Hellenbrand et al.
Journal of Neuroinflammation(2021) 18:284
https://doi.org/10.1186/s12974-021-02337-2

Open Access

REVIEW

Inflammation after spinal cord injury:
a review of the critical timeline of signaling cues
and cellular infiltration
Daniel J. Hellenbrand, Charles M. Quinn, Zachariah J. Piper, Carolyn N. Morehouse, Jordyn A. Fixel andAmgad S. Hanna*

Abstract
Traumatic spinal cord injury (SCI) is a devastating neurological condition that results in a loss of motor and sensory
function. Although extensive research to develop treatments for SCI has been performed, to date, none of thesetreatments have produced a meaningful amount of functional recovery after injury. The primary injury is caused by
the initial trauma to the spinal cord and results in ischemia, oxidative damage, edema, and glutamate excitotoxicity.
This process initiates a secondary injury cascade, which starts just a few hours post-injury and may continue for morethan 6 months, leading to additional cell death and spinal cord damage. Inflammation after SCI is complex and driven
by a diverse set of cells and signaling molecules. In this review, we utilize an extensive literature survey to develop thetimeline of local immune cell and cytokine behavior after SCI in rodent models. We discuss the precise functional rolesof several key cytokines and their effects on a variety of cell types involved in the secondary injury cascade. Furthermore, variations in the inflammatory response between rats and mice are highlighted. Since current SCI treatment
options do not successfully initiate functional recovery or axonal regeneration, identifying the specific mechanisms
attributed to secondary injury is critical. With a more thorough understanding of the complex SCI pathophysiology,
effective therapeutic targets with realistic timelines for intervention may be established to successfully attenuate
secondary damage.
Keywords: Spinal cord injury, Inflammation, Secondary cascade, Macrophages, Cytokines, Microglia, Astrocytes
Background
Every year in North America approximately 12,500 people are paralyzed due to a spinal cord injury (SCI) [1].
After SCI, there is a primary injury caused by the initialtrauma, which compromises neurons and glia and initiates a secondary injury cascade that leads to additional
cell death and spinal cord damage over the subsequentweeks. This overwhelming inflammatory response in
the early phase of injury, combined with the disrupted*Correspondence: ah2904@yahoo.com
Department of Neurological Surgery, School of Medicine and Public
Health (UWSMPH), University of Wisconsin, 600 Highland Ave, Madison,
WI 53792, USA

blood–spinal cord barrier, progressively adds to spinal
cord swelling and damage. The end result in the chronic
stage is a dense glial scar leaving patients with a loss ofboth sensory and motor function below the level of injury[2]. The paralysis often leaves patients unable to care forthemselves, exemplifying the need to develop a treatment
to help retain or restore function after SCI.
Inflammation after SCI is complex and orchestrated by many cell types and numerous inflammatory cytokines including tumor necrosis factor alpha(TNFα), interleukin-1β (IL-1β), and interleukin-6 (IL-6),
among several others. Although there are several positive effects from inflammation after SCI, the extensive
infiltration of immune cells is a principal contributor© The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of thislicence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​
mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Hellenbrand et al. Journal of Neuroinflammation(2021) 18:284

to neural degeneration [3, 4]. These immune cells are
guided to the lesion site from the periphery via cytokinesand chemokines released by microglia, astrocytes, andperipherally derived macrophages (PDMs) within thelesion [5, 6]. Overall, there is a general consensus amongscientists regarding the inflammation process after SCI.
However, there are discrepancies in the inflammation
timeline and the extent of cytokine regulation after injury
in the literature. Delineation of precise functional rolesof cytokines and their timeline of upregulation/downregulation may provide insight into how to regulate acuteinflammatory reactions after SCI [7].
The objective of this review is to conduct an extensive
survey of the literature to develop a precise timeline ofimmune cell and cytokine regulation in the spinal cord
after SCI. Here we discuss the injury timeline with afocus on cellular behavior, cytokine activity, and the corresponding changes that occur to the injury site. The
majority of studies on inflammation after SCI utilize
rodent models. After SCI, mice develop fibrous connective tissue domains throughout the injury site, which differs from rats and other mammals that develop necrotic
lesion cavities encased by reactive astrocytes [8–12].
Thus, we will also discuss differences in cytokine profiles
observed between rats and mice.

Literature review
An electronic search of the Medline database for literature describing animal models of SCI from 1946 to
2021 was performed using the following conditions: SCI(MeSH Terms) AND inflammation (MeSH Terms) OR
secondary injury (MeSH Terms). For the timeline of local
cytokine regulation, the results were further screened
and only included articles utilizing rats or mice withuninjured controls or sham controls for comparison of
local upregulation or downregulation of cytokines within
their experiments (Fig. 1).
Main text
Inflammation after SCI

Neuroinflammation is the activation of the central nervous system’s (CNS) innate immune system in responseto an inflammatory challenge, which is characterized
by a host of cellular and molecular changes within the
CNS. This inflammation is mediated by the upregulationof cytokines/chemokines (Table 1), which are produced
by resident microglia, astrocytes, peripherally derived
immune cells, and endothelial cells. Three inflammatory
cytokines, TNF, IL-1, and IL-6 have been studied extensively and are upregulated within hours after the initial
injury (Table 1, Figs. 2 and 3). This production in inflammatory cytokines and chemokines results in extensive
infiltration of immune cells including microglia, PDMs,

