When the spinal cord is injured, an aggressive, deteriorating environment is created at the lesion site, leading to primary and secondary neuronal loss within the affected area. Although many pathophysiological processes have been studied and discussed as the source of this loss, effective preventative or regenerative treatments remain elusive. Notably, many of the molecules released after SCI, namely ROS, cause macromolecular damage, particularly in neurons, because of their high metabolic rates and diminished spectrum of DNA repair mechanisms [10]. Thus, as neurons are very susceptible to DNA damage, we hypothesized that neuronal loss might be caused by associated genomic stress and the inability to resolve it.

Previous studies have reported contradicting or inconclusive results regarding DNA damage presence in SCI [16, 27]. While prior reports have mainly indicated increased DNA damage using methods such as HPLC, IHC, WB, ELISA, and Comet assay, the results have varied for several reasons. First, different methods of injury induction have been employed, perhaps indicating that the type of lesion or severity of the damage might affect the degree of genomic stress. Second, the investigations have often lacked comparative controls (sham and/or naive mice). Third, various animal models have been employed, making translation between the potentially pathophysiological processes difficult. Fourth, no cell typing was performed (or reported). Finally, the region of analysis was often not defined, perhaps affecting the results (as seen by our studies using entire spinal cord sections). In the study here, we included sham and naive controls, analyzed the kinetics at key time points, examined region variation, and measured neuron-specificity.

In the γH2AX temporal analysis, we observed increased DNA damage (foci) from 1 hpi to 3 dpi and an apoptotic response (pan) from 1 hpi up to 7 dpi in SCI mice. This early response of γH2AX foci in spinal cord cells of SCI mice relative to sham controls, followed by a more extended persisting pan γH2AX response, is in line with previous kinetic patterns of γH2AX foci and pan signal in other cell models [26]. Our study, therefore, indicates that DNA damage is a very rapid response to a traumatic injury of the spinal cord that persists over several days and likely induces cell death. Notably, an increase of γH2AX was detected in sham-treated mice relative to naive controls, possibly indicating that surgically damaging the peri-spinal environment also affects spinal cord integrity. Ozgonul et al. also reported a slight increase in DNA damage in sham mice in the early chronic phase compared to the acute situation [28]. Apoptosis was increased at 3 and 7 dpi compared to naive mice but not compared to sham mice. Although it is unclear why this occurs, there is speculation regarding the effects of isoflurane on the CNS. Isoflurane appears to be associated with neuroprotection in an injured CNS, while under normal conditions, it seems to act as a neurotoxin [29]. Therefore, the anticipated effects described here could be underestimated, resulting in insignificant differences. In addition, it must be noted that only female mice were used in this study due to technical, ethical, and cost considerations. This limitation should be addressed by replicating the study in a mixed-sex experiment.

One broadly studied mechanism of DNA damage induction is (chronic) inflammation. It has been shown in many different pathological frameworks, e.g., chronic gastritis, chronic lung inflammation, and carcinogenesis, that inflammation can cause and increase DNA damage in other cell types [30,31,32,33,34]. However, external inflammation caused by glia and infiltrating immune cells requires several days to reach the injury site. Therefore, other mechanisms must be in play to cause the early DNA damage response in SCI. Possible early mechanisms are paracrine secretion of ROS, reactive nitrogen species (RNS), excessive glutamate, necrotic signals, or conveyance of signals via gap junctions. Additionally, the acute release of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 may contribute to early DNA damage through inflammatory signaling pathways [35, 36].

As 53BP1 has been reported to operate within the same response pathway as γH2AX [23], we conducted 53BP1 labeling as a verification strategy. However, it was found that 53BP1 labeling increased only in the sham-treated animals relative to both SCI and naive mice. Although 53BP1 labeling has proven reliable in in vitro assays [37, 38], an inconsistent labeling pattern between 53BP1 and γH2AX has been seen in other in vivo studies, particularly those involving mouse pathologic tissue [24, 25]. This fact likely reflects functions for 53BP1 outside of the DNA damage response, such as its role in neurogenesis [39], and points to potential problems with using 53BP1 as a general DNA damage marker.

In the spatial analysis of γH2AX, we found that the DNA damage (γH2AX foci) profile resembles a self-propagating wave that begins at the lesion site and affected area and then flows rostrally and caudally after injury before returning to baseline with time. An explanation could be that cells directly neighboring the primary injury are subjected to a battery of hostile molecules released within the affected area that cause locally induced DNA damage. If these recipient cells subsequently die, the next layer of cells will be exposed to damaging signals. These recipient cells will, in turn, experience consequent genomic stress, and this cycle continues until the response pattern has diminished and returned to normal. A similar longitudinal progression was found for apoptotic cells, as identified by DNA degradation. Crowe et al. showed that apoptotic cells increase over time around the injury site and start bifurcating into two waves at 8 dpi, an observation that complements the pattern of reversible, toxic DNA damage reported herein [15]. Microglia could be one source that contributes to this injury spreading, as they play a major role in both the immediate and delayed phases of secondary injury following SCI [40, 41]. In the acute phase, microglia rapidly activate and release pro-inflammatory mediators. Next, they contribute to oxidative stress, the recruitment of other immune cells, and sustained inflammation. Sub-acutely, microglia maintain the inflammation and participate in other processes such as apoptosis, necrosis, glial scar formation, and potentially chronic neuroinflammation [40, 41].

