage) weighing approximately 250 g at the beginning of the experiment were used. The rats were kept on a 12-hour light/12-hour dark cycle at an ambient temperature of 24°C-26°C and a humidity of 65%. Food and water were available ad libitum. For all procedures ethical approval was obtained according to the requirement of the German National Act on the use of Experimental animals, and all animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Surgery
RGC labeling was carried out under anesthesia with ketamine (Ketamin 50 mg/kg intraperitoneally [IP]; Pharmacia GmbH, Erlangen, Germany) and xylazine (Rompun; 10 mg/kg IP; Bayer Vital GmbH, Leverkusen, Germany) by stereotactic injection of 2 μL of carboxylate-modified microspheres (FluoSpheres; Invitrogen GmbH, Karlsruhe, Germany), 0.04 μm, green fluorescent (Invitrogen GmbH) (supplied at 5% solids in water without any sodium azide in the suspension) and/or carboxylate-modified microspheres (FluoSpheres), 0.04 μm, red fluorescent (Invitrogen GmbH) (supplied at 5% solids in water without any sodium azide in the suspension) into the right superior colliculus. For this purpose the cranium was exposed and opened above the tectum, coordinates (oriented to Bregma and Lambda): anterior-posterior 6.9 mm, lateral 1.2 mm. The injections (0.5 μL each) were made at each of the following positions below the dura: 4.0 mm, 3.5 mm, 3.0 mm, and 2.5 mm. Approximately 20 seconds were allowed to elapse between each injection to facilitate dye diffusion. The same procedure was performed for injection of two dyes. 
Partial optic nerve crush was carried out as previously described. 16 18 Briefly, under anesthesia the optic nerve was exposed, leaving both retinal blood supply and dura intact. The nerve was then crushed with a calibrated forceps for 30 seconds with two different crush intensities: “mild” crush (n = 6) was performed with the jaws of the forceps 0.2 mm apart in “rest-position ”; in “moderate” crush (n = 6) the jaws of the forceps were separated by 0.1 mm in “rest-position.” 
Experimental Schedules
Control rats without crush (n = 12) were randomly assigned to two groups: half of the animals (n = 6) received an initial injection of green fluorescent microspheres and 8 days later a second injection with red beads. The other 6 rats received first the red and then the green beads. ICON was performed on day 2, 4, 6, 10, 12, 14, and 16 (with reference to the first injection). In rats with crush (n = 12) the sequence of injection was the same for all animals: red beads were injected 2 days after crush and green beads 7 days after crush. There were two different groups of lesioned animals according to the severity of the impact: half of the rats (n = 6) received a mild crush and the other half was exposed to a moderate crush. ICON was performed on days 4, 9, 11, 13, 15, 17, 19, and 21 after optic nerve crush (ONC) (see Figs. 2, 3). 
ICON Procedure
The ICON method has been described in detail elsewhere. 4 Briefly, rats were anesthetized IP (Ketamin 50 mg/kg, Rompun 10 mg/kg) and phenylephrine (Neosynephrine-POS 2.5%; Ursapharm, Saarbrücken, Germany) was applied onto the surface of the eye to relax the pupil and reduce eye movements (Fig. 1a). Gel (Vidisic; M. Pharma, Berlin, Germany) was applied to a contact lens as an immersion medium to prevent desiccation of the cornea. This Hruby-style −80 diopter plano-concave contact lens (KPC-013, Newport GmbH, Darmstadt, Germany) was placed on the cornea surface to refocus the laser onto the retina. The rat was then placed onto a modified stage of a confocal laser microscope (Odyssey-XL; Noran Instruments GmbH, Bruchsaal, Germany) with the eye being directly placed under the long-range objective so that the fluorescent-labeled RGCs could be visualized. In vivo imaging of RGCs was accomplished by focusing the objective (magnification, ×4) onto the retina. The scanning time of the confocal laser scanning microscope was set at 400 ns (15 pictures per second) with an average of 32 scanning acquisitions. Because fluorescent red and green beads have different excitation and emission wavelengths, cells labeled with the two different dyes could be visualized by changing the laser for excitation and channel filters on the microscope (Fig. 1b). 
