Expression of inflammatory cytokines following acute spinal cord injury in a rodent model

Many therapies that have been developed for acute spinal cord injury (SCI) either influence or are influenced by posttraumatic inflammation. Many such therapies have reportedly produced promising neurologic benefits in animal models of SCI, but demonstrating convincing efficacy in human clinical trials has remained elusive. This discrepancy may be related in part to differences in the inflammatory response to SCI between human patients and the widely studied rodent models. Our objectives were, therefore, to establish the time course of inflammatory cytokine release in the spinal cord of rats after a thoracic contusion, to determine whether the cytokine release was injury dependent, and to correlate these findings with those that we have recently reported for the cerebrospinal fluid (CSF) of human SCI patients. After rodent SCI, GRO (the rat equivalent of IL-8), IL-6, IL-1α, IL-1β, IL-13, MCP-1, MIP1α, RANTES, and TNFα were elevated within the spinal cord, whereas IL-12p70 was decreased. In human SCI, IL-6, IL-8, and MCP-1 were also elevated within the cerebrospinal fluid but at later times than those observed in the rodent spinal cord. IL-6, IL-8, and MCP-1 were released in an injury-dependent manner in both the rodent model of SCI and the human condition. In this regard, similar patterns of expression were observed for a number of inflammatory cytokines after SCI in rodent spinal cords and in human CSF. Such proteins may therefore have potential utility as biomarkers and surrogate outcome measures for evaluating biological response to therapeutic interventions.     

LncRNA Airsci increases the inflammatory response after spinal cord injury in rats through the nuclear factor kappa B signaling pathway

Spinal cord injury(SCI) is a serious traumatic event to the central nervous system. Studies show that long non-coding RNAs(lncRNAs) play an important role in regulating the inflammatory response in the acute stage of SCI. Here, we investigated a new lncRNA related to spinal cord injury and acute inflammation. We analyzed the expression profile of lncRNAs after SCI, and explored the role of lncRNA Airsci(acute inflammatory response in SCI) on recovery following acute SCI. The rats were divided into the control group, SCI group, and SCI + lncRNA Airsci-siRNA group. The expression of inflammatory factors, including nuclear factor kappa B [NF-κB(p65)], NF-κB inhibitor IκBα and phosphorylated IκBα(p-IκBα), and the p-IκBα/IκBα ratio were examined 1–28 days after SCI in rats by western blot assay. The differential lncRNA expression profile after SCI was assessed by RNA sequencing. The differentially expressed lncRNAs were analyzed by bioinformatics technology. The differentially expressed lncRNA Airsci, which is involved in NF-κB signaling and associated with the acute inflammatory response, was verified by quantitative real-time PCR. Interleukin(IL-1β), IL-6 and tumor necrosis factor(TNF-α) at 3 days after SCI were measured by western blot assay and quantitative real-time PCR. The histopathology of the spinal cord was evaluated by hematoxylin-eosin and Nissl staining. Motor function was assessed with the Basso, Beattie and Bresnahan Locomotor Rating Scale. Numerous differentially expressed lncRNAs were detected after SCI, including 151 that were upregulated and 186 that were downregulated in the SCI 3 d group compared with the control group. LncRNA Airsci was the most significantly expressed among the five lncRNAs involved in the NF-κB signaling pathway. LncRNA Airsci-siRNA reduced the inflammatory response by inhibiting the NF-κB signaling pathway, alleviated spinal cord tissue injury, and promoted the recovery of motor function in SCI rats. These findings show that numerous lncRNAs are differentially expressed following SCI, and that inhibiting lncRNA Airsci reduces the inflammatory response through the NF-κB signaling pathway, thereby promoting functional recovery. All experimental procedures and protocols were approved by the approved by the Animal Ethics Committee of Jining Medical University(approval No. JNMC-2020-DW-RM-003) on January 18, 2020.     

