Spinal Cord–Gut–Immune Axis and Its Implications Regarding Therapeutic Development for Spinal Cord Injury

Abstract

Spinal cord injury (SCI) affects ∼1,300,000 people living in the United States. Most research efforts have been focused on reversing paralysis, as this is arguably the most defining feature of SCI. The damage caused by SCI, however, extends past paralysis and includes other debilitating outcomes including immune dysfunction and gut dysbiosis. Recent efforts are now investigating the pathophysiology of and developing therapies for these more distal manifestations of SCI. One exciting avenue is the spinal cord–gut–immune axis, which proposes that gut dysbiosis amplifies lesion inflammation and impairs SCI recovery. This review will highlight the most recent findings regarding gut and immune dysfunction following SCI, and discuss how the central nervous system (CNS), gut, and immune system all coalesce to form a bidirectional axis that can impact SCI recovery. Finally, important considerations regarding how the spinal cord–gut–immune axis fits within the larger framework of therapeutic development (i.e., probiotics, fecal transplants, dietary modifications) will be discussed, emphasizing the lack of interdepartmental investigation and the missed opportunity to maximize therapeutic benefit in SCI.

Introduction

Spinal cord injury (SCI) is defined as temporary or permanent damage to the spinal cord,¹ and is estimated to affect ∼1,300,000 people in the United States.² The average age at SCI in patients is now 43 years of age, which has been a shift over the past few decades from teenagers and young adults.^3,4 Additionally, SCI is three to four times more likely to occur in men than in women, and is likely to continue to do so in the future.² The etiology can be either traumatic or non-traumatic, with traumatic SCI encompassing 90% of all SCI cases, most of which are the result of motor vehicle crashes.^1,2 In traumatic cases, most often the cervical spine is affected (59%) followed by thoracic (32%), and lumbosacral (9%) spine.¹ Additionally, patients with traumatic SCI can have a significantly reduced lifespan, with the risk of mortality increasing with the severity of injury, higher spinal injury level, and increasing patient age at the time of injury.¹ Although paralysis is the most defining feature of SCI, it also results in other debilitating conditions such as cardiovascular disease, metabolic syndrome, bladder and bowel dysfunction, immunodeficiency, chronic pain, anxiety, and depression.^1,5
–7

Bowel dysfunction, in particular, is a common complaint in individuals with SCI, affecting ∼68% of the population.^5,8,9 Symptoms include constipation, nausea, bloating, abdominal pain, and fecal incontinence, with complications of neurogenic bowel including ulceration, delayed gastric emptying, prolonged bowel evacuation, autonomic dysreflexia, bowel obstruction, and bacterial infection.^8

–11 These symptoms of GI distress can last for years following SCI and can greatly impact quality of life, with many SCI patients prioritizing recovery of intestinal function above the ability to walk.^9,12

Immune dysfunction also occurs after SCI. Following injury, a chronic state of inflammation exists at the lesion site, which impairs neuronal recovery within the cord.¹³ In addition to the immune cell changes locally within the injured spinal cord, injury disrupts normal control of all immune organs, including gut-associated lymphoid tissue (GALT), resulting in a condition known as “SCI-induced immune depression syndrome (SCI-IDS).”^5,14 This condition results in a profound, long-lasting decrease in the ability to fight off infection, and contributes to a 37-fold greater risk of death from pneumonia than in able-bodied individuals.⁵ One of the reasons why SCI-IDS continues indefinitely is that the maladaptive plasticity that develops following the injury can lead to excessive production of hormones and neurotransmitters that can kill immune cells.^5,14 Although SCI typically is associated with systemic immunosuppression, it can also paradoxically be involved in systemic trauma-induced autoimmunity (TIA).⁵ TIA is thought to be caused by a non-resolving inflammatory response dominated by innate immune cells.⁵ TIA may also be a response to gut dysbiosis (i.e., increase in segmented, filamentous bacteria) and bacterial translocation from the gut, both of which can lead to autoimmunity.⁵ Chronic changes in immune function following SCI have also been linked to high rates of cardiovascular disease, autonomic dysfunction, pain, depression, increased risk of pneumonia, delayed wound healing, and metabolic syndrome.¹⁵ As such, SCI leads not only to chronic immune reactions locally at the lesion site, but also to profound systemic immune dysfunction resulting in increased risk for infection and death.⁵

Spinal Cord, Gut, and Immune System Communicate as a Bidirectional Axis

Recent SCI studies have proposed that gut dysbiosis and immune dysfunction are related within a “spinal cord–gut–immune” axis ^5,16(Fig. 1). A similar concept has been studied in other central nervous system (CNS) disorders such as stroke,¹⁷ neurodegenerative disorders,^18