Figure 1.
 
ICON-setup. (a) An anesthetized rat placed under the confocal laser scanning microscope. The zoom shows a contact lens placed onto the rat eye to adapt the optics so that individual neurons can be visualized after cells were labeled both with red and green beads as shown in (b). This image was taken in an uninjured rat on day 12 after first injection of red beads (corresponding to day 4 after the second injection of green beads) which was retrogradely transported into the RGCs after injection into the superior colliculus. Scale bar, 50 μm. bv, blood vessel.
Figure 1.
 
ICON-setup. (a) An anesthetized rat placed under the confocal laser scanning microscope. The zoom shows a contact lens placed onto the rat eye to adapt the optics so that individual neurons can be visualized after cells were labeled both with red and green beads as shown in (b). This image was taken in an uninjured rat on day 12 after first injection of red beads (corresponding to day 4 after the second injection of green beads) which was retrogradely transported into the RGCs after injection into the superior colliculus. Scale bar, 50 μm. bv, blood vessel.
To follow the same RGCs over time, the laser was focused (magnification, ×4 objective lens) on a 0.08 mm2 region located at approximately 30° eccentricity (which is approximately 2.0 mm lateral to the optic disc). We determined for each rat as previously described 13 a well-defined area which contained a sufficient number of cells and which could easily be relocated at repeated time points using blood vessel morphology as landmarks. The images were obtained by using a scanning time of 400 ns (15 pictures per second) and an average of 32 scanning acquisitions. 
Results
Double-Labeling of RGCs in Unlesioned Rats
The dye injection method was first verified in naive animals to determine how long it takes for each dye to be transported retrogradely through the optic nerve to the RGCs. The injection of the second dye was carried out to check (1) if double-labeling induces any modification of cell number or appearance, (2) if the presence of one type of fluorescent bead interferes with the visualization of the other, (3) if the double-labeling can be properly visualized at sufficient intensities, (4) if the signal is stable over time, and (5) if the cells could be repeatedly visualized at different sessions (days). 
The uptake rates of the red and green beads followed a very similar temporal pattern: regarding the red labeling partial staining was seen already on postinjection day 2, with increasing numbers of labeled cells until day 6. Of all cells labeled at that time, only 33% were already visible on day 2 after dye injection. At the same time point, in the group injected with green beads, a similar percentage of cells were labeled (28%). On day 4 postinjection, the percentages of red and green labeled RGCs were 69% and 63%, respectively, compared with the number on day 6. Thus, there was a similar gradual increase in the number of red and green labeled cells during the first 6 days. 
After injection of the second dye, the pooled data of the red/green and green/red labeling schemes were used for kinematic analysis of double-labeling. Like the dynamics of the single-labeling, the number of double-labeled cells increased from day 2 through day 6 counted from the time point of the second dye injection. By day 6 after the second injection, 96% of all previously analyzed single-labeled cells were now double-labeled. Thus, those cells that were labeled by one dye were also labeled by the second dye. As with single-labeling, there was almost no change in the extent of labeling after day 6: at day 8 the percentage of double-labeled RGCs was 97%. 
In summary, confirming our previous studies, 19,20 labeled RGCs were detectable already 2 days after injecting the dye into the superior colliculus; the number of stained neurons increased during the first 6 days after dye injection. Furthermore, the second dye injection followed the same time course and uptake. These results demonstrate that it is possible to visualize and study the kinematics of axonal transport in vivo by injecting two different fluorescent dyes into the superior colliculus and to observe the retrogradely transported fluorescent dye in the retina. This knowledge that trafficking and loading of first and second dye are the same was a prerequisite for studying axonal transport after trauma as now described. 
Double-Labeling after ONC
Double-Labeling after Mild ONC.