Prominent microglial activation in the early proinflammatory immune response in naturally occurring canine spinal cord injury

Better understanding of the pathogenesis of spinal cord injury (SCI) is needed for the development of new therapeutic strategies. Spinal cord injury has been investigated in various rodent models, but extrapolation to humans requires the use of a large animal model that more closely mimics human SCI. Dogs frequently develop spontaneous SCI with features that bear a striking resemblance to the human counterpart. We investigated the temporal course of the immune response during naturally occurring canine SCI and in organotypic canine spinal cord slice cultures that are devoid of peripheral immune cells. By immunohistochemistry, the inflammatory response in subacute canine SCI was largely restricted to resident immune cells as demonstrated by activation of major histocompatibility complex class II-expressing microglia/macrophages. By quantitative polymerase chain reaction, there was parallel upregulation of proinflammatory cytokine gene expression (i.e. of interleukin 6 [IL-6] and IL-8 with a trend toward upregulation of tumor necrosis factor) in acute canine SCI. Expression of neuroprotective cytokines (e.g. IL-10) remained unchanged, and transforming growth factor β upregulation was delayed. In organotypic spinal cord slices, there was similar activation of major histocompatibility complex class II-positive microglia and prolonged upregulation of inflammatory cytokines, indicating that resident rather than infiltrating cells play major roles in the postinjury immune response. Thus, canine SCI represents a bridge between rodent models and human SCI that may be relevant for clinical and preclinical treatment studies.     

Neuregulin-1: a novel regulator of glial response in spinal cord injury

<正>Spinal cord injury(SCI)results in a dysregulated microenvironment that is largely driven by the immediate and robust response of resident astrocytes and microglia(Filous and Silver,2016).Activated glial cells initiate a complex innate and adaptive immune response that regulates secondary injury mechanisms with both destructive and supportive impact on     

Attenuation of acute inflammatory response by atorvastatin after spinal cord injury in rats

Spinal cord injury (SCI) is a devastating and complex clinical condition involving proinflammatory cytokines and nitric oxide toxicity that produces a predictable pattern of progressive injury entailing neuronal loss, axonal destruction, and demyelination at the site of impact. The involvement of proinflammatory cytokines and inducible nitric oxide synthase (iNOS) in exacerbation of SCI pathology is well documented. We have reported previously the antiinflammatory properties and immunomodulatory activities of statins (3-hydroxy-3-methylglutaryl [HMG]-CoA reductase inhibitors) in the animal model of multiple sclerosis, experimental allergic encephalitis (EAE). The present study was undertaken to investigate the efficacy of atorvastatin (Lipitor; LP) treatment in attenuating SCI-induced pathology. Immunohistochemical detection and real-time PCR analysis showed increased expression of iNOS, tumor necrosis factor alpha (TNFalpha) and interleukin 1beta (IL-1beta) after SCI. In addition, neuronal apoptosis was detected 24 hr after injury followed by a profound increase in ED1-positive inflammatory infiltrates, glial fibrillary acidic protein (GFAP)-positive reactive astrocytes, and oligodendrocyte apoptosis by 1 week after SCI relative to control. LP treatment attenuated the SCI-induced iNOS, TNFalpha, and IL-1beta expression. LP also provided protection against SCI-induced tissue necrosis, neuronal and oligodendrocyte apoptosis, demyelination, and reactive gliosis. Furthermore, rats treated with LP scored much higher on the locomotor rating scale after SCI (19.13 +/- 0.53) than did untreated rats (9.04 +/- 1.22). This study therefore reports the beneficial effect of atorvastatin for the treatment of SCI-related pathology and disability.     

Tamoxifen reduces infiltration of inflammatory cells,apoptosis and inhibits IKK/NF-kB pathway after spinal cord injury in rats