–23 mood disorders,²⁴ epilepsy,²⁵ and traumatic brain injury (TBI).^26

–29 Following cord injury, damage to the enteric nervous system occurs via decreased vagal nerve input and output, leading to dysfunctions in intestinal transit, expression of nutrient transporters, and the mucosal barrier.³⁰ In this situation, the gut becomes more permeable to the translocation of bacteria to peripheral tissues, resulting in alteration of the gut flora, with more pathogenic bacteria and diminished beneficial bacteria.³⁰ For example, in a mouse model, following SCI there is an increase in Proteobacteria, which consists of endotoxin-producing gram-negative bacteria that was also found to exhibit pro-inflammatory properties through PDE4b.³¹ Further, in 2016, Kigerl and coworkers described that changes in mouse flora had a decrease in Bacteroidales and an increase in Clostridiales starting at 1-week post-SCI.³⁰ Bacteroidales are known to be beneficial microbes in the gut as they can liberate short-chain fatty acids (SCFAs) and gases, and their loss has been associated with gastrointestinal symptoms such as constipation and bloating.⁵ Clostridiales, on the other hand, are thought to be maladaptive, and correlate with worsening of locomotor recovery scores as measured via Basso mouse scale (BMS) scores.³⁰ Minor taxa such as Anaeroplasmatales, Turcibacterales, Proteobacteria, and Lactobacillales have also been shown to be affected post-SCI in mice.^30,32 These findings have also been described in a rat model^33,34 and a porcine model³⁵ of SCI as well as in human patients.^10,36
–38 More specifically, in humans it has been found that in both SCI patients and healthy controls, the most abundant phyla include Firmicutes (mean relative abundance, SCI vs. healthy controls: 50.9% vs. 54.8) and Bacteroidetes (22.8% vs 34.3%), and most abundant families including Ruminococcaceae, Lachnospiraceae, and Bacteroidaceae^38,39 SCI patients, however, showed significant increase in microbiota that are known to be involved in inflammation-based disorders,^40

–43 such as Coriobacteriaceae (3.8% vs. 0.5%), Enterococcaceae (6.3% vs. 0.03%), Lactobacillaceae (2.5% vs. 0.2%), Streptococeae (5.4% vs. 0.6%), Methanobacteriaceae (0.3% vs. 0.002%), Enterobacteriaceae (8.3% vs. 0.5%), and Verrucomicrobiaceae (7.2% vs. 0.4%).³⁸ The group also examined microbiota differences in patients with complete versus incomplete injuries and found that those with complete injuries had decreases in Bacteroides, Faecalibacterium, and Lachnospiraceae, which are thought to have health-promoting properties.³⁸ Similarly, Du and coworkers examined differences in gut flora between higher injuries that abolish sympathetic output to the gut (T4) and lower injuries that preserve autonomic control of the gut (T10) in mice. While examination at the phylum level only demonstrated two significant differences (Firmicutes, which was more abundant in the T10 SCI group, and Actinobacteria, which was more abundant in both T4 and T10 SCI groups than controls), the authors found that there were many more changes at the genus level (11 bacterial genera) between control and SCI groups. The authors, therefore, were able to define three distinct clusters of highly abundant bacteria genera in controls, T4 SCI, and T10 SCI, demonstrating that gut dysbiosis occurs following SCI but may also depend on SCI level.⁴⁴ Zhang and coworkers also examined location of injury with respect to the microbiome in 43 male subjects with chronic traumatic SCI, 20 of whom had cervical SCI and 23 of whom had thoracolumbar SCI.¹⁰ When comparing overall SCI patients with healthy controls, they found that SCI patients had a decrease in intestinal flora diversity with specific decreases in Bifidobacterium and Bacteroides, and increases in Veillonellaceae, Proteobacteria, Firmicutes, and Prevotellaceae.¹⁰ Further, microbial community structure was significantly associated with biomarkers (i.e., glucose, high-density lipoprotein [HDL], creatinine, C-reactive protein) as well as with prolonged time of defecation.¹⁰

FIG. 1.

Spinal cord–gut–immune axis. Trauma to the spinal cord can result in decreased sympathetic output (above L2) or decreased parasympathetic output via the vagus nerve or pelvic splanchnic nerves (below L2) to peripheral organs such as the gut. The loss of neural control over the gastrointestinal tract results in gut microbiome dysbiosis, which in turn alters gut-associated lymphoid tissue (GALT). Dysfunctional GALT can release cytokines, neuroactive metabolites, and neurotransmitters that can act directly upon enteric neurons and the vagus nerve (cranial nerve [CN] X) or travel throughout the blood stream to affect central nervous system (CNS) tissues. These cytokines, neuroactive metabolites, and neurotransmitters can subsequently result in increasing spinal cord injury site inflammation, further worsening injury. Image created by BioRender, Inc.

As we previously discussed, SCI results in altered communication with the gut, leading to dysfunction and maladaptive gut microbiota changes. The gut microbiota is a known modulator of the immune system, particularly with the GALT, which includes the mesenteric lymph nodes and the Peyer's patches in the ileum.^5,16 Kigerl and coworkers studied how SCI specifically induces inflammatory changes within these tissues, and found that in SCI mice there was an increase in B-cells, CD8+ T-cells, dendritic cells, and macrophages as well as an increase in cytokine production (interleukin [IL]-10, transforming growth factor [TGF]β, tumor necrosis factor [TNF]α, and IL-1β) at 1-week post-SCI.³⁰ This resulting increase in GALT inflammation can then further exacerbate the original injury. Mice that demonstrated gut dysbiosis and GALT inflammatory changes had impaired locomotor recovery as assessed by BMS scores as well as having a larger lesion size in the cord with less white matter sparing.³⁰ In addition, when the cord lesion site was examined, it was found to have enhanced inflammatory profiles with an increase in activated macrophages, B-cells, and T-cells.³⁰ More recently, Jing and coworkers found similar results. SCI mice with dysbiosis had more activated astrocytes and microglia, which are known components of the glial scar that inhibits neuronal recovery following SCI⁴⁵ (Table 1). Further, the group found that in these dysbiotic mice, there was a reduction of neurotrophic factors (i.e., brain-derived neurotrophic factor [BDNF], neurotrophin [NT]-3, nerve growth factor [NGF]) that normally function to promote neuronal recovery.⁴⁶

Table 1.

