In our experimental design the mice were separated into five groups. Each group was divided into age-matched cohorts of mice that received intravitreal injection of C+balanced salt solution and control animals that received intravitreal balanced salt solution alone. The caspofungin dose across each group was 1×, 3×, 6×, 10×, or 100× the MIC90 for Aspergillus spp. (0.41–41 μM). Each week for 5 weeks, injections were performed on a different dose group. Seven weeks after the injections, ERGs were recorded and histology samples collected from each group, thereby resulting in a total duration of 12 weeks for the entire study. The ERG a- and b-wave amplitudes for the balanced salt solution–injected control eyes for the different groups were not significantly different from one another (one-way ANOVA: a-wave, F(4,17) = 0.31, P = 0.86; scotopic b-wave Vmax [from Naka-Rushton fit, described later], F(4,17) = 0.10, P = 0.97; mixed b-wave amplitude [2.3 log sc td s], F(4,17) = 0.17, P = 0.94). These observations indicate that our ERG results across the total duration of the study had minimal confounders due to variabilities in the damage caused by the injection procedure, ERG recording settings, differing anesthesia depths, body temperature, and levels of dark-adaptation between sessions. Intravitreal injections did not produce observable histologic changes to the retina in the balanced salt solution–injected control eyes 7-weeks after injection, removing the injection process itself as a significant confounder in the interpretation of our results (see ).
Figure 4. Histopathologic examination of radial sections of mice eyes 7 weeks after intravitreal injection of balanced salt solution or incrementing concentrations of C+balanced salt solution. Superimposed on a phase contrast image are DAPI stained nuclei in various (more ...)
The raw ERGs recorded for those eyes injected intravitreally with 0.41, 1.2, 2.5, or 4.1 μM caspofungin were very similar to those of the mice injected with balanced salt solution alone. However, the mice that received a dose of 41 μM caspofungin showed reduced amplitudes (). The ERGs were analyzed for changes in the photoreceptor-derived, negatively going a-wave measured from the baseline to its trough and for changes in the ON-bipolar derived b-wave, with amplitudes measured from the a-wave trough to the b-wave peak.
Figure 1. Effects of intravitreal caspofungin on the ERG flash response. Representative ERG responses subjects injected with intravitreal balanced salt solution or various doses of C+balanced salt solution to brief flashes of increasing stimulus energies, as indicated, (more ...)
To interpret the ERG data in terms of alteration in retinal physiology after caspofungin injection, we plotted the mean b- and a-wave amplitudes of the C+balanced salt solution and balanced salt solution–injected eyes for each group as a function of stimulus energy, and interpreted their nonlinear monotonic relationship with a fitted Naka-Rushton function47
() as in another study40
is the ERG response amplitude, Vmax
is the maximum amplitude of the response, I0.5
is the flash energy that elicits a half-maximum response, and I
is the flash energy that elicits the response, V
Figure 2. Effects of intravitreal caspofungin on the mean ERG b- and a-wave stimulus-amplitude relationship and scotopic threshold responses. Caspofungin concentrations in micromolar are indicated at the top of each column. Top: Average dark-adapted electroretinogram (more ...)
For examining the scotopic (rod-driven) b-wave, only those responses to flash energies between −3.5 and 0 log sc td s were used to produce the fit, to reduce the effects of the scotopic threshold responses (STRs) in the fit for low energies, and to minimize the influence of the cone-driven responses at higher energies40
(). The parameters of the fit for the C+balanced salt solution and balanced salt solution–injected cohorts for the scotopic b-wave for each group, along with the coefficient of determination (R2
), are detailed in . For examining the mixed rod+cone–driven b-wave, the average amplitudes in response to a high energy stimulus of 2.3 log sc td s were examined (, top, inset; ). The scotopic b-wave Vmax
and the mixed rod+cone-driven b-wave amplitudes of the C+balanced salt solution–injected eyes were not significantly different from their balanced salt solution controls for caspofungin concentrations between 0.41 and 4.1 μM. Both the rod-driven b-wave Vmax
and the mixed rod+cone driven b-wave amplitudes for the C+balanced salt solution–injected eyes that received the 41-μM dose showed a statistically significant reduction of ~59% compared with the balanced salt solution control. The b-wave I0.5
for the C+balanced salt solution–injected eyes that received the 41-μM dose did not show a statistically significant difference compared with the balanced salt solution control. The amplitude–response relationship for the dark-adapted photoreceptor-driven a-wave is shown in , middle. The parameters of the fit for the C+balanced salt solution and balanced salt solution–injected cohorts for the a-wave for each group, along with their coefficient of determination (R2
), are detailed in . The scotopic a-wave Vmax
of the C+balanced salt solution–injected eyes were not significantly different from their balanced salt solution controls for caspofungin concentrations between 0.41 and 4.1 μM. The a-wave Vmax
for the C+balanced salt solution–injected eyes that received the 41-μM dose showed a statistically significant reduction of 61% compared with the balanced salt solution control. The a-wave I0.5
for the C+balanced salt solution–injected eyes that received a dose of 41 μM did not show a statistically significant difference from that of the balanced salt solution control. For both the rod-driven b-wave and a-waves, the I0.5
for the C+balanced salt solution–injected eye that received a dose of 41 μM was similar to that of the balanced salt solution–injected control eyes, indicating that caspofungin at this concentration must have caused damage to the retina without altering light transmission (for example, vitreal hemorrhage or cataracts) or altering photoreceptor sensitivity to light.37
For low flash energies the averaged scotopic threshold responses (STRs) for mice that received 100× caspofungin injection (but not those that received lower doses) showed a statistically significant difference compared with the balanced salt solution–injected controls at criterion times of 110 and 220 ms (t-test, P < 0.05), indicating that at this concentration there was a likelihood of toxicity to the inner retina, proximal to the bipolar cells.
To investigate the probable cause of reduced a- and b-wave amplitudes for the C+balanced salt solution–injected eyes that received 100× caspofungin relative to the control injected only with balanced salt solution, we plotted the b-wave amplitudes for the four highest flash energies as a function of the a-wave amplitude (). Although both the b- and a-waves were reduced in the 100× injected eye, their relative ratios were indistinguishable from those of the control eyes, indicating that the cause of the reduced a- and b-waves was most likely due to the toxic effect of this drug on the photoreceptors.
Figure 3. b-Wave as a function of a- and b-wave to a-wave ratios for the dark-adapted mixed rod-cone ERGs. (A) b-Wave plotted as a function of a-wave for those eyes that received C+balanced salt solution containing 41 μM caspofungin and for the balanced (more ...)
There were no signs of retinal hemorrhages or infection in any injected eye. Light microscopic histologic examination () showed no observable retinal abnormality in eyes injected with balanced salt solution– or C+balanced salt solution–injected eyes that received a dose of 4.1 μM or less. The eyes that were injected with the 41-μM dose were conspicuous for a loss of nuclear staining in the ganglion cell layer. The loss of ganglion cells was seen in large areas of the retina (). We did not observe focal areas of necrosis, localized retinal detachment, or any observable changes in the photoreceptor outer and inner segments, outer nuclear layer, outer plexiform layer, the inner nuclear layer, or the inner plexiform layer.
Figure 5. Histopathologic examination of radial sections through the optic nerve head of mice eyes 7 weeks after intravitreal injection of balanced salt solution or 41 μM (100× MIC90) of C+balanced salt solution. Blue: DAPI-stained nuclei in various (more ...)