In this study, neuroprotective effect of tamoxifen has been explored in spinal cord injury (SCI) in rats by examining factors influencing IKK/NF-kB pathway in SCI in rats. It has been shown in several studies that IKK/NF-kB signaling pathway plays a key role in pathophysiology of SCI. In this study, three groups of rats (n = 17 each) were selected that included, tamoxifen group (here tamoxifen was injected after SCI in rats), SCI group (here only dimethylsulfoxide was administered after inducing SCI in rats) and sham group (here only laminectomy was performed). The effect of tamoxifen (5 mg/kg) on various factors responsible for activation of IKK/NF-kB signaling pathway including NF-kB p65, phosphorylated I-kBα was studied through Western blotting as well as densitometry. The examination of expression of active caspase-3 and myeloperoxidase activity was also carried out through Western blot analysis and densitometry. A comparison of three groups of rats showed that administration of tamoxifen significantly reduced the expression of NF-kB p65 and phosphorylated I-kBα (P < 0.05) compared to control. It also attenuated the expression of active caspase-3 resulting in the reduction of apoptosis, and infiltration of leukocytes to the injury site was also greatly reduced in the group where tamoxifen was administered. Statistical analysis through SPSS 13.0 software showed a significant decrease in the expression of inflammatory factors in groups where tamoxifen was administered. We conclude that tamoxifen possesses the potential neuroprotective effects that can be explored further for future therapeutic techniques in treating spinal cord injuries.     

Dynamics of the inflammatory response after murine spinal cord injury revealed by flow cytometry

Spinal cord injury (SCI) triggers a robust inflammatory response that contributes in part to the secondary degeneration of spared tissue. Here, we use flow cytometry to quantify the inflammatory response after SCI. Besides its objective evaluation, flow cytometry allows for levels of particular markers to be documented that further aid in the identification of cellular subsets. Analyses of blood from SCI mice for CD45 (common leukocyte antigen), CD11b (complement receptor-3), Gr-1 (neutrophil/monocyte marker), and CD3 (T-cell marker) revealed a marked increase in circulating neutrophils (CD45(high):Gr-1(high)) at 12 hr compared with controls. Monocyte density in blood increased at 24 hr, and in contrast, lymphocyte numbers were significantly decreased. Mirroring the early increase in neutrophils within the blood, flow analysis of the spinal cord lesion site revealed a significant (P < 0.01) and maintained increase in blood-derived leukocytes (CD45(high):CD11b(high)) from 12 to 96 hr compared with sham-injured and naive controls. Importantly, this technique clearly distinguishes blood-derived neutrophils (CD45:Gr-1(high):F4/80(negative)) and monocyte/macrophages (CD45(high)) from resident microglia (CD45(low)) and revealed that the majority of the blood-derived infiltrate were neutrophils. Our results highlight an assumed, but previously uncharacterized, marked and transient increase in leukocyte populations in blood early after SCI followed by the orchestrated invasion of neutrophils and monocytes into the injured cord. In contrast to mobilization of neutrophils, SCI induces lymphopenia that may contribute negatively to the overall outcome after spinal cord trauma.     

Heterogeneous astroglial response in the rat spinal cord to long-term portacaval shunt: an immunohistochemical study.

Glial fibrillary acidic protein (GFAP) immunoreactivity has been used to study the astroglial response in the rat spinal cord to long-term portacaval shunt (PCS). The astroglial response in PCS rats is heterogeneous. In general, astrocytes show a loss of GFAP immunoreactivity, as well as shrinking and pyknosis in their nuclei; however, while GFAP reactivity was unchanged in the periependymal region, it was strongly increased in the dorsolateral region of the spinal cord (lateral spinal nucleus, dorsal root entry zone, and the most dorsal region of the dorsal horn). Three possibilities are postulated to explain how astrocytes, in the periependymal and dorsolateral regions, can support the effects of PCS: a) astrocytes related to glutamatergic pathways ought to possess a more efficient ammonia uptake and detoxification system, b) long-term PCS can activate nociceptive pathways (substancePergic fibers), and c) astrocytes located in periependymal and dorsolateral regions can be exposed to lower concentrations of ammonia because of its diffusion into the cerebro-spinal fluid close to these regions.     

The HMGB1 signaling pathway activates the inflammatory response in Schwann cells

Schwann cells are not only myelinating cells, but also function as immune cells and express numerous innate pattern recognition receptors, including the Toll-like receptors. Injury to peripheral nerves activates an inflammatory response in Schwann cells. However, it is unclear whether specific endogenous damage-associated molecular pattern molecules are involved in the inflammatory response following nerve injury. In the present study, we demonstrate that a key damage-associated molecular pattern molecule, high mobility group box 1(HMGB1), is upregulated following rat sciatic nerve axotomy, and we show colocalization of the protein with Schwann cells. HMGB1 alone could not enhance expression of Toll-like receptors or the receptor for advanced glycation end products(RAGE), but was able to facilitate migration of Schwann cells. When Schwann cells were treated with HMGB1 together with lipopolysaccharide, the expression levels of Toll-like receptors and RAGE, as well as inflammatory cytokines were upregulated. Our novel findings demonstrate that the HMGB1 pathway activates the inflammatory response in Schwann cells following peripheral nerve injury.     

