Repeat- and single-blast injury activate the same molecular pathways

We quantified cleaved:total caspase-1 to quantify activation (Fig. 1a, b). The levels were comparable to sham at 2 weeks post injury (Fig. 1b). The ratio was increased by 40% in the single- and 37% in the repeat-blast retinas at 4 weeks post injury as compared to shams (Fig. 1b). Levels of the inflammasomes, NLRP1, and NLRP3 were unchanged from shams (data not shown). Since these proteins could still be active without their levels being increased, this does not rule out their involvement11. We performed a 24-plex cytokine ELISA, and for both blast groups we only detected increases in IL-1α and IL-1β (Fig. 1c, d). The levels of IL-1α were unchanged from shams at 2 weeks, but increased 138% at 4 weeks after single injury (Fig. 1c). After repeat injury, IL-1α levels were elevated at both 2 and 4 weeks by 55% and 72%, respectively, above shams (Fig. 1c). The levels of IL-1β were unchanged from shams at 2 weeks after either injury (Fig. 1d). However, 4 weeks after either injury, IL-1β levels increased 51% and 106%, respectively, above shams (Fig. 1d). Since IL-18 was not represented on the multiplex ELISA, we assessed it separately (Fig. 1e). IL-18 levels increased 78% and 56%, respectively, above shams at 2 weeks after either single or repeat injury and returned to sham levels at 4 weeks (Fig. 1j). We also measured levels of thioredoxin interacting protein (TXNIP), a protein that activates the inflammasome in response to ROS. Its levels were unchanged after injury (data not shown); however, this does not exclude the possibility of increased activation by phosphorylation.

Fig. 1: The inflammasome pathway is activated in the retina after single or repeat blast injury to the eye. a Western blot of caspase-1 in sham, single injury, and repeat injury mice at 4 weeks after injury. b Quantification of cleaved to total caspase-1 showing an increase at 4 weeks in both single and repeat injury groups. c Quantification of IL-1α after single or repeat blast. IL-1α is increased at 4 weeks after a single blast and at both 2 and 4 weeks after a repeat blast. d Quantification of IL-1β after single or repeat blast showing an increase at 4 weeks after either injury. e Quantification of IL-18 after single or repeat blast showing a transient increase at 2 weeks after injury for both groups. This experiment was repeated twice. n = 5 for all groups. **p < 0.01, ***p < 0.001, #p < 0.0001 Full size image

ROS is a primary driver of secondary axon degeneration after ocular trauma

We manipulated levels of retinal vitamin C (VitC) and vitamin E (VitE) in order to examine the contribution of ROS to trauma-induced inflammasome activation, neuronal degeneration, and vision loss. The low-VitC diet provides sufficient levels to avoid scurvy, but does not saturate the sodium-dependent VitC transporter, type 2 at the blood–brain barrier14. This dose may also be clinically relevant since significantly depleted or frankly deficient serum VitC levels (< 28 μM) are observed in up to 50% of otherwise healthy populations (for a review see15). Deficiency is greater in smokers16,17 and persistent hypovitaminosis for VitC is observed in veterans18. Blast had no effect on endogenous retinal VitC levels (Fig. 2a). We detected a 50% and 40% decrease in retinal VitC levels in sham and blast low-VitC mice, respectively, as compared to controls (Fig. 2a). The retinas of high-VitE-diet mice contained comparable levels of VitC as controls (Fig. 2a). This was expected because wild-type mice can moderate VitC synthesis according to the requirement. VitC in these diets is provided in excess to ensure sufficient VitC is available to recycle oxidized VitE from its radical form19. The retinas of high-VitE mice contained 70% (sham) and 90% (blast-injured) more α-tocopherol (VitE) than control mice, p < 0.05 (Fig. 2b). Blast had no effect on VitE content (Fig. 2b).

Fig. 2: Diets alter tissue levels of VitC, VitE, and superoxide after blast. a Quantification of retina ascorbic acid (VitC) levels. Retinas of Gulo-/- mice provided a low-VitC content diet had less ascorbic acid, *p < 0.05. Retinas of wild-type mice fed a high-VitE diet contained normal levels of ascorbic acid. b Quantification of retina α-tocopherol (VitE) levels. Retinas of wild-type mice fed a high-VitE diet contained higher levels of α-tocopherol. c, d Representative images of DHE fluorescence (superoxide levels) in the retinas of sham (c) and repeat blast-exposed (d) mice. e Quantification of retina DHE fluorescence in normal, low-VitC, and high-VitE-diet mice. Levels are increased in the 2 and 4 weeks after blast injury in both the control and low-VitC mice, but not in the high-VitE mice. f Western blots of SOD2 levels in retinas from sham, and 2- or 4-week post-blast mice on control, low-VitC, or high-VitE diets. g Quantification of SOD2 levels. SOD2 levels are decreased at 4 weeks after injury in both normal-diet and low-VitC-diet mice, but not the high-VitE mice. Low VitC retinas contained increased levels of SOD2 in sham and 2 week post-blast mice, suggesting an endogenous compensatory effect of the low-VitC diet. This experiment was repeated twice. n = 5 for all groups. *p < 0.05, **p < 0.01, ***p < 0.001, #p < 0.0001 Full size image

