Kentaro Hayakawa

Phosphorylated neurofilament subunit NF-H as a biomarker for evaluating the severity of spinal cord injury patients, a pilot study

Study design:A pilot cross-sectional study of patients with acute cervical spinal cord injury (SCI).Objectives:The precise evaluation of the severity of SCI is important for developing novel therapies. Although several biomarkers in cerebrospinal fluid have been tested, few analyses of blood samples have been reported. A novel biomarker for axonal injury, phosphorylated form of the high-molecular-weight neurofilament subunit NF-H (pNF-H), has been reported to be elevated in blood from rodent SCI model. The aim of this study is to investigate whether pNF-H values in blood can serve as a biomarker to evaluate the severity of patients with SCI.Setting:Tokyo Metropolitan Bokutoh Hospital and National Rehabilitation Center, Japan.Methods:This study enrolled 14 patients with acute cervical SCI. Sequential plasma samples were obtained from 6 h to 21 days after injury. Patients were classified according to American Spinal Injury Association impairment scale (AIS) at the end of the follow-up (average, 229.1 days). Plasma pNF-H values were compared between different AIS grades.Results:In patients with complete SCI, pNF-H became detectable at 12 h after injury and remained elevated at 21 days after injury. There was a statistically significant difference between AIS A (complete paralysis) patients and AIS C (incomplete paralysis) patients.Conclusions:Plasma pNF-H was elevated in accordance with the severity of SCI and reflected a greater magnitude of axonal damage. Therefore, pNF-H is a potential biomarker to independently distinguish AIS A patients (complete SCI) from AIS C–E patients (incomplete SCI). However, further studies are required to evaluate its utility in predicting prognosis of patients in the incomplete category.

In vivo messenger RNA introduction into the central nervous system using polyplex nanomicelle.

Messenger RNA (mRNA) introduction is a promising approach to produce therapeutic proteins and peptides without any risk of insertion mutagenesis into the host genome. However, it is difficult to introduce mRNA in vivo mainly because of the instability of mRNA under physiological conditions and its strong immunogenicity through the recognition by Toll-like receptors (TLRs). We used a novel carrier based on self-assembly of a polyethylene glycol (PEG)-polyamino acid block copolymer, polyplex nanomicelle, to administer mRNA into the central nervous system (CNS). The nanomicelle with 50 nm in diameter has a core-shell structure with mRNA-containing inner core surrounded by PEG layer, providing the high stability and stealth property to the nanomicelle. The functional polyamino acids possessing the capacity of pH-responsive membrane destabilization allows smooth endosomal escape of the nanomicelle into the cytoplasm. After introduction into CNS, the nanomicelle successfully provided the sustained protein expression in the cerebrospinal fluid for almost a week. Immune responses after mRNA administration into CNS were effectively suppressed by the use of the nanomicelle compared with naked mRNA introduction. In vitro analyses using specific TLR-expressing HEK293 cells confirmed that the nanomicelle inclusion prevented mRNA from the recognition by TLRs. Thus, the polyplex nanomicelle is a promising system that simultaneously resolved the two major problems of in vivo mRNA introduction, the instability and immunogenicity, opening the door to various new therapeutic strategies using mRNA.

Lipopolysaccharide preconditioning facilitates M2 activation of resident microglia after spinal cord injury