All data concerning mild ONC are presented in Figure 2, which shows the labeling pattern of the retinal ganglion cells and their respective fate in a time series. 
Figure 2.
 
Schematic diagram of labeling dynamics after mild crush (0.2 mm). The boxes give the numbers of RGCs labeled with red beads or with both markers on every data collection point. Double-labeled cells are differentiated in “persisting” (vertical color arrangement) and “newly appearing” (horizontal color arrangement). The numbers of dead cells represent these individual fluorescent cells which have disappeared since the last ICON sessions. The reduced numbers of red labeled cells during the course of the experiment result from cells disappearing (dying) and cells becoming double-labeled. The two curves on the left side of the diagram illustrate the temporal pattern of cell death (sum of all cells having disappeared since the last ICON session) in parallel with the retrograde axonal transport (sum of all cells which are newly labeled since the last ICON analysis). nr, number.
Figure 2.
 
Schematic diagram of labeling dynamics after mild crush (0.2 mm). The boxes give the numbers of RGCs labeled with red beads or with both markers on every data collection point. Double-labeled cells are differentiated in “persisting” (vertical color arrangement) and “newly appearing” (horizontal color arrangement). The numbers of dead cells represent these individual fluorescent cells which have disappeared since the last ICON sessions. The reduced numbers of red labeled cells during the course of the experiment result from cells disappearing (dying) and cells becoming double-labeled. The two curves on the left side of the diagram illustrate the temporal pattern of cell death (sum of all cells having disappeared since the last ICON session) in parallel with the retrograde axonal transport (sum of all cells which are newly labeled since the last ICON analysis). nr, number.
Injection of First Dye (Red)—Labeling Dynamics and Death Rates.
Four days after mild ONC (which corresponds to 2 days after injection of the red beads) we observed a pronounced cell labeling with red fluorescence (190 cells in total). Clearly, cells that had died or lost their axon connections in the first 2 days could not be labeled. Until day 21 post ONC, no newly red-labeled cells appeared, but several of the red labeled cells disappeared (presumably they died). Between days 9 to 11, and 17 to 21 post ONC we noted increased death rates of 27%, 11% to 15%, respectively, in the red-labeled cell population, whereas between all other data collection points (days 4 to 9, 11 to 13, 13 to 15, and 15 to 17) only 0 to 3% of red cells died. Thus, we noticed two “waves of cell death.” 
Injection of Second Dye (Green)—Double-Labeling Dynamics.
The injection of the second (green) dye did not lead to such a massive and immediate (double-) labeling as seen after injection of the red dye: on day 9—2 days after the injection of the second dye—only 17 cells with combined green and red/green fluorescence were detectable. This is significantly less than the 190 red-labeled cells 2 days after the first (red) dye injection. However, unlike in control animals without lesions (see above), even beyond post injection day 6, new green and red/green double-labeled cells appeared in the ONC group. But also unlike in unlesioned animals the second (green) dye never labeled all cells stained with the first (red) dye. Only 58% of the surviving cells containing the first dye injected (red) were double-labeled at the end of the experiment (i.e., 14 days after injection of the second [green]) dye; for comparison: in control animals 96% double-labeling was seen on day 6 postinjection of the second dye). 
The number of newly appearing double-labeled cells was decreased on days 11, 13, and 15, but recovered to increased labeling rates again on days 17 through 21. This can be taken as a sign that axonal transport was temporarily suppressed with a delayed recovery at postoperative days 17 through 21. 
Injection of Green Dye—Cell Death Dynamics of Double-Labeled Cells.
Interestingly, this partially restored axonal transport did not guarantee cell survival. Rather, on the contrary: except for post lesion period days 9 to 11, the death rate was always higher in the double-labeled population in comparison with the single red labeled neurons. For example, 32% of all double-labeled cells counted on day 19 died by day 21, but only 15% of single red labeled cells died. This shows that cell death rate was doubled in those cells with recovered axon transport and points toward the possibility of a retrogradely transported cell death signal that originates from the deafferented target. 
