Background
There are over 250,000 people in the USA currently living with a spinal cord injury (SCI), and over 15,000 new cases are documented every year [
1]. These injuries cause permanent motor, autonomic, and sensory function loss [
2]. Reduced cell viability and neuron, astrocyte, and oligodendrocyte loss are seen acutely after SCI [
3]. White matter and axon loss, glial scar formation, and inflammation are seen chronically after injury, in the days to months following onset [
3].
SCI is largely studied in the young adult population due to the high propensity of injuries from athletic and military injuries and car accidents; however, there has been a notable increase in age of onset in recent decades [
4]. In the 1970s, the average age at onset of injury was 29, but the average age has now increased to 42 [
1]. Aging tissue shows progressive DNA damage beginning by the age of 40, causing increased expression of genes involved in stress response and repair, including immune and inflammatory genes [
5]. Further, aging causes changes to hormones and cellular function associated with increased co-morbidities, lengthened recovery time, reduced functional recovery, and prolonged chronic inflammation after SCI [
6].
One notable change to the cellular environment with aging is an increase in oxidative stress caused by the buildup of reactive oxygen species (ROS) over time [
7,
8]. The superoxide producing NADPH oxidase (NOX) enzyme is a major source of ROS [
9]. The NOX2 isoform has been shown to be the most responsive to injury and is present on microglia, the primary inflammatory cell type of the central nervous system [
10]. Microglia are found in a resting state in uninjured tissue, with ramified cell body and long processes. When activated, they take on a pro-inflammatory state with an amoeboid-like cell shape, releasing cytotoxic and inflammatory mediators [
11]. Activated microglia are found in the spinal cord cellular environment within 24 h post-injury and have a maintained, low-level activated presence at 60 days post-injury (dpi) [
11].
In animal models of traumatic brain injury (TBI), aged microglia show alterations to morphology and activation compared to younger microglia [
12‐
14]. In uninjured aged brain tissue, microglia demonstrate a hypertrophic or partially activated cell shape, considered a “primed” activation state [
14]. When injured, these aged cells show a hyperactive response to injury compared to younger cells, resulting in extended chronic inflammation [
14]. Previous work has demonstrated that in the spinal cord, compared to the brain, there is a significantly greater inflammatory response and recruitment of macrophages and neutrophils to trauma [
15‐
17]. Therefore, findings in the aging brain may not accurately indicate what will occur in the spinal cord.
Few studies have examined how age influences microglial activation and/or oxidative stress in the spinal cord [
18‐
21] and the role NOX2 plays in this process [
22]. Work in our lab has characterized the role of NOX2 in SCI [
23], demonstrating that inhibition of NOX2 improves functional recovery and decreases oxidative stress and inflammation in a young adult rodent model of SCI [
24]. Previous work on age-related cellular alterations after spinal cord injury have been performed in mice [
22] and aged female rats [
19], while in this paper we examine a middle-aged male rat model of SCI as the average age of onset of human SCI is 42 years old. Based on previous work showing age-related alterations after injury in the brain [
12,
14,
25] and spinal cord [
18,
19,
21,
26], we investigated microglia, NOX2 components, and ROS prior to and chronically after injury (30 dpi). We now show that microglial and NOX2 gene expression and ROS production are increased in middle-aged rats, demonstrating age-related alterations in the spinal cord.
Discussion
This study demonstrates that aging alters the inflammatory environment in the 12-month-old rat spinal cord. Uninjured 12-month-old, middle-aged rats show increased microglial activation and NOX2 cytosolic subunit and pro-inflammatory gene expression and increased DNA oxidation when compared to 3-month-old, young adult rats. At 30 dpi, middle-aged rats show decreased functional recovery and increased lesion volume, microglial activation, NOX2 component and inflammatory gene expression, and protein oxidation and nitrosylation compared to young adult rats.
Previous work has shown increasing oxidative stress with age [
8]. We now demonstrate that this increase is obvious in the CNS, with increased markers of oxidative stress in the middle-aged rat spinal cord both before and after injury. In naïve middle-aged rats compared to young adult, increases in the DNA peroxidation marker 8-OHdG, but not of protein carbonylation (Oxyblot) and nitrosylation (3-NT), were observed. As mutations in DNA have been found by early middle age [
5], DNA peroxidation may be one of the first signs of increasing ROS production, as part of a pro-inflammatory environment in an aging tissue [
14]. As protein modifications occur further downstream than DNA, age-related increases in protein carbonylation and nitrosylation markers may be more indicative of a hyperactive inflammatory response to injury.
The increased ROS production found in middle-aged rats coincides with our results showing increased microglial activity with age. Increased staining of the microglial marker Iba1 was coupled with increased pro-inflammatory gene expression of CD86 in middle-aged rats, suggesting that microglia are present in aged tissue in a pro-inflammatory state. Further, staining demonstrates alterations in microglial phenotype as defined previously [
11], with 3-month-old rats showing a non-activated phenotype with lengthy processes and a small cell body and 12-month-old rats showing a partially activated microglial phenotype, with an engorged, rounder shaped cell body. These findings in the spinal cord are similar to previous findings in the brain [
14], suggesting an age-related pattern of the inflammatory response that is not impacted by regional differences between the spinal cord and the brain [
17].