Key Studies in Fecal Microbiota Transplant to Improve Gut Dysbiosis Following SCI

References	Purpose	Model	Methods	Results	Limitations/Implications
Brechmann et al., World J. Gastroenterol., 2015⁸⁴	To treat severe recurrent C. difficile infections secondary to antibiotic treatment of pneumonia	Human	Case study Colonoscopic FMT from healthy relative	FMT eradicated C. difficile infection and patient remained C. difficile infection for remainder of follow-up period of 12 weeks	FMT can improve bowel functioning in a patient with SCI FMT comes with risks (i.e., systemic inflammation response syndrome)
Jing et al., Microbiome., 2021⁴⁶	To determine if FMT can improve neurological recovery and gut dysbiosis following SCI	Mouse	Randomized; mice were treated with FMT for four weeks via oral gavage following SCI after a two-week period of antibiotics; FMT were from healthy female mice in same living conditions	Improved locomotor ability and recovery of the neuromuscular unit Revival of neuronal cell bodies and synapses Maintained the integrity of the intestinal barrier Restored the gut microbiome and revived gut mobility	Demonstrated that FMT can improve GI and neuronal functioning in an SCI mouse model Proposed that FMT works via downregulation of the inflammatory pathway IL-1β/NF-κβ
Jing et al. Microbiol. Spectr., 2022⁴⁵	To examine whether FMT may impact the SCI microenvironment	Mouse	Randomized; mice were treated with FMT via oral gavage for four weeks following SCI after a two-week period of antibiotics; FMT were from healthy female mice in same living conditions	Reversed spinal cord ischemia and promoted blood vessel repair Protects against blood-spinal cord-barrier disruption Reduced neuroinflammation at the SCI site Reversed the downregulation of neurotrophic factors.	FMT may restore neurological recovery through re-vascularization and enhanced expression of neurotrophic factors, particularly through β-alanine producing bacteria
Schmidt et al., PloS One, 2020³³	To study how FMT impacts SCI-induced dysbiosis and the development of anxiety	Mouse	Randomized; mice were treated with FMT daily up to 2 days post-injury through oral gavage	FMT prevented gut dysbiosis FMT prevented development of anxiety-like behavior FMT did not affect neuronal recovery at SCI lesion site	FMT may also have beneficial psychological effects in SCI

C. difficile, Clostridium difficile; FMT, fecal microbiota transplant; SCI, spinal cord injury.

Although these emerging data have indicated an intricate connection among the CNS, gut, and immune system in SCI, the underlying mechanisms that drive this pathogenesis remain largely undetermined. As briefly mentioned earlier, recently Myers and coworkers demonstrated that PDE4b, an endotoxin-responsive cyclic adenosine monophosphate (cAMP)-specific phosphodiesterase (PDE), plays a significant role in gut dysbiosis, induced neuroinflammation and white matter loss following SCI.³¹ PDE4b accounts for the greatest proportion of cAMP-dependent PDE activity in the CNS, currently making the molecule a highly researched target for disorders such as SCI.⁴⁷ Using a thoracic contusion mouse model, SCI led to shifts in gut microbiota, especially an increase in the phylum Proteobacteria, which consists of endotoxin-harboring, gram-negative bacteria that can trigger PDE4b on macrophages.³¹ This was accompanied by increased systemic inflammatory marker and monocyte/macrophage marker CD14, as well as increased endoplasmic reticulum stress response (ERSR) markers and inflammation at the SCI epicenter.³¹ Deletion of PDE4b demonstrated an increase in oligodendrocyte mRNA expression as well as a decrease in activated astrocytes, macrophage/microglia, and the proinflammatory mediator cyclooxygenase (COX)2, which resulted in white matter sparing and recovery of hindlimb locomotion following injury.³¹ Additionally, deletion of PDE4b prevented gut dysbiosis, bacterial overgrowth, and endotoxemia following SCI.³¹ These findings indicate that PDE4b may be a suitable drug target, and current efforts to utilize PDE4b inhibitors are underway.⁴⁸ Rong and coworkers also recently proposed a mechanism behind how gut dysbiosis promotes inflammation and exacerbates cord injury through the toll-like receptor (TLR)4/ myeloid differentiation primary response gene (MyD)88 signaling pathway.⁴⁹ TLR4 is expressed on the membranes of microglia and is thought to participate in SCI through the release of growth factors, chemokines, cytokines, and other metabolites.⁵⁰ Gut dysbiosis has been shown to be related to inflammatory changes, including TLR4 activation.⁵¹ Activation of TLR4 can turn on MyD88,⁵² which leads to enhanced astrocyte and myeloid proliferation and exacerbation of the spinal cord lesion site.⁵³ As TLR4 is activated by lipopolysaccharide (LPS), which is a component of gram-negative bacteria, Rong and coworkers postulated that this pathway could be involved in the spinal cord–gut–immune axis.⁴⁹ Using an SCI mouse model, they found that the TLR4/MyD88 pathway was upregulated in both spinal cord and colon tissues following SCI, which correlated with microbiota dysbiosis. Interestingly, the group also found that a fecal microbiota transplant (FMT) from previously injured SCI mice could exacerbate this pathway, promoting inflammation and worsening neuronal death within the spinal cord. Overall, whereas these two mechanisms—PDE4b and TLR4/MyD88—appear to play a significant role within the spinal cord–gut–immune axis, discovering the molecular pathways that make up this axis are still very needed fields of research.