Page 2 of 16

and neutrophils, which continue the production of additional inflammatory mediators. Although the degree ofneuroinflammation depends on the extent of the primary stimulus or insult, the primary insult often results
in an overreaction of the inflammatory process after CNS
injury leading to additional cell death [13].
Although the present review focuses primarily on thelocalized inflammatory response to SCI in rodent models, it is important to note that there is systemic inflammation occurring after SCI. Due to the inability tosample the spinal cord parenchyma of human patients
after injury, many are investigating whether human blood
serum and/or cerebrospinal fluid (CSF) cytokine samples may be used as biomarkers to provide insight into
injury severity, thereby influencing treatment decisions[14–16]. Kwon et al. [14] found that at 24 h after injury,
cytokines IL-6, IL-8, and MCP-1, along with structuralproteins, such as GFAP, tau, and S100β, were present insignificantly higher concentrations than corresponding
serum samples in human patients. In the CSF of a rat
SCI model 6 h after injury, TNFɑ, IL-2, IL-10, IL-17ɑ,and IFN-γ concentrations were significantly increased[17], and serum concentrations of TNFɑ, IL-1β, and IL-6
have been shown to remain elevated in rat throughout
the first week following SCI [18–20]. This is similar to the
local upregulation of these cytokines that occurs within
the injured spinal tissue (Figs. 2 and 3). Interestingly, theupregulation of proinflammatory cytokines TNFɑ and
IL-1β persists in the serum 28 days after SCI in a rat,
while IL-4 is downregulated at 28 days and there is no
significant change in IL-10 at this timepoint [21]. However, Ogurcov et al. [15] found a decreased concentration of both IL-1β and IL-10 in serum of human patients
14 days after SCI. Furthermore, they report the elevationof IL-4 and IFN-γ levels at this timepoint [15]. In general,
analysis of cytokine in CSF and serum display similar patterns as the local response in injured spinal tissue (Figs. 2
and 3).
Locally, the primary trauma after SCI, injures the glial
cells in the spinal cord and damages blood vessels causing
ischemia. The death of nearby glial cells and the ischemia
are the initial structural and cellular damage that triggers the secondary injury cascade. The primary cytokinesthat have been studied extensively within this cascadeare listed in Table 1 in terms of the cells that secrete
them, receptors they bind to, and their effects after SCI.
Below, we discuss the timeline of this cascade in terms ofcytokine/chemokine regulation and cellular infiltration.
Within the first hour post‑injury

Ischemia, oxidative damage, edema and glutamate
excitotoxicity all start minutes after SCI and contribute to substantial secondary damage [22–25]. CellHellenbrand et al. Journal of Neuroinflammation(2021) 18:284

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Fig. 1 Flowchart displaying how articles were screened to be analyzed for assessing cytokine/chemokine regulation after SCI. All studies used were
performed in rats and mice and assessed local cytokine/chemokine regulation at specific times post-injury compared to uninjured or sham controls

Hellenbrand et al. Journal of Neuroinflammation(2021) 18:284

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Table 1 Cytokines and chemokines involved in inflammation after SCI
Names
Aliases

Cells that secrete the cytokine/chemokine, receptors they bind to, and their effects after SCI

IL-1α
IL-1F1

Released via activated microglia and PDMs largely in response to disease, infection, or inflammatory events [52]Binds to IL-1R1 [52]Required for neutrophil recruitment during cell death-induced sterile inflammation [128]IL-1βIL-1F2

Produced by activated microglia and PDMs as a proprotein, which is proteolytically processed to its active form by caspase 1 (CASP1/ICE)[52, 129–131]Binds to the IL-1R [129]Plays a more substantial role than IL-1α post-SCI [52, 132, 133]
Stimulates inflammatory mediators including prostaglandins, cyclooxygenase 2, and phospholipase A2 [52, 129]IL-2

Produced by activated T-cells [134]
Binds to IL-2R complex, which consists of IL-2Rα, IL-2Rβ, and the common γ-chain subunits [134]Contributes to the proliferation of T-helpers [87]Responsible for initiating the proper response of memory T-cells to invading pathogens [135]IL-4

Produced by T-helper cells, eosinophils, basophils, and mast cells [136]
Binds to IL-4Rɑ, which will either dimerize with the common cytokine-receptor γ-chain and produce the type-1 signaling complex, or with
IL-13Rα1 and produce the type-2 signaling complex [136, 53]
Increases microglia/macrophages expressing antigens characteristic of an anti-inflammatory M2 phenotype [70]
Increases the number of oligodendrocytes and neuronal markers βIII-tubulin and NeuN after SCI, suggesting a role in neuroprotection
[137]

IL-5

Produced by hematopoietic and non-hematopoietic cells, including granulocytes, T-cells, and natural helper cells [138]Binds to IL-5R and stimulates B-cell growth as well as increases immunoglobulin secretion (primarily IgA). Is also a key mediator in eosinophil activation [139, 140]

IL-6

Expressed in astrocytes, microglia and PDMs, and neurons [37]Binds to IL-6R, which exists either as a membrane-bound receptor or a soluble receptor [141]Activates inflammation and is a strong recruiter of immune cells after SCI [142]Acts on neural stem cells to induce their differentiation into astrocytes [143]

IL-8
CXCL8
GRO (therat analogue)