Interestingly, our gene expression experiments at 30 dpi showed an increase in both pro-inflammatory- and anti-inflammatory-related genes, suggesting that chronically after injury, all microglial activities are elevated, not only pro-inflammatory as seen in uninjured tissue. Previous work using aged mouse and female rat models of traumatic injury has shown a decrease in anti-inflammatory signaling and an increase in pro-inflammatory signaling at 24 h and 7 dpi [
18,
19,
21,
25]. In a traumatic brain injury model with 23-month-old mice, a similar increase in both pro- and anti-inflammatory gene expression was observed at 24-h post-injury [
35].Our findings at 30 dpi suggest that a change in activation type may occur chronically post-injury that leads to a more active overall inflammatory response. Further, Morganti et al. [
35] found that acute gene expression alterations were strongly dependent on peripheral monocyte infiltration; it is possible that the 30 dpi data in our study is more microglial related due to the reduction in macrophage invasion at this later time point [
36]. Future work should evaluate these earlier time points and contribution of peripheral and central inflammatory cells in 12-month-old rats to fully characterize our rodent model.
Increased gene expression of the activated NOX2 cytosolic subunit p47
PHOX but not the membrane components p22
PHOX or gp91
PHOX suggests that NOX2 expression is increased in middle-aged naïve rats, corresponding with the observed pro-inflammatory microglia. At 30 dpi, gene expression of both NOX2 membrane components p22
PHOX and gp91
PHOX and the cytosolic subunit p47
PHOX are elevated in middle-aged rats compared to young adults, suggesting that microglial NOX2 is “primed” prior to injury and activated more fully with injury. This could play a role in the increased ROS production we see in a middle-aged spinal cord. We and the others have shown that NOX2 is involved in microglial ROS production [
22‐
24].
Our lesion volume experiments at 30 dpi showed that at the epicenter of the injury, the lesion volume was not significantly different by age, but was significantly increased in middle-aged rats compared to young adults in sections of the spinal cord both rostral and caudal to the injury, demonstrating an increased volume farther from the lesion epicenter in middle-aged rats. Our results coincide with previous work showing increased lesion volume in aged female rats after SCI [
19] and in a mouse model of TBI [
25]. These findings demonstrate that injury severity is increased in middle-aged rats, which may be a result of the exaggerated inflammatory response leading to increased oxidative stress on cells. Control of motor function is distributed throughout the spinal cord with multiple interacting tracts, and recovery of function observable in injury models is largely due to the ability of the spinal networks to compensate for some loss of tissue [
37]. Interestingly, previous work has shown that the increase of lesion volume and functional deficits is not completely linear, but with increasing injury severity, once a threshold proportion of tract is lost, loss of function is seen [
38,
39]. The increased lesion volume found in our middle-aged rats would impact a greater number of tracts in the spinal cord, likely surpassing that threshold and contributing to the delayed functional recovery we observed.
Interestingly, naïve middle-aged rats also show functional impairments, with altered stepping patterns with wider toe spread and longer stride length compared to young adult rats. Functional performance was more prominently affected in middle-aged rats than young adult after contusion SCI, at all time points examined out to 30 dpi. Middle-aged rats compared to young adult rats showed age-related delays in motor function with delayed general functional recovery, as demonstrated by lower scores on the BBB test, and worsened ability to complete the footprint task. These results agree with previous work in mouse and male and female rat models of aged SCI [
19,
21,
40]. These previous studies demonstrate that 18-month-old female rats showed elevated base of support during walk both before and after injury [
19], similar to our studies, while 12-month-old male rats consistently demonstrate impaired BBB function by 7 dpi [
40]. In contrast, 14-month-old male mice did not demonstrate functional impairment before injury, although this may be due to the use of only the locomotor score, similar to the BBB; however, a significant reduction in locomotor score was noted after injury [
21], as in our study.
Interestingly, footprint analysis in middle-aged rats after SCI are opposite of what was seen in naïve, showing decreased stride lengths and narrower toe spread at 30 dpi versus increased stride length and wider toe spread in naïve. Previous work in mouse models of SCI have shown that both stride length and toe spread decrease significantly after injury [
41‐
43] and have suggested that the decrease may be indicative of sensory motor function deficits in the hind limbs due to injury. Thus, our findings of impaired recovery and altered stepping pattern in footprint analysis may be associated with an age-related decrease in precise motor control in the hind limbs.
Conclusions
Overall, the results of our study suggest that increasing age leads to a pro-inflammatory environment, with alterations to microglial activation, NOX2 enzyme component expression, and an increase in oxidative stress in the cellular environment that may contribute to worsened outcomes after SCI. The initiating factor in these effects and the cause/effect relationship between each remains unclear and is an important consideration for the field moving forward. It is clear that there are intimate relationships between inflammation, oxidative damage, and NOX2, and the current work contributes to understanding that all three play a role in motor function and post-injury recovery. However, more work is needed to understand which, if not all three, the essential component is. We currently hypothesize that age-related changes to ROS production caused by increased microglial activation and NOX2 expression are associated with an exaggerated chronic inflammatory response to injury.
With the rise in SCI in the aging population, the results of this study suggest that increased inflammatory response may be an important factor to be considered in therapeutic interventions after injury and that age should be considered when developing a therapeutic treatment plan. Further, previous work has demonstrated that inhibition of the NOX enzyme can decrease oxidative stress and inflammation. Due to the association of NOX2 with both ROS production and microglial activity, our findings suggest a potential avenue for NOX2 inhibitory treatment options to quell the exaggerated inflammatory response and should be a topic of interest for future research.