Important Considerations and Future Directions in the Treatment of SCI

Since the 1990s, treatment for SCI has been largely unchanged. Surgical decompression remains the standard treatment, with optimal timing now suggested to be within 24 h.^54,55 Blood pressure goals for a mean arterial pressure (MAP) >85–90 mm Hg are also suggested for at least 5–7 days post-injury.⁵⁶ Steroids such as methylprednisolone have also played a role in the treatment of SCI, however their use has fallen out of favor.^57

–60 Although these therapies have remained the standard approaches to SCI clinically, in recent years researchers have been actively testing other means, such as stem cells^61

–67 and biomaterial scaffolds, to restore function to SCI individuals.⁶⁸ These therapies, although exciting, are mostly focused on restoring neurological function, but as discussed earlier, SCI has other consequences such as bowel and immune dysfunction. In the past few years, therapies such as probiotics, FMT, and dietary modifications have become of increased interest in addressing these more distal manifestations of SCI (Tables 1–3).

Table 3.

Dietary Modifications to Reverse Gut Dysbiosis Following SCI

References	Purpose	Model	Methods	Results	Limitations/Implications
Li et al., Arch. Phys. Med. Rehabil., 2022¹¹²	To evaluate is a LC/HP diet has any beneficial effects in restoring gut symbiosis	Human	Randomized, parallel-controlled trial; 8-week period of LC/HP diet or no intervention	LC/HP diet improved microbiome composition in 8 weeks	LC/HP diet increases bacteria in fiber metabolism to improve constipation LC/HP diet reduces bacteria associated with cardiometabolic disorders
Cameron et al., Spinal Cord, 1996¹²⁴	To assess the effect of increased dietary fiber in bowel function in individuals with SCI	Human	Clinical trial with three-weeks of increased fiber intake via consuming Kellogg's All Bran daily	High fiber diet increased mean colonic transit time with a fourfold delay at the rectosigmoid colon	Participants remained on their prior bowel regimens including suppositories/enemas, aperients without any restrictions on alterations Changed dietary recommendations to encourage a low-fiber diet
Allison et al., J. Spinal Cord Med., 2019¹²⁸	To determine how a previously reported anti-inflammatory diet affects chronic inflammation in SCI individuals	Human	Randomized, clinical trial with intervention of a three-month MD	MD reduced proinflammatory cytokines and increased anti-inflammatory nutrients	MD may have beneficial effects in SCI including reducing risk for neuropathic pain, depression, metabolic and cardiovascular disease Limited by small sample size
Bigford et al., Spinal Cord Ser. Cases, 2017¹³⁰	To improve BMI and known risk factors for cardiometabolic disease in SCI patients	Human	Case series with a 6-month MD/CT program with support followed by a 6-month maintenance phase with minimal support	MD/CT resulted in weight loss MD/CT reduced cardiometabolic risk factors	Limited by only three SCI patient experiences Program requires intensive interventions that may not be feasible for all patients
Rong et al., Front. Cell. Neurosci., 2022¹³⁵	To determine if UA, a previously known neuroprotective and anti-inflammatory pentacyclic triterpenoid, could treat neuronal recovery in SCI	Mouse	Randomized with intervention of daily intragastric administration of UA versus corn oil (control) following SCI	UA treatment increased body weight and muscle mass Inhibited inflammation Improved neuronal recovery and locomotion UA restored gut dysbiosis	UA treatment may combat SCI progression via improvement of spinal cord-gut-immune axis Effects of UA in human patients remain unclear

BMI, body mass index; LC/HP, low-carbohydrate, high-protein; MD, Mediterranean diet; MD/CT, Mediterranean diet and circuit training, SCI, spinal cord injury; UA, Ursolic acid.