A novel spinal pathway and other connections to the spinocerebellum in the pigeon.

Avian dorsal column nuclei do not project to the cerebellum. Injections of fluorescent tracers into the spinocerebellum of homing pigeons (Columba livia) disclosed a group of neurons located rostral to the dorsal column nuclei which receives spinal primary afferents, as confirmed by double-labeling experiments. Since this group has some similarities to the mammalian group x (location medial to the restiform body, spinal afferents, efferents to the cerebellum), this name was adopted for the pigeon. Further brainstem nuclei projecting to anterior or posterior spinocerebellum and with some relevance to transmission of spinal signals are described.     

Descriptive epidemiology of primary tumors of the spinal cord and spinal meninges in Los Angeles County, 1972-1985

This report presents data on the distribution of 462 primary tumors of the spinal cord and spinal meninges (both benign and malignant) diagnosed among residents of Los Angeles County from 1972 to 1985. Incidence rates of gliomas, meningiomas, nerve sheath tumors, and all histologic types combined are presented for specific age, sex, and ethnic groups. The highest rates are seen for meningiomas in women (age-adjusted rate 3/million/year compared to 1/million/year for the other two histologic types in women and for each of the three types in men). Proportional incidence ratios for spinal tumors are elevated among men and women born in Eastern Europe and among Jewish residents of Los Angeles County. The incidence rates appear not to relate to the social class.     

Release of cytokines by brain endothelial cells: A polarized response to lipopolysaccharide

Brain endothelial cells (BECs) comprise the blood-brain barrier (BBB) and are an active part of the neuroimmune system, responding to and transporting cytokines. BECs also have the ability to secrete neuroimmune substances, including cytokines. A unique feature of the BEC is its polarization, with its luminal (blood-facing) and abluminal (brain-facing) cell membranes differing in their lipid, receptor, and transporter compositions. This polarization could have functional consequences for neuroimmune communication. We postulated (i) that cytokine secretion from the luminal or abluminal membranes could differ under baseline or stimulated conditions and (ii) that an immune challenge from one side of the BBB could result in cytokine release from the other. We used an in vitro BBB model of mouse BECs cultured as monolayers to investigate cytokine secretion into luminal and abluminal chambers. Our major findings in these studies were: (i) the first demonstration that interleukin (IL)-1alpha, IL-10, and granulocyte-macrophage colony-stimulating factor are secreted from BECs and confirmation of the secretions of IL-6 and tumor necrosis factor-alpha, (ii) that constitutive and lipopolysaccharide (LPS)-stimulated secretion of cytokines is polarized in favor of luminal secretion, and (iii) that response to neuroimmune stimulation is also polarized as exemplified by the finding that abluminal LPS more robustly induced secretion of IL-6 than did luminal LPS. Overall, these findings support the BBB as an important source of cytokines. Furthermore, the BBB can respond to immune challenges received from one side of the neuroimmune axis by releasing cytokines into the other.     

A novel substance P pathway linking the dorsal and ventral horn in the upper lumbar segments of the rat spinal cord

We report here the presence of a novel substance P pathway existing at a segmental level in the upper lumbar region (L₁/L₂) of the rat spinal cord. Substance P-containing fibres were seen directly linking a number of discrete areas of the rat spinal cord, including dorsal and ventral regions. These fibres were found closely associated with a specific group of motoneurones, the mediolateral motor nucleus in the ventral horn. This motoneurone group appeared as a ‘focusing’ nucleus around which substance P-containing tracts appeared to congregate.An intrinsic spinal cord origin of substance P-containing neurones in these interconnecting pathways is suggested as deafferentation achieved either by section of appropriate dorsal roots, or pharmacologically by neonatal treatment with capsaicin, had no effect on the appearance of the nucleus in the ventral spinal cord or of the associated substance P-containing tracts. Likewise, mid-thoracic lesions of the spinal cord (to determine supraspinal contributions) including hemisection, dorsolateral funiculus lesions and lesions of the region around the central canal, induced no change. The significance of these interconnecting substance P-immunoreactive fibre tracts is not known. However, the intimate association of substance P-containing fibres with a specific group of motoneurons indicates that they may modulate motor function.     