Produced by a wide variety of cells including monocytes, endothelial cells, T-cells, and macrophages [144]
Binds to CXCR1 and CXCR2 receptors [52]Induces chemotaxis in neutrophils and granulocytes [52]Upregulated for at least 14 days after SCI and strongly correlates with the extent of injury [87, 49, 38, 14]

IL-10

Produced by monocytes, B-cells, dendritic cells, natural killer cells, and T-cells [145]Binds to IL-10R heterotetramer complex made of two IL-10R1 molecules and two IL-10R2 molecules [146]
Downregulates several pro-inflammatory cytokines and inflammatory species [56]Provides trophic support to neurons through downregulation of pro-apoptotic factors and upregulation of anti-apoptotic factors [56]IL-12
IL-12ɑ
P35

Produced by dendritic cells, macrophages, monocytes, neutrophils, microglia cells, and B-cells [147]
Binds to IL-12R, which consists of the IL-12Rβ1 and IL-12Rβ2 chains [148]IL-12(p70) expresses nitric oxide synthase and TNFα in microglia and PDMs [149]IL-13

Produced by T-cells, dendritic cells, and activated Th2 cells [150]
Binds either to IL-13Rɑ1 or IL-13Rɑ2 [53, 151]
Involved in the production of transforming growth factor beta (TGF-β) [151]IL-17ɑ
IL-17
CTLA-8

Produced by several types of cells including T-cells, dendritic cells, and macrophages [152]
Binds to the A and C subunits of IL-17R [152]IL-17 knockout mice showed increased locomotor function after SCI suggesting a role in regulating secondary degeneration of neuraltissue [153]

TNF-ɑ
TNF
TNFSF2

Produced by microglia, PDMs, astrocytes, oligodendrocytes, monocytes, and neurons [37, 39]
Two active forms are transmembrane TNFɑ and soluble TNFɑ [154]Soluble TNFα is released by regulated cleavage of transmembrane TNFɑ by TNFɑ-converting enzyme (TACE/ADAM17) [154]
Binds to TNFR1 and TNFR2 [154]Affects cell proliferation, differentiation, apoptosis, immunity, and inflammation [37]Recruits macrophages to injury site [37]IFN-γ

Produced by γẟ T-cells and leads to the activation of macrophages [95, 155]
Induces secretion of IL-10 from microglia and PDMs [155]Can act synergistically with GM-CSF to promote the production of cytokines [156]

GM-CSF
CSF2

Produced by macrophages, mast cells, T-cells, fibroblasts, and endothelial cells in response to immune activation and cytokines that mediate inflammation [157]
Causes differentiation of hematopoietic progenitor cells into granulocytes, macrophages, and dendritic cells [158]Can stimulate the proliferation of bone marrow stem cells and reduce leukocyte apoptosis, as well as cause microglia to proliferate andchange their morphology [158]
GM-CSF-induced activation of microglia may promote functional recovery and axonal regeneration by release of brain-derived neurotrophic factor or by phagocytosis of myelin debris [159, 160]

Hellenbrand et al. Journal of Neuroinflammation(2021) 18:284

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Table 1 (continued)
Names
Aliases

Cells that secrete the cytokine/chemokine, receptors they bind to, and their effects after SCI

MCP-1
CCL2

Secreted by activated T-cells, astrocytes, microglia, and monocytes [161]
Binds to CCR2 [106]Activates and recruits mononuclear phagocytes, T-cells, and B-cells [162]MIP-1ɑCCL3

Secreted by activated T-cells, astrocytes, microglia, and monocytes [161]Promotes chemotaxis of monocytes and T-cells [161]Expression induced by pro-inflammatory stimulators, such as LPS, TNFα and IL-1β, and neuronal injury [163]Can enhance the production of other pro-inflammatory cytokines via CCR1, CCR4, and CCR5 [163]Impacts CNS inflammation through regulation of macrophages and astrocytes [163]

RANTES
CCL5
TCP228

Produced by astrocytes and is primarily involved in promoting migration of M2 macrophages [51]Expression induced by IL-1 and macrophage migration inhibitory factor [51]Binds to CCR1, CCR3, and CCR5 [164]
Associated with T-cell activation in SCI. Chronic T-cell activation subsequently contributes to neurodegeneration and inhibits repair of
injured tissues [165]Induces the migration and recruitment of a wide variety of cells including T-cells, dendritic cells, natural killer cells, eosinophils, basophils,
mast cells, and endothelial progenitor cells [51]May help amplify inflammatory response by facilitating the recruitment of inflammatory cells to the injury [51]

TGF-β1

Produced by lymphocytes, macrophages, and dendritic cells [166]
Binds to TGF-βR1, TGF-βR2, and TGF-βR3 [167]
Activates glial cells and phagocytes to form connective tissue and extracellular matrix [168]Influences the differentiation, proliferation, and state of activation of leukocytes [166]
Known to suppress expression of MHC class II antigen [166]permeabilization, pro-apoptotic signaling and ischemic
injury due to the destruction of the microvascular supply cause additional cell dysfunction and death just minutes after SCI [26, 27]. There is also intracellular calcium
dysregulation in both neurons and glia, resulting in theactivation of calpains, which can cause mitochondrial
dysfunction and cell death [25, 28, 29]. Microglia, theresident immune cells, quickly respond to the injury
and their early response is protective [30–32]. However,microglia rapidly change to proinflammatory cells andrelease cytokines setting off a cascade of events that leadto an infiltration of peripheral immune cells [12]. The
activated microglia retract their cytoplasmic processes
and become indistinguishable in terms of morphology
from the PDMs, which enter from damaged blood vessels[33–36]. Within 30-min post-injury, microglia and astrocytes begin expressing inflammatory cytokines IL-1β
and TNFɑ mRNA [37]. Pineau and Lacroix showed that
TNFɑ mRNA positive cells, including microglia, astrocytes, oligodendrocytes, and neurons, peaked just 1 h
after mice sustained a spinal cord contusion [37].
Cytokine signaling from 1‑h up to 3‑h post‑injury