Probiotics

Probiotics are bacteria that can regulate immune function, improve intestinal barrier function, and generate organic acids as well as antimicrobial products.¹² As previously discussed, Kigerl and coworkers recently demonstrated the benefit of administering probiotics following SCI in mice³⁰ (Fig. 2 and Table 2), specifically the medical-grade probiotic “VSL #3,” containing eight distinct lactic acid bacteria, mostly Lactobacillus and Bifidobacterium, which are known to produce butyrate and other SCFAs.³⁰ The clinical reasoning behind the probiotic's benefit is thought to come from the replacement of deficient bacteria, as butyrate-producing bacteria are reduced in individuals with SCI.³⁰ In addition, these bacteria can produce neurotransmitters such as serotonin, dopamine, and γ-Aminobutyric acid (GABA).³⁰ These neuro-metabolites have been shown to pass the blood–brain barrier, and mice receiving rescue probiotics showed significantly reduced SCI lesion size and demonstrated functional improvement.³⁰ Kigerl et al. additionally noted that probiotics significantly increased the population of regulatory T cells, which may account for the improvement in reducing SCI pathology.³⁰ Finally, the authors suggest that probiotics have maximal clinical benefit early post-SCI. First, initiating antibiotic-induced dysbiosis 2 weeks after injury (after the onset of maximal gut permeability and time of plateau in functional recovery) did not result in further locomotion impairment, suggesting that dysbiosis has its maximal effect prior to 2 weeks and that probiotics should be given before this critical time point.³⁰ Second, and perhaps more importantly, the probiotic treatment increased numbers of lactic acid-producing bacteria, which conferred neuroprotection at the SCI lesion site.³⁰ Although the article laid significant groundwork for the idea that probiotics can improve neurological function and SCI pathology, using probiotics is not a new idea in this patient population. Individuals with SCI often take probiotics (i.e., Bifico) to relieve gastrointestinal symptoms.¹² Additionally, a recent randomized clinical trial using a Lactobacillus casei Shirota (LcS) probiotic drink was completed in SCI patients, which demonstrated that probiotics could reduce the incidence of antibiotic-associated diarrhea⁶⁹(Table 2). A similar study is also currently being conducted with a multi-species probiotic, Ecologic AAD, in the Netherlands⁷⁰ (NTR 5831; http://www.trialregister.nl/trialreg/index.asp) with results expected in the next few years (Table 2). Another study in Switzerland is scheduled to study the use of probiotics to improve the health of para-athletes, with outcomes including gastrointestinal function, hand grip strength, and intestinal microbiome characterization⁷¹ (Table 2).

FIG. 2.

Current therapeutic strategies targeting gut dysbiosis in spinal cord injury (SCI). Probiotics, fecal microbiota transplant (FMT), and dietary changes constitute the most common techniques being studied to reverse gut dysbiosis following SCI. Kigerl and coworkers demonstrated that medical-grade probiotic VSL #3 given daily to mice following a spinal cord contusion injury (a) improved locomotor recovery as assessed through the Basso Mouse Scale (BMS) score and (b) reduced lesion size at the SCI epicenter. (Reproduced with author and journal's permission, ©2016 Kigerl et al. Originally published in J Exp Med https://doi.org/10.1084/jem.20151345). Jing et al. treated mice following a spinal cord contusion injury with a healthy donor FMT which showed (c) improved locomotion, (d) improvement in reduction of motorneurons within the injury site, (e) faster gastrointestinal transit time, and (f) reversed gut dysbiosis. (Reproduced with author and journal's permission, ©2021 Jing et al. Originally published in Microbiome https://doi.org/10.1186/s40168-021-01007-y). Rong and coworkers discovered that dietary changes utilizing Ursolic acid (UA) given to mice following spinal cord contusion injury (g) improved locomotor recovery, (h) increased microbiome diversity, and (i) promoted neuronal survival and synaptic regeneration (Reproduced with author and journal's permission, ©2022 Rong et al. Originally published in Front Cell Neurosci https://doi.org/10.3389/fncel.2022.872935). Image created by BioRender, Inc.

Table 2.

Recent Studies Investigating Probiotics to Target Gut Dysbiosis Following SCI

Reference	Purpose	Model	Methods	Results	Limitations/Implications
Kigerl et al., J. Exp. Med., 2016³⁰	To determine if gut dysbiosis affects the recovery of neurological function following SCI To test whether probiotics have a beneficial effect in SCI	Mouse	Medical-grade probiotic VSL #3 (mostly contains Lactobacillus and Bifidobacterium) delivered daily post-injury versus no intervention	Probiotics improved locomotor recovery Probiotics increased T-regulatory cells and DCs in MLNs Probiotics reversed gut dysbiosis	Probiotics can improve gut dysbiosis, immune dysfunction, and neuronal recovery Probiotics may have maximal effect within two weeks of injury
Wong et al., Br. J. Nutr., 2014⁶⁹	To determine if commercially produced probiotic could reduce the incidence of AAD/CDAD	Human	Lactobacillus casei Shirota (LCS) daily probiotic drink; No control group	Probiotics could reduce ADD	Randomized, controlled trial is needed to validate the findings
Faber et al., Spinal Cord, 2020⁷⁰	To investigate whether a probiotic can prevent AAD in patients with SCI To measure probiotics effects on bowel mobility, nausea, and quality of life	Human	Randomized trial with daily probiotic drink or placebo for antibiotic course duration and three-weeks post-antibiotic use	N/a	Trial will address Wong et al. study proposing that probiotics can prevent AAD in SCI
Glisic et al., Pilot Feasibility Stud., 2022⁷¹	To assess the feasibility of probiotic and prebiotic intervention in athletes with SCI	Human	Single-center randomized crossover trial for four weeks of daily dose of 3 g of probiotics or 5 g of prebiotic supplementation to normal diet, with a four-week washout period	N/a	Will be assessing neurological functioning measures (e.g., handgrip strength) in addition to bowel functioning