Final Outcome.
On the last day (21) post ONC, 44% of all cells counted at the first ICON analysis post ONC (day 4) were still alive. Of these surviving neurons 42% were still single red-labeled and 58% were double-labeled. On day 21 after the lesion nine cells were only green-labeled (10% of all cells alive). 
Double-Labeling after Moderate ONC.
All data concerning moderate ONC are presented in Figure 3. Injection schedule and data collection points were identical with the procedures for the mild ONC. 
Figure 3.
 
Schematic diagram of labeling dynamics after moderate crush (0.1 mm). The systematic of the illustration is the same as in Figure 2. The boxes give the numbers of RGCs labeled with red beads or with both markers on every data collection point. Double-labeled cells are differentiated in “persisting” (vertical color arrangement) and “newly appearing” (horizontal color arrangement). The numbers of dead cells represent these individual fluorescent cells which have disappeared since the last ICON sessions. The reduced numbers of red labeled cells during the course of the experiment result from cells disappearing (dying) and cells becoming double-labeled. The two curves on the left side of the diagram illustrate the temporal pattern of cell death (sum of all cells having disappeared since the last ICON session) in parallel with the retrograde axonal transport (sum of all cells which are newly labeled since the last ICON analysis).
Figure 3.
 
Schematic diagram of labeling dynamics after moderate crush (0.1 mm). The systematic of the illustration is the same as in Figure 2. The boxes give the numbers of RGCs labeled with red beads or with both markers on every data collection point. Double-labeled cells are differentiated in “persisting” (vertical color arrangement) and “newly appearing” (horizontal color arrangement). The numbers of dead cells represent these individual fluorescent cells which have disappeared since the last ICON sessions. The reduced numbers of red labeled cells during the course of the experiment result from cells disappearing (dying) and cells becoming double-labeled. The two curves on the left side of the diagram illustrate the temporal pattern of cell death (sum of all cells having disappeared since the last ICON session) in parallel with the retrograde axonal transport (sum of all cells which are newly labeled since the last ICON analysis).
Similarities with Labeling after Mild ONC.
Like in the mild crush group we found significant axonal transport on days 2 to 4 post ONC (injection/analysis of red beads) but no further red labeling. Also similar to the mild crush group axonal transport decreased after day 7 (injection of second [green] dye) and recovered partly toward the end of our experiment (day 21). 
Differences to Double-Labeling after Mild Crush.
However, there are also differences in the details of the axonal transport decline/recovery dynamics and neuronal cell death between mild and moderately lesioned animals: unlike the mildly injured rats, there was a complete interruption of axonal transport for several days (no new green labeling from days 11 to 15) and there was no cell death detectable at all during this period. 
Final Outcome.
On the last day of assessment (day 21 post ONC), 66% of all cells counted at the first ICON session on day 4 post ONC were still alive. Of these surviving neurons 63% were still single red-labeled and 37% were double-labeled (in contrast to mild crush, where more double-labeled cells than single-labeled cells were present on day 21 post ONC). As in the mild crush group, a few green-only cells appeared during the course of the experiment (24% of all cells alive). 
Discussion
We established a combination of an in vivo double-labeling and an in vivo double-tracing approach by applying two fluorescent dyes in living animals. We also were able to detect the temporal/spatial pattern of labeling repetitively in vivo. The goal was to study the relationship between the fate of the cell (death/survival) and the recovery of axonal transport in living tissue. 
In comparison to the kinematics of (double)-labeling in unoperated animals, optic nerve damage significantly influenced the axonal transport dynamics:
  1.  
    There were two waves of increased retrograde axonal transport: within the first days after ONC and approximately 3 weeks post lesion;
  2.  
    There was a delayed wave of increased cell death around 3 weeks post ONC; and
  3.  
    Recovery of axonal transport was not associated with improved survival but rather enhanced cell death.