Using colocalization studies for mRNA after SCI in
mice, Pineau and Lacroix showed that during the first
few hours, proinflammatory cytokines IL-1β, TNFɑ, andIL-6 were being synthesized by microglia/macrophages,
astrocytes, and neurons [37]. Using various techniques
to measure TNFα in these early hours after SCI, fiveother studies all observed significant increases in TNFɑlevels (Fig. 2B) [38–42]. There were also five studies thatmeasured IL-1β using various techniques in these earlyhours after SCI and three of these studies observed a significant increase in IL-1β (Fig. 2A) [38, 40, 41]. However,two experiments utilizing enzyme-linked immunosorbent assays (ELISAs) to measure IL-1β did not observe an
increase at this early timepoint [42, 43], which may be theresult of the discrepancy in time needed to synthesize thefull protein and the time needed for it to be proteolytically processed to its active form by caspase 1 [44]. There
were very similar trends in the early IL-6 upregulation tothat of IL-1β (Fig. 2C) [38, 40–42]. The proinflammatory
cytokines TNFɑ, IL-1β, and IL-6 are presumed to be thepredominant players early in the injury timeline. Thus,
these three cytokine levels were the most frequently analyzed in these early hours after SCI (Fig. 2).
Cytokine signaling and cellular reactions from 3‑h
up to 6‑h post‑injury

At this early stage of inflammation, the majority of the literature agrees that the primary inflammatory cytokines,
TNFɑ, IL-1β, and IL-6, are all significantly upregulated(Fig. 2) [37, 38, 40, 45–48]. Pineau and Lacroix observedthat at 3 h after SCI in mice, the number of cells expressing the TNFɑ mRNA transcript at the lesion epicenter
was significantly upregulated compared to uninjured
controls. However, the number of cells had decreased
by 66% compared with the 1-h timepoint [37]. Tworesearch groups did not observe a significant increasein TNFɑ protein expression at 4 h after a rat contusion,

Hellenbrand et al. Journal of Neuroinflammation(2021) 18:284

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Fig. 2 Depiction of cytokine regulation following SCI in rodent models. A literature search was conducted and relevant data regarding significant
cytokine regulation was collected at various timepoints. Data is presented as a percentage of studies that found significant changes in cytokine
protein or mRNA expression levels compared to sham or naïve controls (p < 0.05). The number of papers used for each timepoint is listed at the
top of each bar. A Changes in IL-1β levels after SCI [17, 21, 37, 38, 40–43, 45–47, 66–68, 85, 87–89, 94, 106, 107, 169–187]. The proinflammatory
cytokine IL-1β shows consistent upregulation in the acute phase following SCI. However, there are some discrepancies as to whether IL-1β remains
upregulated several days after injury and the second surge 14 days was only observed in mice. B Changes in TNFɑ levels after SCI [17, 36–42,
45–48, 66–69, 85, 87–89, 94–96, 107, 121, 169–180, 182–189]. The majority of studies show an upregulation of the proinflammatory cytokine TNFɑ
immediately following SCI and persisting several days after injury. C Changes in IL-6 levels after SCI [17, 37, 38, 40–42, 45, 47, 66–68, 84, 85, 87–90, 93,
95, 96, 107, 121, 173, 174, 176, 178, 183, 184, 186–189]. Consistent upregulation of the proinflammatory cytokine is seen in the first 24 h following
injury before returning to baseline levels by 7-day post-injury. D Changes in IL-1ɑ levels after SCI [17, 38, 40, 66, 84, 87, 90, 95]. The proinflammatory
cytokine IL-1ɑ is upregulated in a similar manner to its isoform IL-1β, though IL-1β plays a more significant role following SCI [132, 133]. E Changes in
IFN-γ levels after SCI [17, 38, 48, 87, 90, 95]. The relative change in IFN-γ expression following SCI remains controversial, as shown by the conflicting
data presented. It appears to be upregulated in mice and downregulated in rats after 24 h. F Changes in MCP-1 levels after SCI [17, 38, 47, 84, 85,
87–90, 93, 121]. While there is some debate surrounding the regulation of the MCP-1 chemokine immediately after injury (1 h to < 6 h), nearly all
data collected shows that MCP-1 expression levels elevate quickly and remain upregulated for several days. G Changes in IL-10 levels after SCI [17,21, 38, 46, 47, 66, 85, 87–90, 96, 170, 173–176]. A delayed response is seen with IL-10 showing mixed results until upregulation at 3–7 days after
injury. The anti-inflammatory cytokine returns to baseline levels by 14 days. H Changes in IL-4 levels after SCI [7, 17, 38, 47, 48, 66, 70, 87]. While somestudies show increased expression of the anti-inflammatory IL-4, most researchers did not observe a change in IL-4 levels. I Changes in IL-13 levels
after SCI [17, 38, 47, 48, 87, 90]. Previous studies display conflicting data surrounding the regulation of IL-13 after injury, where it was upregulated inmice and downregulated in rats 3 days hours post-injury