AAD, antibiotic-associated diarrhea; CDAD, Clostridium difficile-associated diarrhea; DCs, dendritic cells; MLNs, mesenteric lymph nodes; SCI, spinal cord injury

Although limited currently in SCI, probiotics have been used in other diseases such as inflammatory bowel disease (IBD),⁷² stroke,⁷³ and TBI,^74

–77 and more recently, in traumatic peripheral nerve injury (TPNI).⁷⁸ Building off of recent findings that probiotics could improve neurological function in SCI,³⁰ Rodenhouse and coworkers utilized antibiotic-induced dysbiosis and medical-grade VSL #3 probiotic-induced enrichment models to explore the role of the gut microbiome in TPNI.⁷⁸ They found that with probiotic treatment, an increase in butyrate- producing bacteria could partially restore gut dysbiosis. Even more interesting, they found that both pre-injury and post-injury delivery of probiotics promoted or rescued nerve function, respectively, suggesting that gut dysbiosis does impact TPNI recovery and can benefit from probiotic therapy. This study supports the findings from Kigerl and coworkers in that probiotics can be used for nerve regeneration.^30,78 Future work is needed to validate these findings and to elucidate optimal use of probiotic treatment in both central and peripheral nerve injuries.

FMT

FMT is the process in which the gut flora from the feces of healthy individuals is isolated and transplanted into the digestive tract of patients in order to alleviate disease.¹² FMT has been shown to be beneficial in numerous CNS diseases such as ischemic stroke,⁷⁹ Parkinson's disease,^80,81 Alzheimer's disease,⁸² multiple sclerosis,⁸³ and more recently, SCI. In 2015, a clinical case report demonstrated that a patient with acute SCI quadriplegia obtained benefit from FMT for the treatment of severe recurrent Clostridium difficile infections as a result of antibiotic treatment for pneumonia⁸⁴ (Table 1). The patient was initially treated with antibiotics, including metronidazole, vancomycin, fidaxomicin, rifaximin, and tigecycline; however, when these therapies failed, the patient underwent a colonoscopic FMT from his healthy son. Following FMT, the patient did not develop another C. difficile infection for the remainder of the study follow-up period of 12 weeks. It is of note that the patient was classified as an American Spinal Injury Association (ASIA) impairment scale grade C prior to the FMT, but the authors unfortunately did not evaluate any changes in motor function following FMT.

There are animal studies that do suggest that FMT in SCI can provide further benefit, however. Jing and coworkers recently demonstrated that FMT in SCI mice improves locomotion and gastrointestinal functions possibly because of the anti-inflammatory functions of SCFAs⁴⁶ (Fig. 2 and Table 1). Mice demonstrated significantly increased hindlimb locomotor function starting at 14 days from FMT and increased grip strength starting at 28 days. When recorded motor-evoked potentials at 4 weeks post-SCI were analyzed, SCI mice treated with FMT demonstrated a greater recovery of the neuromuscular unit in comparison with SCI mice without FMT. These results were supported through immunostaining of spinal cord sections, which demonstrated an increase in neuronal cell bodies and synaptic regeneration following FMT treatment in SCI animals. When assessing the GI tract, Jing et al. found that FMT maintained the integrity of the intestinal barrier through upregulation of tight junction proteins, revived gastrointestinal motility, and restored the richness of the intestinal microbiota in SCI mice. More specifically, FMT showed a reversal of the reduction of Firmicutes phylum but no differences within Bacteroidetes; the increase in Firmicutes correlated with locomotor recovery. At the genus level, FMT reversed the reduction of beneficial Blautia, Anaerostipes, and Lachnospiraceae_NK4A136, and reversed the increase of detrimental bacteria Bilophila. When analyzing mechanisms behind how FMT functions, the group proposed that FMT increases bacteria that produce SCFAs that are immunosuppressive through the IL1β/nuclear factor (NF)- $κ$ B signalling pathway; reduction of this inflammatory pathway may result in restoration of gastrointestinal and neuronal function.

Fecal transplants do come with risk; although usually benign, there is an increase in risk of infection and even death, and factors such as the physical condition and receptivity of the patient should be addressed prior to transplant.¹² For example, the 65-year-old patient described earlier showed a systemic inflammation response syndrome 4 days after FMT that was without a clear source, and contributed to his recent FMT. The patient was given antibiotics (i.e., metronidazole, vancomycin) and an additional enema for 7 days, which led to the improvement of his symptoms. When analyzing donor characteristics for FMT in ulcerative colitis, Kump and coworkers found that the taxonomic composition of the donor's intestinal microbiota is a major factor in predicting the success of therapy, with higher bacterial richness and high abundance of Akkermansia muciniphilia, unclassified Ruminococcaceae, and Ruminococcus spp. were more likely to induce remission.⁸⁵ Additionally, Schmidt and coworkers demonstrated that FMT from healthy rats prevented both SCI-induced microbiota changes and the development of anxiety-like behavior³³ (Table 1). However, more recently they found that the physical and mental state of the donor are important factors when considering the efficacy of FMT.⁸⁶ When using FMT from uninjured rats with anxiety-like behaviors at baseline and reduced proportion of Lactobacillus in their stool into healthy SCI rats, they found that the recipients had increased intestinal permeability, induced anxiety-like behavior, and long-term alterations in their immune profiles. The recipients also did not demonstrate any motor recovery following FMT from these less- healthy donors. It is also known that other factors such as age^87
–89 and diet^90