The fact that there is preserved trafficking between days 2 to 4 post ONC is surprising, as so far mainly the deteriorating effects of impaired/interrupted axonal transport after traumatic brain injury have been observed. 21 However, our data suggest that during this early time-window axonal transport may be even accelerated or, another possibility, slow transport mechanisms are specifically destroyed. These hypotheses are based on the observations that maximum labeling was accomplished already 2 days after injection (in unlesioned animals it took 6 days until maximum labeling was achieved). This effect is unlikely due to completely interrupted axonal transport after day 2 as we did see retrograde transport in the very same cells when a second dye (green) was later injected. 
Results from other laboratories investigating peripheral nerve injury 22 are in line with our hypothesis of a preserved/enhanced axonal transport after injury. For example, retrograde axonal transport of growth factors is accelerated after injury and this increased trafficking is not only restricted to the growth factor protein family but applies more generally to axonal transport of different substances. 23,24 Similar dynamics of axonal transport are actually also seen after nerve crush damage in the central nervous system. 3,25 However, labeled biologic or injected artificial tracers did not cross the lesion site. Traumatic axonal injury is even used as a “stop-crush” model where the interrupted axonal transport is used as a tool to quantify the kinetics of accumulating substances. 26 However, on the other hand, it has been demonstrated that different substances are retrogradely transported by different mechanisms. 
The fluorescent dyes used in the present study are basically nanoparticles. It might be that such nanoscale beads are transported by different mechanisms—maybe independent of the microtubules/axonal cytoskeleton which is already severely deranged during the first days after ONC and which causes blockage of other tracers. This assumption of different ways/kinetics of axonal transport is supported by data showing that, for example, modifications of the horseradish peroxidase lead to different trafficking kinetics. 27 We therefore propose that early after ONC a window is open for retrograde (nonclassical) axonal transport, but this window is not only temporally limited but also restricted to selected chemical/particular entities. Another possible explanation of retrograde RGCs labeling in a 2-day period is that the posttraumatic “transport window” is exclusively open for the fast transport mechanism. This would mean that we actually do not see the effects of acceleration/enhancement but a “cutoff” of the slow transport. Clearly, this issue requires further study. 
However, it is now also widely accepted that traumatic axon injury is a progressive event and does not only lead to an immediate but also delayed axonal disconnection. 28 As this process is probably dependent on the characteristics and severity of the damage, 29 the functional disturbance may vary widely and the fact that we observed preserved axonal transport on day 2 to 4 post ONC may be a situation that is unique to our neurotrauma crush model. 
In addition to the effect of a measurable transport early after ONC our data reveal a decreased/interrupted trafficking (with a minimum at approximately 2 weeks after lesion) and a partial recovery of axonal transport at 3 weeks post ONC. This temporal pattern of decrease and subsequent recovery is in good agreement with earlier data from our laboratory. Here we showed initial damage to be followed by repair after ONC with classical histologic/immunohistochemical approaches. 25 Also in that study indicators of axonal transport and integrity transiently decreased and then recovered at 3 weeks. Similar trends within this 3-week time window have been found with other parameters: visual function is lost transiently after a lesion but spontaneously improves within a few weeks, 30,31 cellular activity in the deafferented tectum is decreased after traumatic nerve injury but recovers within this period, 32 and structural repair of the axon has been shown with immunohistochemical techniques 3 weeks after ONC. 33 Especially these immunohistochemical studies provide interesting results regarding the recovery mechanisms of axonal transport. It was shown that the structure of neurofilament-H (an element of the axonal cytoskeleton) was partially repaired on day 21 after ONC and that this was accompanied by a recovery of axonal transport. Therefore, the temporal profile of RGC labeling in the present study confirms the hypothesis that there is internal axon repair or regeneration of the damaged cytoskeleton within 3 weeks after ONC which is why axonal transport can recover as well. 