Hellenbrand et al. Journal of Neuroinflammation(2021) 18:284

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Fig. 3 Line graph displaying the percentage of studies that observed significant upregulation in IL-1β, TNFα, IL-6, and IL-10. The majority ofinvestigators observe a significant upregulation of the three most investigated inflammatory cytokines early after injury and TNFα and IL-1β remain
upregulated. The anti-inflammatory cytokine IL-10 lags further behind and the majority of investigators show it peaking around 1-week post-injury

which suggests a return to baseline after a rapid onset of
TNFɑ [38, 40]. Although the upregulation of IL-1β andIL-6 lags slightly behind that of TNFɑ and IL-1ɑ, mostof the literature agrees that IL-1β and IL-6 are significantly upregulated within this 3–6-h time window [37,38, 40, 42, 45–47]. The expression of growth-related
oncogene (GRO), the rat analogue of IL-8, is also significantly upregulated at 4-h post-injury [38, 45, 49, 50].
The cytokine regulated upon activation, normal T-cell
expressed and presumably secreted (RANTES), which
induces migration and recruitment of T-cells, eosinophils, basophils, and leukocytes, has been shown to besignificantly upregulated 4-h post-injury, although it is
not as prevalent in the literature [38, 47, 51]. This early
upregulation in inflammatory cytokines is produced predominantly by microglia and astrocytes and leads to therecruitment of peripheral immune cells.
Although anti-inflammatory cytokines are capableof reducing proinflammatory cytokines, they are generally present at low levels or absent after SCI. IL-4
and IL-13 are related anti-inflammatory cytokines thathave been shown to induce alternative macrophageactivation [52–55], and IL-10 is an anti-inflammatory
cytokine that has been shown to downregulate proinflammatory cytokines [56, 57]. Four hours after SCI
in mice, IL-10 was significantly upregulated [47], yet,at this same time post-injury in rats IL-10 was not

upregulated [38, 46]. In contrast, IL-13 was upregulated in rats [38, 48] but not in mice [47]. A numberof researchers have shown that there are differencesin the adaptive immune system between mice and rats
as well as between different strains of mice, which
may explain the discrepancies in anti-inflammatory
cytokine regulation [12, 58–60].
The majority of the literature shows that in this 3–6-h
post-injury period there is an upregulation in inflammatory cytokines, which are produced largely by local
microglia and astrocytes, leading to further recruitment and proliferation of microglia and astrocytes andthe recruitment of peripheral immune cells (Fig. 4).
Neutrophils begin to appear 4–6 h after SCI and produce oxidative and proteolytic enzymes to sterilize andprepare the area for repair [61, 62]. However, overwhelming numbers of neutrophils result in tissue damage [61].
Lipid peroxidation, the process by which free oxygen
radicals react with the polyunsaturated fatty acids ofmembrane lipids, leads to 4-hydroxynonenal (4-HNE)
formation and disrupts cell membrane fluidity, metabolic processes, and ion transport systems [63, 64].
4-HNE itself is neurotoxic and can bind to cellular proteins and damage their structural and functional integrity [65]. Xiong et al. [65] also observed that byproductformation such as 4-HNE increases as soon as 3-h postinjury and remains high for 2 weeks.

Hellenbrand et al. Journal of Neuroinflammation(2021) 18:284

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and IL-10 [81, 82], and have elevated expression of key
enzymes involved in synthesizing pro-resolving mediators that actively turn off inflammation [83]. In vivo, macrophages in general are not fully inflammatory (M1) or
anti-inflammatory (M2), but rather exist on a continuum
in which their roles and phenotype are dictated by the
extracellular environment [57]. As early as 6 h after SCI
in mice, Kigerl et al. [12] observed antigen-presenting
MHCII cells in the dorsal roots, meninges, and within asubset of perivascular spaces.
Fig. 4 Primary injury after SCI causes cell membrane disruption andrupture of blood vessels leading to secondary injury with extensive
upregulation of cytokines/chemokines and infiltration of immune
cells. Microglia, the resident macrophages, are early responders andbecome indistinguishable in terms of morphology from peripherally
derived macrophages. In rats, neutrophils peak early in the injury ataround 24 h and gradually decrease over the next 7–10 days, while
lymphocytes peak at lower levels and at a much later time. Pericytes
also infiltrate later and interact with microglia around the edges of
the injury and this entire injury site is encased by activated astrocytes