–93 affect the gut microbiota and should be considered before FMT. Finally, it is important to note that the Food and Drug Administration (FDA) has not formally recognized the use of FMT. Even more so, the FDA released a public safety announcement on March 13, 2020 warning of potential multi-drug-resistant bacteria being transferred to individuals after receiving FMT and the death of a couple patients following treatment.⁹⁴ Close investigation into these instances was conducted, which demonstrated that one patient who died following FMT was not only shown to be positive for Shigatoxin-producing Escherichia coli (STEC) but also had numerous chronic medical conditions that could have contributed to their death.⁹⁵ The other patient who died following FMT was found to be negative for STEC, and the FDA later released that they do not believe the death to be related to the FMT.⁹⁵ Either way, there is no federal regulation of FMT. The current standards of donor selection for FMT in clinical trials, however, consist mostly of screening for any known infectious agent,^{85,96

–101} and more recently, dysbiosis risk,¹⁰¹ but may not consider other characteristics of the donor. These studies, however, suggest that further screening may be warranted to provide the maximal benefit for patients. Heavy screening for individual donors for FMT may limit their availability, however, as one study pointed out that only 10% of potential donors ended up being qualified for donation.¹⁰¹ An alternative solution is to pool samples of vetted donors as a “stool biobank” for future use.¹⁰² Finally, although FMT has been utilized in clinical trials for other diseases such as C. difficile,^100,103,104 ulcerative colitis,^105,106 Crohn's disease,¹⁰⁷ irritable bowel syndrome (IBS),^108,109 and obesity,^110,111 no trials of FMT for SCI have been conducted at the time of this review.

Dietary Modifications

Metabolic disorders have similar gut microbiota profiles to what is observed following SCI.^112,113 For example, decreased Bacteroidetes and increased Firmicutes occur in both obesity and type 2 diabetes¹¹⁴ as well as in SCI.⁹ In addition, patients with SCI have a greater abundance of Lachnospiraceae and Acidaminococcaceae,¹¹⁵ which have been implicated in glucose metabolism.^116

–119 It is also known that many individuals with SCI develop metabolic disorders, which has shown that the combination leads to decreased microbiome diversity and more dysbiosis (i.e., decreased Clostridiales, increased Akkermansia) when they are compared with SCI individuals with normal glucose tolerance.¹²⁰ Knowing that gut dysbiosis can occur in both metabolic disorders and SCI has led researchers to consider modifying diet to restore balance to the gut microbiota and improve overall health. Li et al. recently published a study investigating the use of a low-carbohydrate, high-protein diet in SCI to improve metabolism and cardiovascular health¹¹² (Table 3). Patients who underwent dietary modifications had increased Bacteroides thetaiotaomicron, Coprococcus 3, and Fusicantenibacter, which are bacteria that are involved in fiber digestion and metabolism.^121,122 Coprococcus 3 was shown to be reduced in SCI individuals with severe constipation,¹²³ suggesting a link among fiber intake, Coprococcus 3, and bowel function in SCI.¹¹² Although the authors of this study did not specifically examine bowel function, the concept of fiber intolerance in SCI is not a new concept. Cameron and coworkers in 1996 conducted a clinical trial in which participants who consumed daily bran in their diets had worsening bowel transit times, suggesting that increasing fiber intake may have a detrimental effect¹²⁴ (Table 3). Following these findings, Stoffel and coworkers reported that a high fiber diet worsened constipation by prolonging colonic transit times in SCI individuals and suggested that patients adhere to a low-fiber diet.¹²⁵ Yeung and coworkers proposed that the ideal consumption of fiber in SCI patients is ∼15 g/day to manage neurogenic bowel, whereas ≥25 g/day of fiber results in worsening of constipation.¹²⁶

