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Kinetics of inflammatory markers following traumatic injuries to the spinal cord.

Danuta Radzioch1, 2*, Gabriella Wojewodka1, 3, Samuel David1, 2, Ioli Makriyianni3, Mohan Radhakrishna1, 3, Stefan Parent4, 5 and Jean Ouellet1, 6
  • 1 McGill University, Canada
  • 2 Montreal General Hospital Research Institute, Canada
  • 3 Montreal General Hospital, McGill University Health Center, Canada
  • 4 Hôpital Sacré-Coeur de Montréal, Canada
  • 5 Université de Montréeal, Canada
  • 6 Shriners Hospital Canada, Canada

ABSTRACT

Inflammation following traumatic spinal cord injuries precedes the wound healing process, however, excessive inflammation may lead to irreversible neuronal damage. In this study we assessed whether there are differences in elevation of inflammatory markers in patients with spinal cord injuries (SCI) as compared to those with spinal fractures (SF) without CNS damage.

Patients with SCI and SF were recruited to the study. Blood samples were collected at 1, 2, 3, 8-16, 25-49 days and 1-2 years post injury. We analyzed inflammatory markers in blood plasma (IL-1β, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-13, IL-17A, IFNα2, IFNγ, and TNFα) and assessed the differences in their levels between patients with SCI and SF injuries. Here we report preliminary data on 7 SCI and 10 SF patients.

IL-6 levels were higher in SCI than SF patients from days 1 to 3, whereas at 8-16 days post injury, IL-6 became significantly lower in SCI patients than SF (p = 0.0428). At day 2, IL-10 levels were significantly higher in SCI patients compared to SF (p = 0.0341). IFNγ levels were higher in SCI and SF patients at day 3 (p = 0.0216). At 8-16 days post injury , IL-17A and IL-8 were higher in SCI than SF patients (p = 0.0079 and p = 0.0033, respectively).

SCI patients had significantly higher levels of inflammatory markers than SF patients from the first day of injury suggesting that SCI patients may benefit from therapies reducing systemic inflammation immediately following SCI injury.

INTRODUCTION

Injuries to the spinal cord may cause debilitating and permanent motor damage. The inflammatory process plays a crucial role in neuronal repair. Neutrophils are rapidly recruited following trauma to the spinal cord. They start appearing within 2 to 3 hours post injury and their peak concentration can be observed between 1 to 3 days (1). Macrophage recruitment follows neutrophil infiltration and the increased amount of macrophages may persist for months (1, 2). Unabated, the inflammatory process may impede neuronal repair leading to secondary tissue damage. Unlike spinal cord injuries, spinal fractures result in little CNS damage. We hypothesized that the expression of inflammatory markers following these two types of injuries have different kinetics, explaining in part the difference in patient recovery. In this report, we describe preliminary data from our study assessing the expression of cytokines following trauma causing spinal cord injury and spinal fracture.

METHODS

Study Protocol. Patients with an isolated spinal cord injury (SCI) or isolated spinal fracture (SF) were recruited from the Montreal Sacré-Coeur Hospital (Montreal, Qc, Canada) and the Montreal General Hospital (Montreal, Qc, Canada) emergency departments. Here we report preliminary data on 7 SCI and 10 SF patients. The study was approved by the institutional review boards for both study sites. Patients and family members were asked to give consent to participate in the study. The inclusion criteria for isolated SCI included: up to 65 years of age, complete American Spinal Injury Association (ASIA) A grade of injury, incomplete (ASIA) B, C or D. The inclusion criteria for isolated SF included: up to 65 years of age, ASIA E (no spinal cord deficit), cervical, thoracic, and lumbar burst fractures with no spinal cord injury that have had conservative treatment or surgical intervention. Exclusion criteria included: older than 65 years of age, presence of tumors, other major trauma such as in long bones (femur, tibia, fibula, humerus, ulna, radius), traumatic brain injury, Type B or C pelvic fractures, Cauda Equina Syndrome, pregnancy, metabolic diseases and more than 72 hours post injury.

Sample collection and cytokine analysis. Blood samples were collected at 1, 2, 3, 8-16, 25-49 days and 1-2 years post injury in EDTA coated tubes. Samples were spun at 3000 rpm at 4°C for 10 min for plasma isolation. We analyzed inflammatory markers in blood plasma (IL-1β, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-13, IL-17A, IFNα2, IFNγ, and TNFα) with the MILLIPLEX® Map multiplex assay (Millipore, Mississauga, ON, Canada) using the MAGPIX® multiplexing instrument (Millipore) according to manufacturer’s instructions. Data was analyzed with the MILLIPLEX® Analyst software, version 4.2 (Millipore).
Statistical analysis. Data were analyzed using the GraphPad Prism 5 (GraphPad Software, LaJolla, CA, USA). The Mann-Whitney test was used to determine statistical differences between the two patient groups at each time point. Significance was set at p < 0.05.