Cytokine signaling and cellular reactions from 6‑h
up to 12‑h post‑injury

Almost all of the literature shows that the upregulation inproinflammatory cytokines TNFɑ, IL-1β, and IL-6 continues through the 6–12-h timeframe post-injury (Figs. 2
and 3) [17, 37–39, 41, 42, 66–69]. There is also a significant increase in monocyte chemoattractant protein 1(MCP-1) [17], macrophage inflammatory protein 1-alpha(MIP-1ɑ) [17], RANTES [17], GRO [17, 50], and C–X–C
motif chemokine ligand 1 (CXCL1) [69]. The majority of
the literature shows that IL-4, IL-10, and IL-13 remain atbaseline levels (Fig. 2G–I) [17, 38, 66, 70]. Ultimately, thisinflammatory environment continues the recruitment
and proliferation of microglia and astrocytes and therecruitment of peripheral immune cells. Apoptosis fromthis exacerbated inflammation process after SCI peaks
near 8 h in neurons and around 24 h in glial cells [71, 72].
During these early hours after injury, polymorphonuclear leukocytes are the predominant infiltrating cells[73], and over-activation of these cells continue to cause
tissue destruction through the release of significant
amounts of neurotoxins including reactive oxygen species (ROS), reactive nitrogen species (RNS), chemokines,
and enzymes [4, 61, 74, 75]. Popovich et al. [32] also
showed that lymphocytes begin to accumulate aroundblood vessels in gray matter as early as 6 h after SCI in
rats.
After SCI, activated microglia and PDMs have the capability to take on different functional phenotypes [33, 57,
63, 76–80]. Resolution-phase macrophages are enrichedwith major histocompatibility complex II (MHC) expression [81], express anti-inflammatory cytokines TGF-β1

Cytokine signaling and cellular reactions from 12‑hup to 24‑h post‑injury

The majority of the literature continues to show elevated levels of TNFɑ, IL-1β, and IL-6 (Fig. 2A–C). There
are also increased levels of GRO [50, 84], IL-7 [48], andchemokines MCP-1 [7, 85], and MIP-1α [84]. IL-4, IL-10,
IL-13 and TGF-β1 all remain at baseline levels (Fig. 1G–I)[41, 70, 85]. Although IL-4 was not upregulated, FrancosQuijorna et al. [70] observed an upregulation of IL-4Rɑ
on microglia and macrophages.
Cells expressing IL-1β and cells expressing IL-6 bothpeaked at 12-h post-injury [37]. Infiltrating leukocytes
also produce IL-1β, TNFɑ, IL-6, and LIF 12-h post-injury[37]. The infiltrating neutrophils, which phagocyte andclear debris, secrete proteases, elastase, myeloperoxidase, and release reactive oxygen species, peak at 24-h
post-injury [86]. The byproduct formation of 4-HNE also
peaked at 24 h and remained elevated for 2 weeks [65].
Cytokine signaling and cellular reactions from 24 h
up to 7‑day post‑injury

Pineau and Lacroix’s colocalization studies showed that
TNFɑ mRNA levels 2-day post-injury had returned to thelevels detected in mice that had received laminectomy
only [37]. The number of IL-1β positive cells continued
to decrease at 2-, 4- and 7-day post-injury, and from24 h up to 4 days, the average number of cells expressing the IL-6 transcript progressively decreased to become
almost nonexistent at 7 days [37]. Despite the decrease
observed in the number of cells expressing mRNA, several researchers observed levels that were significantly
higher than their uninjured controls at 1 day (TNFɑ
77.8%, IL-1β 79.2%, IL-6 88.2%), 3 days (TNFɑ 80.1%,IL-1β 68.8%, IL-6 73.7%), and 7 days (TNFɑ 64.3%, IL-1β
41.7%, IL-6 23.1%) after SCI (Figs. 1 and 2). These trends
were similar in the literature for both rats and mice after
SCI. GRO was also significantly elevated on day 1 [50,84], day 3 [84, 87], and day 7 [87].
There is significant upregulation of chemokines
throughout this first week recruiting monocytes,
T-cells, and dendritic cells to the injury site. MIP-1α
is upregulated on day 1 [84], day 3 [84, 87], and day 7

Hellenbrand et al. Journal of Neuroinflammation(2021) 18:284[87]. When tested, CXCL1 expression is significantly
increased on day 3 [69, 88, 89] and day 7 [86] andRANTES is significantly upregulated on day 3 [47, 88–90]. Previous studies have shown that MCP-1 mRNA
is increased in mice 1-day post-injury and returns tobaseline before 7-day post-injury [91, 92]. However,here there are some conflicting results 7-day postinjury. Although studies looking at mRNA MCP-1α levels 7-day post-injury observed a significant increase inmice [93] and rats [85], a study using multiplex assay
7-day post-injury in rats observed a significant decreasein MCP-1α levels (Fig. 2F) [87]. MIF and COX2 were
inducibly expressed after SCI in rats and peak levels
were observed on day 4 [94].
Some cytokines displayed contrasting behaviors
between rat and mouse models during the 24-h to 7-day
period. In mice, IFN-γ levels were significantly increased
1 day [48, 95], 3 days [48], and 7 days [48] after injury,
but IFN-γ levels in rats were significantly decreased at
3 days [87] and 7 days [87] following SCI (Fig. 2E). Levelsof IL-2 and IL-5 were significantly decreased in rats on
day 3 and day 7 post-injury [87], where in mice there wasno change in IL-2 levels [88], and a significant increasein IL-5 levels 3-day post-injury [89]. IL-13 was also significantly decreased 3 days and 7 days after SCI in rats[87]. However, in mice IL-13 was significantly upregulated 1 day and 3 days after SCI [48, 90] and returned tobaseline levels 7-day post-injury (Fig. 1I) [48]. Similarly,
IL-4 levels were not changed 1 day [38, 66], 3 days [7, 66,87], and 7 days [7, 87] after SCI in rats, but were significantly increased 1 day, 3 days, and 7 days post-injury inmice (Fig. 2H) [48].
The majority of researchers did not observe a significant increase in the anti-inflammatory cytokine
IL-10 until 3-day post-injury, which remained elevateduntil 7 days after SCI (Fig. 2G). There were also several
researchers that did not observe changes in IL-10 levels
after SCI in both mice [90] and rats [38, 66]. TGF-β1 wassignificantly increased 3-day [41] and 7-day post-injury[41, 96].
Using flow cytometry after SCI in rats, Beck et al. [97]demonstrated a time-dependent multiphasic responseof cellular inflammation, where the initial phases of cellular inflammation were composed of an early peak ofneutrophils 1-day post-injury, followed by a peak ofmacrophages/microglia 7-day post-injury, and T-cells
peaking at 9-day post-injury. This timeline of cellularinflammation in rats with SCI is in agreement with otherliterature for neutrophils [61, 62], macrophages [62, 98,99], and lymphocytes [98] (Fig. 4). After SCI in mice,neutrophils enter the injured spinal cord within 6 h, but
surprisingly do not reach peak numbers until 14-day
post-injury and persist for up to 6-week post-injury [12].