Dietary choices are a well-known modifier of the gut microbiota composition.^90

–93 In addition to the low-carbohydrate, high protein diet,¹¹² a Mediterranean diet rich in cereals, low-fat dairy products, olive oil, and fish and low in high-fiber fruits and vegetables has also been recommended in SCI.¹²⁷ Allison and coworkers found that treating SCI patients with a 3-month Mediterranean diet had anti-inflammatory effects (reductions in the pro-inflammatory cytokines interferon- $γ$ , IL1-β, and IL-6)¹²⁸ (Table 3), which are known to contribute to immune dysfunction and a chronic low-grade inflammatory state.¹²⁹ More specifically, they found that nutrients with established anti-inflammatory properties, such as vitamin A, carotenoids, omega-3 fatty acids, and zinc negatively correlated with these pro-inflammatory cytokines. Bigford and coworkers additionally used a Mediterranean diet to improve cardiovascular health in combination with circuit resistance training¹³⁰ (Table 3). They found that with these interventions, patients with SCI had a significant loss of body mass and effectively reduced component risk factors for cardiometabolic syndrome and diabetes (i.e., fasting triglycerides). In terms of the gut microbiome, a Mediterranean diet has also been shown to increase Bacteroidetes and decrease the presence of Firmicutes and Lachnospiraceae,¹³¹ which have been shown to be decreased and increased, respectively, following SCI.^30,115 Mediterranean diets have been used to reverse gut dysbiosis in other conditions such as Alzheimer's disease,¹³² IBD,¹³³ colorectal cancer,¹³³ and obesity.¹³⁴ Additional evidence to suggest that a Mediterranean diet may improve gut dysbiosis in individuals in SCI comes from the recent study by Rong and coworkers¹³⁵ investing the use of ursolic acid, a known component of olive oil, in a SCI mouse model^136,137(Fig. 2 and Table 3). Mice demonstrated increased body weight and muscle weight following ursolic acid treatment as well as inhibition of the inflammatory response and improved neuronal recovery. The treatment also restored dysbiosis by increasing the abundance of Muribaculaceae, Lachnospiraceae, and Alloprevotella in the gut. Overall, these studies suggest that dietary changes may improve not only bowel functioning but also cardiovascular health and neuronal recovery, emphasizing the need to treat SCI health more broadly instead of solely focusing on reversing paralysis.

Combination Therapy

Efforts to combine multiple therapeutic approaches in SCI have shown greater success than single therapies alone. For example, calcineurin inhibitors, cyclosporin or FK506 (tacrolimus), have been used to support the survival of transplanted fibroblasts and promote greater recovery of axons after SCI in rats than when fibroblasts are transplanted alone.^13,138 Granulocyte-colony stimulating factor (GCSF) has been used to enhance the therapeutic benefits of methylprednisolone,¹³⁹ neural stem cells,^140,141 mesenchymal stem cells,¹⁴² and peripheral blood stem cells.¹⁴³ Fingolimod (FTY720), a sphingosine structural analog, has been infused with poly(lactic-co-glycolic acid) (PLGA) nanoparticles for local delivery to the spinal cord and co-delivered with PuraMatrix™ embedded neural stem/progenitor cells within an SCI mouse model.¹⁴⁴ The drug minocycline, which has been shown to have beneficial effects on inflammation and gut microbiota,³⁴ has been combined with multiple therapies (i.e., mesenchymal stem cells,¹⁴⁵ olfactory ensheathing cells,¹⁴⁶ hydrogel,¹⁴⁷ or PLGA¹⁴⁸ nanoparticles) to improve neuronal function in SCI. The collagen-based NeuroRegen scaffold is currently being investigated in combination with mesenchymal stem cells in a clinical trial (n = 30; NCT02688049) based off of successful preclinical results.^149,150 Many other combinations exist that can be more expertly reviewed elsewhere.^{151

–159} As for combination treatments targeting the microbiome and SCI, there are no current studies to date. As previously described in this review, the spinal cord injury site, immune system, and gut microbiota are interconnected on an axis, one that could benefit from a multi-faceted treatment strategy. In a multi-modal approach to IBD, which is thought to demonstrate similar brain–gut–immune axis interactions, mesenchymal stem cell therapy and have been combined to show improved clinical benefit over either treatment alone.¹⁶⁰ With limited FDA-approved treatment options for SCI, the lack of intersectional investigation regarding the microbiome and SCI demonstrates a missed opportunity to maximize treatment efficacy.

Conclusion

SCI is a devastating condition that extends beyond paralysis to include gut dysbiosis and immune dysfunction. Damage to the spinal cord can result in diminished output to the gut, leading to microbiome dysbiosis and bowel malfunction. The altered gut environment can then activate GALT, which can release cytokines, neuroactive metabolites, and neurotransmitters that travel via the bloodstream to CNS tissues or can directly activate the vagus nerve, leading to worsening of the inflammation at the SCI lesion epicenter. This circle of events is known as the “spinal cord–gut microbiome–immune axis.” Previous treatment modalities for SCI have focused mostly on reversing paralysis, but SCI comes with other devastating consequences such as bowel and immune dysfunction. More recent efforts have been focused on treating these more distal manifestations of SCI. Probiotics, FMT, and a Mediterranean diet offer treatment options targeted to improve gut dysbiosis and immune function. Remarkably, these interventions also have been shown to improve neuronal recovery at the SCI site. Future investigation into these therapies as well as the combination of these approaches are necessary as the field advances in providing a more holistic approach to SCI treatment.

Footnotes

Acknowledgments

The authors thank the Rush Medical College Alumni Association for sponsoring this project through the Rush Medical College Dean's Research Fellowship.

Authors' Contributions

K.D.R. developed the idea in detail, reviewed the literature, and wrote the manuscript. B.T.D. and R.G.F. provided valuable feedback and revision of the manuscript.

Funding Information

This project was sponsored through the Rush Medical College Dean's Research Fellowship.

Local institutional funding for medical students so no grant number available. More institution information:

Dean's 2020 Summer Research Fellowship, Rush Medical College Alumni Association, Rush University, Chicago, IL.

Author Disclosure Statement

No competing financial interests exist.

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