RESULTS AND DISCUSSION

Inflammation plays an important role in tissue repair following any type of injury. In the case of spinal trauma, the timing of inflammatory cell influx and cytokine expression is sensitive (3). For instance, soon after spinal trauma, macrophages are polarized towards a M1 phenotype inducing the expression of pro-inflammatory cytokines. This is followed by a brief period of polarization towards M2 macrophages, which may have anti-inflammatory effects in spinal trauma. Eventually, the polarization returns to predominantly M1 macrophages, with minimal presence of M2 macrophages (4). Our analysis of the expression of inflammatory markers revealed differences between SCI and SF patient groups post spinal trauma (Figure 1). Within 24 hours of injury (day 1), there were no statistically significant differences between the two groups, although IL-6 had the tendency to be higher in SCI patients (Figure 1D). At day 2, IL-6 and IL-10 were higher in SCI patients than SF (Figure 1D and G). The higher levels of IL-10 suggest a brief shift towards anti-inflammatory mechanisms however not sustained at day 3. IFNγ levels in SCI patients were significantly higher in SCI patients as of day 3 (p = 0.0216, Figure 1B) which may induce prolonged activation of pro-inflammatory pathways. Significantly higher levels of IL-8 and IL-17A (p = 0.0033 and p = 0.0079, respectively) were observed in SCI patients at 8-16 days post-injury (Figure 1F and J). Interestingly, IL-17 was found to impair recovery from spinal contusions in mouse models and IL-17 knock-out mice had improved recovery of locomotor function compared to wild-type controls (5). Our previous studies using a mouse model of spinal cord injury demonstrated improved locomotor function recovery and reduced cytokine expression following treatment with fenretinide, a Vitamin A derivative (6). Our preliminary results suggest that SCI patients have higher levels of inflammatory markers following injury. Further studies are needed to determine whether they may benefit from anti-inflammatory therapies, such as fenretinide.

CONCLUSION

SCI patients had significantly higher levels of inflammatory markers than SF patients from the first day of injury. These could be a target for therapy immediately following SCI injury.



FIGURE LEGEND

Figure 1. Kinetics of inflammatory markers in SCI and SF patients. Blood plasma was analyzed from 7 SCI (red) and 10 SF (blue) patients at 1. 1 day (SCI n = 3; SF n = 8), 2. 2 days (SCI n = 7; SF n = 9), 3. 3 days (SCI n = 7; SF n = 10), 4. 8-16 days (SCI n = 7; SF n = 9), 5. 25-49 days (SCI n = 6; SF n = 5), 6. 1-2 years after trauma (SCI n = 2; SF n = 2). A) IFNα2 levels were higher in SCI patients than SF. B) IFNγ levels were higher in SCI and SF patients at day 3 (p = 0.0216). C) IL-1β levels were higher in SF patients than SCI patients on Day 1. D) IL-6 levels were higher in SCI than SF patients from days 1 to 3 whereas at 8-16 days post injury, IL-6 became significantly lower in SCI patients than SF (p = 0.0428). E) IL-7 levels were higher in SCI patients at later time points. F) At 8-16 days post injury, IL-8 was higher in SCI than SF patients (p = 0.0033). G) At day 2, IL-10 levels were significantly higher in SCI patients compared to SF (p = 0.0341). H) IL-12p70 levels were higher in SF patients on day 2, but were higher in SCI patients at later time points. I) IL-13 levels were barely detectable in all samples excepting high levels in one patient at 1-2 years post injury. J) At 8-16 days post injury, IL-17A was higher in SCI than SF patients (p = 0.0079). K) TNFα levels were similar in SCI and SF groups.

Figure 1
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References

1 Fleming JC, Norenberg MD, Ramsay DA, et al. The cellular inflammatory response in human spinal cords after injury Brain 2006;129:3249-69.
2 Chang HT Subacute human spinal cord contusion: few lymphocytes and many macrophages Spinal Cord 2007;45:174-82.
3 David S, Kroner A Repertoire of microglial and macrophage responses after spinal cord injury Nat Rev Neurosci 2011;12:388-99.
4 Kigerl KA, Gensel JC, Ankeny DP, et al. Identification of two distinct macrophage subsets with divergent effects causing either neurotoxicity or regeneration in the injured mouse spinal cord J Neurosci 2009;29:13435-44.
5 Hill F, Kim CF, Gorrie CA, et al. Interleukin-17 deficiency improves locomotor recovery and tissue sparing after spinal cord contusion injury in mice Neurosci Lett 2011;487:363-7.
6 Lopez-Vales R, Redensek A, Skinner TA, et al. Fenretinide promotes functional recovery and tissue protection after spinal cord contusion injury in mice J Neurosci 2010;30:3220-6.

Keywords: Spinal Cord Injuries, Inflammation, Cytokines, Spinal Fractures, Kinetics, Chemokines, clinical study

Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013.

Presentation Type: Abstract

Topic: Immune-mediated disease pathogenesis

Citation: Radzioch D, Wojewodka G, David S, Makriyianni I, Radhakrishna M, Parent S and Ouellet J (2013). Kinetics of inflammatory markers following traumatic injuries to the spinal cord.. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.01182

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Received: 31 Jul 2013; Published Online: 22 Aug 2013.

* Correspondence: Prof. Danuta Radzioch, McGill University, Montreal, Canada, danuta.radzioch@mcgill.ca