Another interesting aspect of our study is that this recovery of axonal transport was not associated with increased neuronal survival; rather the contrary was seen: the extent of delayed cell death was similar (moderate crush group) or even higher (mild crush group) in the population of cells with recovered trafficking (i.e., which were double labeled). Although ongoing neuronal death has been detected already in previous studies, 4,19,20 the precise temporal pattern of cell loss was not systematically analyzed. The current results suggest that restoration of axonal transport does not prevent delayed neuronal death after diffuse traumatic brain injury but rather accelerates it. This might point toward the existence of a cell death signal in the deafferented target, but such a potential association between restoration of nerve function and cell degeneration clearly needs to be explored further. 
A secondary cell death scenario has already been described previously with experiments using classic labeling/staining techniques. They found signs of “secondary degeneration” (i.e., delayed death) as a function of the severity of the primary insult. 34  
On the one hand, the increased cell death rate during the period of restoration of axonal transport is rather surprising as it is generally assumed that retrograde transport of growth factors (e.g., brain-derived neurotrophic factor [BDNF]) may be beneficial to prevent cell death caused by apoptosis. 35,36 On the other hand, there are multiple sources of growth factors (e.g., Müller cells 37 ) and it is not exactly clear whether the RGCs die from a lack of trophic supply after ONC. The existing data actually do not seem to support the hypothesis that a preserved axonal transport or even the existence of the axon is a condition sine qua non for neuron survival. Experiments using paclitaxel (Taxol; Sigma Aldrich Chemie GmbH, Munich, Germany), an axonal transport inhibitor, showed that this changed the state of the deranged cytoskeleton for the worse and deteriorated axonal trafficking. 25,38 Nevertheless, neurons appeared to have survived even better when this compound was applied. 38 Furthermore, studies of the pathophysiology of optic nerve cut report that a certain percentage (10% to 15% 39 ) of RGCs survive, even though no retrograde supply via axonal transport is possible. In a recent elegant study, Leung et al. 40 followed the slow degenerating process of RGCs after mild ONC. They showed that although neurons lost their axons within 3 weeks post lesion, some of these cells were still alive more than 2 months later. In summary, the data of all these studies are compatible with the concept that while axonal damage may trigger retrograde cell death, repair of axonal transport does not support neuronal survival. But, to the contrary, restored trafficking may actually accelerate cell death. 
We would like to point out that the current protocol was designed to analyze the dynamics of labeling/axonal transport post ONC in vivo. It was not designed to compare the absolute number of labeled (surviving) cells, as this depends on the individual retinal region chosen for imaging. However, the fact that after mild crush we find 190 red cells labeled and in the moderate crush group we find only 97 red RGCs 2 days after the first dye injection is in line with the assumption that after a more severe lesion axonal transport is more impaired or, alternatively, impaired in more neurons. 
In summary, our study demonstrates the usefulness of in vivo tracing studies with two different dyes and subsequent in vivo ICON imaging as a sensitive tool to relate cell death with the axon transport ability of individual cells. In our model of diffuse and incomplete nerve trauma axonal transport recovers within 3 weeks after the impact. We take this as a sign of intrinsic axon repair. 
Our second main finding is a delayed, secondary wave of neuronal death which not only occurred despite the axonal transport repair but actually perhaps because of it. Thus, recovered axon transport does not enhance neuronal survival but, to the contrary, actually increases cell death. This possibility of an axon transport-dependent cell death suggests the existence of a delayed cell death signal (“kiss of death”) which may be transported retrogradely from the deafferented target. 41 Uncovering such factors will likely lead not only to new insights into mechanisms of neuronal death but also provide new clues in our search for means to enhance restoration (recovery) of vision. 
Footnotes
 Supported by the Ministry of Education and Research (BMBF, 01ZZ0407) and Sibylle Assmus Foundation (PH-N).
Footnotes
 Disclosure: S. Prilloff, None; P. Henrich-Noack, None; B.A. Sabel, None
The authors thank Steffi Matzke for administrative assistance and Uta Werner for excellent technical assistance. 
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Figure 1.