Page 9 of 16

Although axons begin to retract within 2-day postinjury due to the initial trauma, there is a later phaseof axon retraction caused by activated macrophages[63, 100–104]. Bisicchia et al. [105] demonstrated that
autophagosomes mediating the initiation of autophagy
accumulate in axotomized neurons 1 day after SCI
and remain high from 3 to 5 days before falling slightly
7-day post-injury. By 5-day post-injury, activatedmicroglia and PDMs are the predominant inflammatory cells, where phagocytic macrophages are mostly
located in areas of necrosis and microglia are at themargins. Lendate et al. [99] showed that microglia arehighly dynamic, proliferate extensively the first week
after SCI, and form a dense cellular interface at the border of the lesion between reactive astrocytes and infiltrating PDMs (Figs. 4 and 5).
Cytokine signaling and cellular reactions from 7‑day
up to 14‑day post‑injury

A second surge was observed in cells expressing TNFɑand IL-1β 14 days after SCI in mice [37]. This second
upregulation of TNFɑ and IL-1β appears to only occur
in mice [37, 48, 106], and not rats [87, 107] (Fig. 2A,
B). IL-6 appears to be returning to baseline in bothrats and mice by 14-day post-injury [37, 87, 90, 93,107] (Fig. 1C). Mukhumedshina et al. [87] measured
cytokine levels 14 days after SCI in a rat and observed
a significant increase in IL-1ɑ, GRO, IL-2, and MIP-1ɑ,and a significant decrease in levels of IL-13, IL-2, IL-5,
IL-18, IL-17ɑ, and GM-CSF. IL-1ɑ, IL-4, IL-12, IL-15,
RANTES, MIP-1ɑ, MCP-1, and IL-7 are significantly
upregulated in mice 14-day post-injury [48, 90, 93].
Beyond day 14 post-injury, there is less data measuring
cytokine levels for comparison (Fig. 2).
Although there is proliferation of oligodendrocyte
progenitor cells and maturation over the first 2-weekpost-injury, the end result is still improper remyelination [63, 108, 109]. The response of microglia and
PDMs are similar after SCI in mice, rats, and humans,
where peak numbers are reached around 7-day postinjury and remain in the injury site for months after
injury [57, 97–99, 110, 111]. In contrast, lymphocyte infiltration is much larger in mice than in rats orhumans and peaks at a much later timepoint (14-day
post-injury) in mice [98, 110, 111]. Also unique to miceis the infiltration of fibroblast-like cells and the formation of fibrotic tissue matrix [110]. It is suggested thatthe infiltration of fibroblast-like cells coupled with thelarger T-cell infiltration may be responsible for minimizing the development of cysts in mice [110, 111].

Hellenbrand et al. Journal of Neuroinflammation(2021) 18:284

Page 10 of 16

Fig. 5 Diagram displaying cellular activation/infiltration after rat SCI. After the primary insult, there is a much larger secondary injury with extensive
infiltration of immune cells. Neutrophil infiltration peaks 24-h post-injury and decreases over the next week. Infiltrating lymphocytes accumulate
around blood vessels in gray matter as early as 6 h and T-cell specific lymphocytes peak around 9-day post-injury. Microglia are activated, retracting
their cytoplasmic processes and becoming indistinguishable in terms of morphology from the infiltrating PDMs. The astrocytes also become
reactive and retract the cytoplasmic processes and migrate to the lesion. Although initially the astrocytes aid in tissue repair, they eventually
become scar-forming astrocytes and begin to wall off the ensuing inflammation. The final glial scar is compartmentalized with infiltrating
immune cells in the center, microglia interacting with pericytes around the edges, and astrocytes encapsulating the entire tissue containing the
inflammatory cells