Inhibition of inflammatory cytokines after early decompression may mediate recovery of neurological function in rats with spinal cord injury

A variety of inflammatory cytokines are involved in spinal cord injury and influence the recovery of neuronal function. In the present study, we established a rat model of acute spinal cord injury by cerclage. The cerclage suture was released 8 or 72 hours later, to simulate decompression surgery. Neurological function was evaluated behaviorally for 3 weeks after surgery, and tumor necrosis factor α immunoreactivity and apoptosis were quantified in the region of injury. Rats that underwent decompression surgery had significantly weaker immunoreactivity of tumor necrosis factor α and significantly fewer apoptotic cells, and showed faster improvement of locomotor function than animals in which decompression surgery was not performed. Decompression at 8 hours resulted in significantly faster recovery than that at 72 hours. These data indicate that early decompression may improve neurological function after spinal cord injury by inhibiting the expression of tumor necrosis factor α.     

Acute inflammatory responses to mechanical lesions in the CNS: differences between brain and spinal cord

Lesion-induced inflammatory responses in both brain and spinal cord have recently become a topic of active investigation. Using C57BL/6J mice, we compared the tissue reaction in these two central nervous system (CNS) compartments with mechanical lesions of similar size involving both grey and white matter. This evaluation included the quantitative assessment of neutrophils, lymphocytes and activated macrophages/microglia, as well as astrocyte activation, upregulation of vascular cell adhesion molecules (ICAM-1, VCAM-1, PECAM) and the extent of blood-brain barrier (BBB) breakdown. Time points analysed post-lesioning included 1, 2, 4 and 7 days (as well as 10 and 14 days for the BBB). We found clear evidence that the acute inflammatory response to traumatic injury is significantly greater in the spinal cord than in the cerebral cortex. The numbers of both neutrophils and macrophages recruited to the lesion site were significantly higher in the spinal cord than in the brain, and the recruitment of these cells into the surrounding parenchyma was also more widespread in the cord. The area of BBB breakdown was substantially larger in the spinal cord and vascular damage persisted for a longer period. In the brain, as in spinal cord, the area to which neutrophils were recruited correlated well with the area of BBB breakdown. It will be of interest to determine the extent to which the infiltration of inflammatory cells contributes, either directly or indirectly, to the vascular permeability and secondary tissue damage or, conversely, to local tissue repair in the brain and the spinal cord.     

Increased peripheral benzodiazepine binding sites and pentraxin 3 expression in the spinal cord during EAE: relation to inflammatory cytokines and modulation by dexamethasone and rolipram

We have studied the mRNA expression of pentraxin 3 (PTX3) and the binding of the peripheral-type benzodiazepine receptor (PBR) ligand, [3H]-PK11195, in the spinal cord of Lewis rats where EAE was actively induced. PTX3 was induced during the active phase of EAE (day 10-14), it remained high up to 30 days and disappeared only 60 days later. Similarly, PK11195 binding peaked at day 14-17 during the recovery and it disappeared by day 60. On the other hand, the levels of TNF and IL-6 in the spinal cord were elevated at the peak and at the onset of clinical signs and returned to non-detectable by day 14-17. Dexamethasone abolished all these changes, while treatment with rolipram, delayed the appearance of the disease and then decreased its severity. However the peaks of TNF, IL-6, PBR and PTX3 levels in spinal cord were only delayed, but not reduced, by rolipram treatment. In conclusion, we show two types of inflammatory changes in EAE: acute, short term changes (TNF and IL-6), that correlate with the disease; and effects such as PTX3 expression and PK11195 binding that last longer after recovery from the disease.