Blast caused an increase in dihydroethidium (DHE) fluorescence, a marker of superoxide, as compared to shams (Fig. 2c, d). An increase of over 50% was detected at 2 and 4 weeks after repeat blast in control-diet mice (Fig. 2e). A similar increase was detected in retinas from low-VitC-diet mice (Fig. 2e). The lack of additional increase suggests that we may have reached the technical maximum of the assay. In contrast, fluorescence remained at sham levels in the retinas of high-VitE-diet mice at both 2 and 4 weeks after blast (Fig. 2e). Quantification by quadrant did not yield a regional effect (data not shown). Since the primary source of superoxide is the mitochondria, we measured levels of the mitochondrial superoxide dismutase (MnSOD/SOD2) (Fig. 2f). SOD2 levels were decreased by 72% at 4, but not 2 weeks after injury (Fig. 2g). Low-VitC diet mice exhibited a 286% increase over control shams regardless of injury (Fig. 2g). However, they exhibited a decrease in SOD2 at 4 weeks after blast to almost undetectable levels (Fig. 2g). Treatment with high VitE prevented the decrease in SOD2 after blast (Fig. 2g).

In the low-VitC mice, the decreased antioxidant capacity resulted in a 79% and 129% increase in the levels of cleaved:total caspase-1 at 2 and 4 weeks after blast, respectively, as compared to the low-VitC shams (Fig. 3a, b). This is an earlier and greater increase than in wild-type mice (Fig. 1b, 3b). The retinas of low-VitC-diet mice also exhibited a 97% increase in IL-1α levels as compared to wild-type mice regardless of injury, demonstrating a higher baseline inflammatory state (Fig. 3c). No further increase was detected after blast (Fig. 3c). Similarly, the levels of IL-1β in the low VitC retinas were elevated as compared to sham retinas regardless of injury and no additional increase was detected after blast (Fig. 3d). IL-18 baseline levels were also increased in the retinas of low-VitC-diet as compared to sham levels in retinas of control-diet mice (Fig. 3e). In addition, there was a further increase in IL-18 levels at 2, but not 4 weeks after blast as compared to shams (Fig. 3e). In contrast, high-VitE-diet mice maintained levels of cleaved:total caspase-1, IL-18, IL-1α, and IL-1β at sham levels after blast (Fig. 3a-e).

Fig. 3: Diets alter activation of the inflammasome pathway after injury. a Western blots of cleaved and total caspase-1 in sham and post-blast mice treated with a high-VitE or low-VitC diet. b Quantification of cleaved to total caspase-1 shows a greater increase in low-VitC retinas than control-diet mice. There was no increase in activated caspase -1 in the retinas of mice on a high-VitE diet. c, d Quantification of IL-1α (c) and IL-1β (d) shows that levels are elevated in retinas of mice on a low-VitC diet regardless of injury. There was no increase in IL-1α or IL-1β levels in the retinas of high-VitE-diet mice. e Quantification of IL-18 levels also shows an overall elevation in retinas from mice on a low-VitC diet. In addition, these mice also had a transient increase at 2 weeks after injury as in control-diet mice. There was no increase in IL-18 in the retinas from the high-VitE-diet mice. This experiment was repeated twice. n = 5 for all groups. *p < 0.05, **p < 0.01, ***p < 0.001, #p < 0.0001 Full size image

The ON of sham mice contained axons with condensed myelin and clear axoplasms along with glial cells with thin processes (Fig. 4a). At 2 weeks after injury many different degenerative profiles were evident, including vacuolization (arrows) and hyper-myelination (arrowheads) (Fig. 4b). At 4 weeks after injury, there were still many degenerative profiles present in the ON and many small axons were missing (Fig. 4c). The ON from low-VitC mice exhibited glial hypertrophy in addition to degenerating profiles at both time points (Fig. 4d, e). In contrast, the ON from high-VitE mice contained many fewer degenerating axons and the glial morphology appeared more similar to that of shams at 2 weeks post injury (Fig. 4f). At 4 weeks post injury, there were more axon profiles and smaller glia than in the control diet of 2 week post-injury ON (Fig. 4g). However, some axon degeneration was detected in the high-VitE-treated mice at 4 weeks after injury (Fig. 4g). Axon transport was measured by quantifying fluorescence in the superior colliculus after intravitreal injection of fluorescently labeled cholera toxin B (CTB; Fig. 4h-l). Transport loss was apparent at 2 weeks after injury, with a greater loss in the medial region of the superior colliculus (Fig. 4i). The same pattern of loss, but with greater deficit, was detected at 4 weeks post injury (Fig. 4j). Much of the transport loss was prevented with the high-VitE diet, with the exception of far medial loss (Fig. 4k, l).