 
ICON-setup. (a) An anesthetized rat placed under the confocal laser scanning microscope. The zoom shows a contact lens placed onto the rat eye to adapt the optics so that individual neurons can be visualized after cells were labeled both with red and green beads as shown in (b). This image was taken in an uninjured rat on day 12 after first injection of red beads (corresponding to day 4 after the second injection of green beads) which was retrogradely transported into the RGCs after injection into the superior colliculus. Scale bar, 50 μm. bv, blood vessel.
Figure 1.
 
ICON-setup. (a) An anesthetized rat placed under the confocal laser scanning microscope. The zoom shows a contact lens placed onto the rat eye to adapt the optics so that individual neurons can be visualized after cells were labeled both with red and green beads as shown in (b). This image was taken in an uninjured rat on day 12 after first injection of red beads (corresponding to day 4 after the second injection of green beads) which was retrogradely transported into the RGCs after injection into the superior colliculus. Scale bar, 50 μm. bv, blood vessel.
Figure 2.
 
Schematic diagram of labeling dynamics after mild crush (0.2 mm). The boxes give the numbers of RGCs labeled with red beads or with both markers on every data collection point. Double-labeled cells are differentiated in “persisting” (vertical color arrangement) and “newly appearing” (horizontal color arrangement). The numbers of dead cells represent these individual fluorescent cells which have disappeared since the last ICON sessions. The reduced numbers of red labeled cells during the course of the experiment result from cells disappearing (dying) and cells becoming double-labeled. The two curves on the left side of the diagram illustrate the temporal pattern of cell death (sum of all cells having disappeared since the last ICON session) in parallel with the retrograde axonal transport (sum of all cells which are newly labeled since the last ICON analysis). nr, number.
Figure 2.
 
Schematic diagram of labeling dynamics after mild crush (0.2 mm). The boxes give the numbers of RGCs labeled with red beads or with both markers on every data collection point. Double-labeled cells are differentiated in “persisting” (vertical color arrangement) and “newly appearing” (horizontal color arrangement). The numbers of dead cells represent these individual fluorescent cells which have disappeared since the last ICON sessions. The reduced numbers of red labeled cells during the course of the experiment result from cells disappearing (dying) and cells becoming double-labeled. The two curves on the left side of the diagram illustrate the temporal pattern of cell death (sum of all cells having disappeared since the last ICON session) in parallel with the retrograde axonal transport (sum of all cells which are newly labeled since the last ICON analysis). nr, number.
Figure 3.
 
Schematic diagram of labeling dynamics after moderate crush (0.1 mm). The systematic of the illustration is the same as in Figure 2. The boxes give the numbers of RGCs labeled with red beads or with both markers on every data collection point. Double-labeled cells are differentiated in “persisting” (vertical color arrangement) and “newly appearing” (horizontal color arrangement). The numbers of dead cells represent these individual fluorescent cells which have disappeared since the last ICON sessions. The reduced numbers of red labeled cells during the course of the experiment result from cells disappearing (dying) and cells becoming double-labeled. The two curves on the left side of the diagram illustrate the temporal pattern of cell death (sum of all cells having disappeared since the last ICON session) in parallel with the retrograde axonal transport (sum of all cells which are newly labeled since the last ICON analysis).
Figure 3.
 
Schematic diagram of labeling dynamics after moderate crush (0.1 mm). The systematic of the illustration is the same as in Figure 2. The boxes give the numbers of RGCs labeled with red beads or with both markers on every data collection point. Double-labeled cells are differentiated in “persisting” (vertical color arrangement) and “newly appearing” (horizontal color arrangement). The numbers of dead cells represent these individual fluorescent cells which have disappeared since the last ICON sessions. The reduced numbers of red labeled cells during the course of the experiment result from cells disappearing (dying) and cells becoming double-labeled. The two curves on the left side of the diagram illustrate the temporal pattern of cell death (sum of all cells having disappeared since the last ICON session) in parallel with the retrograde axonal transport (sum of all cells which are newly labeled since the last ICON analysis).
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