Cytokine signaling and cellular reactions greaterthan 14‑day post‑injury

Astrocytes are the predominant subtype of glial cells
in the central nervous system that work to maintainneurons as well as the blood spinal cord barrier [112].
Although initially reactive astrocytes migrate to thelesion and aid in the tissue repair process after SCI,
they eventually become scar-forming astrocytes andform a glial scar around the lesion [112]. This thick layerof astrocytes surrounding the injury site is characterized by cellular hypertrophy, process extension, and theincreased expression of intermediate filaments, such as
glial fibrillary acidic protein (GFAP) (Fig. 5) [112].
T-cells are responsible for cell mediated adaptive
immunity [113]. Infiltration of T-cells into the injured
spinal cord occurs at different times depending on thespecies and strain of animal [110, 111]. After SCI, T-cellproliferation and cytokine production is further inducedby chemokines CXCL10 and RANTES [113–116]. It is
still a controversial topic on whether T-cells cause secondary degeneration or mediate wound repair after SCI[111, 113, 117, 118]. Gonzalez et al. [119] neutralizedHellenbrand et al. Journal of Neuroinflammation(2021) 18:284

Page 11 of 16

the chemoattractant CXCL10 and showed that it limited T-cell infiltration and improved tissue preservation and functional recovery, suggesting a negative rolefor T-cells. After SCI in rats, Wu et al. [120] observedthat the dominant T-cells were cytotoxic T-cells (> 90%)
with small numbers of regulatory T-cells (< 10%), which
again suggests more of a destructive role in regard to tissue recovery. Furthermore, Lui et al. [121] showed that
T-cell-derived perforin destroys the blood spinal cord
barrier after SCI, resulting in an infiltration of inflammatory cytokines aggravating secondary injury. After SCI in
mice, T-cell infiltration peaks around 14 days, decreases
for approximately 2 weeks, then increases again overthe next few weeks to reach amounts similar to the levelat 14-day post-injury, and are still detected for at least
42-day post-injury [12, 120, 122–125].
After SCI, pericytes, the main source of the scar connective tissue, enter the lesion area with blood vessel
sprouts and form the stromal component of the scar tissue [126]. Pericytes can also be activated by trace amines
to locally constrict the vasculature [127]. Thus, pericytes
regulate the capillary tone and blood flow in the spinal
cord after injury. After SCI in mice, the number of pericyte-derived cells peaked at 2 weeks, which was shown tobe more than a 25-fold increase, and then decreased after
4 months before leveling off and remaining for at least
7 months [126]. The final glial scar is compartmentalizedwith infiltrating immune cells in the center, microglia
interacting with pericytes around the edges, and astrocytes surrounding the periphery (Fig. 5) [99, 126].

Abbreviations
4-HNE: 4-Hydroxynonenal; CASP1/ICE: Caspase-1/interleukin-1 converting
enzyme; CXCL1: C–X–C Motif Chemokine Ligand 1; CNS: Central nervous
system; CSF: Cerebrospinal fluid; COX: Cyclooxygenase; ELISA: Enzyme-linked
immunosorbent assay; GFAP: Glial fibrillary acidic protein; GM-CSF: Granulocyte–macrophage colony-stimulating factor; GRO: Growth-related oncogene;
IFN-γ: Interferon gamma; IL: Interleukin; MCP-1: Monocyte chemoattractant
protein 1; MeSH: Medical Subject Headings; MHC: Major histocompatibility
complex; MIF: Macrophage migration inhibitory factor; MIP-1ɑ: Macrophageinflammatory protein 1-alpha; NK: Natural killer; PDMs: Peripherally derived
macrophages; RANTES: Regulated upon activation, normal T-cell expressedand presumably secreted; RNS: Reactive nitrogen species; ROS: Reactive
oxygen species; SCI: Spinal cord injury; TACE/ADAM17: Tumor necrosis factor
alpha converting enzyme; TGF-β: Transforming growth factor beta; TNFɑ:Tumor necrosis factor alpha.

Conclusions
Over the past few decades, there have been a plethora ofresearchers studying the complex inflammatory processthat follows SCI. This research has helped to define thecritical timeline of cell infiltration and cytokine profiles
that occur after SCI. The majority of this work has been
performed using rodent models. Although there aremany similarities in both rats and mice after SCI, differences exist in terms of neutrophil infiltration, lymphocyte infiltration, fibrotic scarring, and an absence of cysts
in mice. Overall, these rodent models have served well in
defining the cytokines, chemokines, and reactive species
involved after SCI.
To date, there is no successful treatment for SCI
patients to either help retain function after the injury,or regain function through regeneration after the injury.
Many scientists are in agreement that adapting/minimizing the secondary injury that occurs after SCI would be a
premiere target for treating SCI patients. Thus, researchneeds to continue in this area to discover new mechanisms to modulate inflammation after SCI.

Consent for publication
Not applicable.

Acknowledgements
Not applicable.
Authors’ contributions
All authors had full access to all the data in the study and take responsibility
for the integrity of the data and the accuracy of the data analysis. Literature
research was performed by: DJH, CMQ, ZJP. CNM, and JAF. Analysis andinterpretation of data: ASH, DJH, CMQ, and ZJP. Drafting of the manuscript
and critical revision of the article: DJH, CMQ, ZJP and ASH. All authors read andapproved the final manuscript.
Funding
This work was supported by National Institutes of Health under Award Number R56NS117935.
Availability of data and materials
The data sets used and analyzed during the current study are included within
the article and its additional files. All material used in this review are frompublicly available articles.

Declarations
Ethics approval and consent to participate
Not applicable.

Competing interests
The authors declare that they have no competing interests.
Received: 17 August 2021 Accepted: 30 November 2021

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