Fig. 4: High-VitE diet prevents injury-induced axon degeneration and axon transport deficits. a-g Representative brightfield micrographs of ON from a sham (scale represents 20 μm and applies to all micrographs); b 2-week post-blast (arrow indicates re-myelination; arrowhead indicates Wallerian degeneration); c 4 week post-blast (note the loss of small axons); d 2 week post-blast low-VitC-diet; e 4 week post-blast low-VitC-diet; f 2-week post-blast high-VitE-diet; and g 4 week post-blast high-VitE-diet mice. h-l Representative heat maps of fluorescently tagged CTB in the superior colliculus of sham (h), 2 week post-blast (i), 4 week post-blast (j), 2 week post-blast high-VitE-diet (k), and 4 week post-blast high-VitE-diet (l) mice. Medial (M), lateral superior colliculs (L), inferior (I), nasal (N), superior (S), and temporal (T) regions of the retinotopic map on the superior colliculus. m Quantification of total axons at 2 and 4 weeks after blast exposure in all diet groups as compared to their respective shams. Total axons were preserved in the high-VitE-diet group only. n Quantification of degenerative axons at 2 and 4 weeks after blast exposure in all diet groups as compared to their respective shams. Axon degeneration was more sustained in the low-VitC-diet group and was lowest overall in the high-VitE-diet group. o Quantification of cross-sectional ON glial area at 2 and 4 weeks after blast exposure in all diet groups as compared to their respective shams. Baseline glial area was elevated in the low-VitC-diet group and was unchanged after injury. The increase in glia area was less than in controls in the high-VitE-diet group. p Quantification of axon transport based on CTB fluorescence levels in the superior colliculus. Axon transport was preserved in the mice on the high-VitE diet. This experiment was repeated twice. n = 5 for all groups. *p < 0.05, **p < 0.01, ***p < 0.001, #p < 0.0001 Full size image

Two weeks after injury there was 37% axon degeneration and a loss of approximately 50% of axons (Fig. 4m, n). At 4 weeks after injury, there were both fewer total axons and fewer degenerative profiles (Fig. 4m, n). Mice on the low-VitC diet did not exhibit a statistically significant loss of axons until 4 weeks post injury (Fig. 4m). However, these mice had degenerative profiles at both time points suggesting prolongation of the window of axon degeneration (Fig. 4n). Glial area was increased to the same level in the ON of low-VitC mice regardless of injury (Fig. 4o). In combination this may suggest lack of appropriate phagocytosis of degenerating axons in the low-VitC-diet group, thus explaining the higher numbers of total and degenerating axons detected in these mice as compared to those on a control diet. The total axons in the ON from the high-VitE mice was similar to that in shams at both post-injury time points (Fig. 4m). Axon degeneration in the high-VitE mice was comparable to shams at 2 weeks after injury (Fig. 4n). However, at 4 weeks after injury 10% axon degeneration was detected in the high-VitE-treated mice (Fig. 4n). Glial area remained near sham levels at both time points in the high-VitE-diet mice (Fig. 4o). There was a 58% reduction in axon transport at 2 weeks after injury, but no additional decrease at 4 weeks (Fig. 4p). Transport in the high-VitE-diet mice was not different from that of sham, despite a trend towards the lower transport (Fig. 4p).

The visual evoked potential (VEP) waveform was diminished at 4 weeks after injury in control and low-VitC-diet mice but not in high-VitE-diet mice (Fig. 5a). The VEP N1 amplitude decreased by 27% and 40% at 2 and 4 weeks after injury, respectively, as compared to shams (Fig. 5b). It was also decreased by 37% and 49% at 2 and 4 weeks after injury, respectively, in the low-VitC-diet mice (Fig. 5b). In contrast, the amplitude in the high-VitE-diet mice was similar to shams (Fig. 5b). The VEP N1 latency was increased after injury in mice on normal or low-VitC diet (Fig. 5c). In contrast, the latency was comparable in all high-VitE-diet groups (Fig. 5c).