Another feature of the DNA damage wave is the greater spread and higher presence of DNA damage caudally. This might result from Wallerian degeneration, where the distal segments of the severed spinal axons degenerate, releasing intracellular contents and myelin debris into the extracellular space [42]. This process creates a toxic environment rich in ROS and inflammatory cytokines, which can cause further DNA damage. Additionally, myelin breakdown and microglia activation amplify the local inflammatory response, perpetuating oxidative stress and cytotoxic effects. When considering the early cell death (pan γH2AX cells) profile, the increased signal is restricted to the affected area up to 7 dpi. Therefore, neurons at the affected area seem more prone to early cell death after DNA damage, perhaps due to a more toxic environment as a result of oxidative stress. Contrarily, neurons outside the affected area appear more capable of withstanding DNA damage and mediating survival.

The location of DNA damage following SCI has not been studied before, but a more caudal degradation of the spinal cord has been previously established by Ohnishi et al. [43]. They showed that rostral degeneration is an immediate process exacerbated by oxidative stress, while caudal degeneration is delayed and associated with deficits in the glycolytic pathway. Limited energy supplies have more implications than cell death alone, as DNA repair processes require a lot of energy. Cellular metabolism can, therefore, affect the levels of genomic stress by limiting the available substrates necessary for efficient DNA repair activity [44]. Thus, having compromised energy supplies in the caudal regions of SCI could adversely affect genome maintenance mechanisms, decreasing the capacity of cells experiencing genotoxic threats from resolving induced DNA damage.

Traumatic brain injury (TBI) is the cortical homolog of an SCI. Thus, TBI likely exhibits pathophysiological processes similar to SCI. In TBI, DNA damage, as assessed by e.g., 8-oxoguanine/ γH2AX immunofluorescence or a polymerase I mediated biotin dATP nick-translation assay, has been established as part of the injury pathophysiology in both humans and rodent models [45]. In mouse models involving controlled cortical impact injury, TBI animals exhibited increased DNA damage as early as 15 min post-injury up to 7 dpi [45, 46]. Moreover, γH2AX has even been considered a marker of brain damage [47]. Given our findings, the broader applicability of DNA damage in SCI research requires further investigation.

Focusing on neuronal cells, our experiments showed that DNA damage is present at 1 dpi and that early cell death starts at 1 hpi up to 3 dpi. Above, we note that in the general cell population, elevated levels of DNA damage (γH2AX foci) at 1 hpi and 1 dpi (and 3 dpi vs. naive) precede signs of early cell death (pan γH2AX). In the neuronal population, the DNA damage is limited to 1 dpi. However, it is still proceeded by prolonged elevated early death (pan) markers and apoptosis (ClCasp3) at 3 dpi. This suggests that neuronal cells are more sensitive to cell death following less severe (measurable) levels of DNA damage. This characteristic might be explained by the specific set of DNA repair pathways that neurons possess compared to the broader battery of DNA repair systems available to dividing cells, such as glia. Due to their post-mitotic character, neurons rely primarily on pathways like error-prone non-homologous end-joining (NHEJ), single-strand break repair (SSBR), base excision repair (BER), and nucleotide excision repair (NER). Conversely, major high-fidelity pathways that are intimately connected to DNA replication, such as homologous recombination (HR) and mismatch repair (MMR), are absent in their classic forms [10]. Therefore, neurons might accumulate more DNA damage in a faster timeframe, making them more susceptible to DNA damage-induced cell death, potentially via transcription-blocking neurodegenerative mechanisms [48]. This was shown previously in the retina, where retinal neurons are more sensitive to DNA damage than glia [49]. Therefore, it seems that acute action is needed to avoid DNA damage and associated early cell death to preserve neuronal tissue.

Since DNA repair seems inadequate following an SCI, a promising therapeutic tactic would be enhancing DNA repair systems to decrease neuronal loss. In preliminary data mining work investigating DNA repair response pathways, we have seen an upregulation of mechanisms involved in oxidative DNA damage repair with a peak at 3 dpi (unpublished work; Fig. 6). This result correlates nicely with the elevated levels of DNA damage reported at early time points in this paper. Looking to the future, strategies that selectively inactivate hyperactive responses or stimulate underperforming mechanisms could serve as effective therapeutic interventions. One venture targeting the DNA damage response that has shown some success in SCI is Nicotinamide Riboside (NR), a compound that supports poly(ADP-ribose) polymerase (PARP) and strand-break repair mechanisms. A recent study shows that NR increases functional recovery and reduces spinal tissue loss [50], supporting targeting genome maintenance pathways as a therapeutic approach.

Summary of the results. DNA damage occurs at early time points post-SCI (1 hpi to 3 dpi). DNA repair follows the onset of DNA damage at 1 dpi and remains upregulated up to 28 dpi at least. Apoptosis starts at 1 dpi and remains high until at least 28 dpi. Neuron-specific apoptosis has a slower onset but follows general apoptosis patterns from 7 dpi