Fig. 5: High-VitE diet prevents injury-induced decrease in the VEP N1 amplitude and latency. a Representative waveforms from sham, and 4 week post-injury mice on control, low-VitC, or high-VitE diets. b Quantification of the VEP N1 amplitude shows a decrease in both at 2 and 4 weeks after injury in mice on the control or low-VitC diets. Mice on the high-VitE diet show no decrease in amplitude at either time point. c Quantification of the VEP N1 latency shows increased latency that is statistically significant at 1 month after blast in both control and low-VitC-diet groups. There was no change in latency after blast in the high-VitE group. A total of 23 sham, 24 2-week-injury, 19 4-week-injury, 21 high-VitE sham, 10 high-VitE 2-week-injury, and 19 4-week-injury mice were used. *p < 0.05, ***p < 0.001, #p < 0.0001 Full size image

Ketogenic diet (KD) prevents the injury-induced increases in IL-1α and superoxide, but not IL-1β, and preserves the optic nerve and vision after repeat blast injury

Mice fed the KD lost weight during the first week but regained overtime, and ultimately weighed the same as the ketogenic control-diet (KCD) mice (Fig. 6a). In order to confirm that the KD induced ketosis, we measured ketone body levels in the blood (Fig. 6b). Ketone bodies were increased in mice on the KD as compared to the KCD at the end of the study (Fig. 6b). Glucose levels were similar between groups (data not shown). None of the above parameters were affected by injury (Fig. 6a, b).

Fig. 6: KD prevents increase in IL-1α and superoxide, but not IL-1β, and preserves axons and VEP. a Average mouse weight over duration of the study. Mice on the KD lost weight over the course of the first week, but regained this weight over time. Both diet groups had comparable weight at the beginning and end of the study. b Quantification of ketone body levels in serum of mice fed a KD or KCD. The KD increased ketone plasma levels regardless of blast exposure. c Representative western blot of SOD2. d Quantification of SOD2 levels in retinas from sham or post-blast mice fed control (KCD) or KD (Keto) diet. The KD prevented the blast-induced decrease in SOD2. e Quantification of DHE fluorescence in the retinas of mice on the KD or KCD. The KD prevented the increase in superoxide levels. f Quantification of cleaved to total caspase-1 in retinas from sham or post-blast mice fed a KCD or KD. The KD prevented activation of caspase-1. g Quantification of IL-1α levels in retinas from sham or post-blast mice fed a KD or KCD. The KD prevented the blast-induced increase in IL-1α. h Quantification of IL-1β levels in retinas from sham or post-blast mice fed a KD or KCD. Levels were increased similarly in both groups. i-k Brightfield micrographs of representative optic nerve cross-sections from KCD sham (i), KCD 4 weeks post-blast (j), and KD 4 weeks post-blast (k) mice. l Quantification of ON cross-sectional glial area in all groups showing an increase in glial area only in the KCD group. m Quantification of total axons in all groups showing a decrease only in the KCD group. n Quantification of CTB fluorescence in the superior colliculus (percent axon transport) in mice from all groups showing a decreased loss in transport in the KD group. o, p Quantification of VEP N1 amplitude (o) and latency (p) in sham and post-blast mice on all diets showing preservation of waveforms in the KD group. A total of 5 samples were used for the biochemistry and axon histology. A total of 8 KCD sham mice, 10 KCD blast mice, 5 KD sham mice, and 11 KD blast mice were used for the VEPs. *p < 0.05, **p < 0.01, ***p < 0.001 Full size image

We measured SOD2 and superoxide levels in these mice (Fig. 6c-e). SOD2 was decreased in the retinas of mice on the KCD after blast (Fig. 6d). The KD prevented the decrease in SOD2 levels (Fig. 6d). Similarly, superoxide levels were increased in the KCD, but not KD, mice (Fig. 6e). Further, mice on the KCD exhibited increased cleaved:total caspase-1, IL-1α, and IL-1β at 4 weeks after blast (Fig. 6f–h), consistent with our results in mice on normal chow (Fig. 1). In contrast, the KD prevented the increase in cleaved:total caspase-1 and IL-1α (Fig. 6f, g). However, it did not prevent the increase in IL-1β (Fig. 6h).

The ON of 4 week post-blast mice on the KD had fewer degenerating profiles and a more normal glial phenotype than KCD mice (Fig. 6i-k). The glial area was increased post blast in the KCD ON, but not in the KD ON (Fig. 6l). Similarly, total axons were decreased in KCD mice after blast as compared to shams (Fig. 6m). Total axons were similar in KD and shams at 4 weeks post-blast (Fig. 6m). Surprisingly, axon transport was decreased in both groups after blast, although there was partial protection by the KD (Fig. 6n).

The VEP N1 amplitude decreased by 44% in the mice on the KCD as compared to its sham group (Fig. 6o). In contrast, there was no difference in the N1 amplitude of KD and sham mice (Fig. 6o). The N1 latency was increased in KCD, but not in KD mice (Fig. 6p).