The inflammatory response following spinal cord injury (SCI) has both harmful and beneficial effects; however, it can be modulated for therapeutic benefit. Endotoxin/lipopolysaccharide (LPS) preconditioning, a well‐established method for modifying the immune reaction, has been shown to attenuate damage induced by stroke and brain trauma in rodent models. Although such effects likely are conveyed by tissue‐repairing functions of the inflammatory response, the mechanisms that control the effects have not yet been elucidated. The present study preconditioned C57BL6/J mice with 0.05 mg/kg of LPS 48 hr before inducing contusion SCI to investigate the effect of LPS preconditioning on the activation of macrophages/microglia. We found that LPS preconditioning promotes the polarization of M1/M2 macrophages/microglia toward an M2 phenotype in the injured spinal cord on quantitative real‐time polymerase chain reaction, enzyme‐linked immunosorbent assay, and immunohistochemical analyses. Flow cytometric analyses reveal that LPS preconditioning facilitates M2 activation in resident microglia but not in infiltrating macrophages. Augmented M2 activation was accompanied by vascularization around the injured lesion, resulting in improvement in both tissue reorganization and functional recovery. Furthermore, we found that M2 activation induced by LPS preconditioning is regulated by interleukin‐10 gene expression, which was preceded by the transcriptional activation of interferon regulatory factor (IRF)−3, as demonstrated by Western blotting and an IRF‐3 binding assay. Altogether, our findings demonstrate that LPS preconditioning has a therapeutic effect on SCI through the modulation of M1/M2 polarization of resident microglia. The present study suggests that controlling M1/M2 polarization through endotoxin signal transduction could become a promising therapeutic strategy for various central nervous system diseases.

Hes1 functions downstream of growth factors to maintain oligodendrocyte lineage cells in the early progenitor stage

Expansion of the progenitor pool of oligodendrocytes (OLs) is a critical process for obtaining appropriate amounts of mature myelin-forming OLs in the developing and regenerating central nervous system. In vitro, fibroblast growth factor-2 (FGF2), together with platelet-derived growth factor (PDGF), is required to expand oligodendrocyte progenitor cells (OLPs) in an unlimited manner, maintaining them in the early progenitor stage. However, the intracellular mechanisms that prevent OLP maturation remain elusive. In order to investigate these mechanisms, we established a mouse OLP primary culture, which enabled us to undertake biochemical analyses. We found that the suppressive effects on maturation of early OLP to the late O4(+) progenitor by PDGF+FGF2 treatment was abrogated by Mek inhibitor, while transfecting cells with a constitutively active Mek1 construct prevented OLP maturation, suggesting that the Mek-Erk pathway is implicated in the effects of the growth factor treatment. The activation of Mek-Erk pathway promoted proliferation of OLP suggesting that cell cycle progression has suppressive effects to the maturation of OLP. Furthermore, molecular screening using DNA microarrays revealed that Hes1, a negative regulator of bHLH transcription factors, is one of the downstream molecules induced by PDGF+FGF2 treatment. We confirmed that forced activation of Mek-Erk pathway is sufficient to induce Hes1 expression and that Hes1, in turn, exerts suppressive effects on the maturation of OL lineage by itself. Our observations thus indicate that Mek-Erk pathway plays pivotal role in preventing early OLP maturation to late OLPs and the effect is mediated by cell cycle progression as well as Hes1 induction.

Treatment of spinal cord injury by an advanced cell transplantation technology using brain-derived neurotrophic factor-transfected mesenchymal stem cell spheroids

Curing spinal cord injury (SCI) is challenging because of the onset of multiple and irreversible pathological responses to such injury. To suppress the responses, we employed an advanced cell transplantation technology integrating three-dimensional spheroid cell transplantation with non-viral gene transfection using biodegradable polycations. Brain-derived neurotrophic factor (BDNF)-transfected mesenchymal stem cell (MSC) spheroids were transplanted at thoraces level (Th9) to SCI region in mice. BDNF-transfected MSC spheroid transplantation led to a significantly enhanced recovery of hindlimb motor function in acute phase of SCI with myelinated axons preserved at the SCI region, while use of either technology in isolation, BDNF transfection or spheroid culture, exerted only a limited therapeutic effect, demonstrating the importance of integrated approaches. Secretion of endogenous therapeutic proteins, such as anti-inflammatory factors, was greater in MSC spheroids than in monolayer culture MSCs, and these factors appeared to act synergistically alongside BDNF secretion in SCI treatment. This study forms a basis for cell therapy regulating complex pathophysiologic processes.

Phosphorylated neurofilament subunit levels in the serum of cervical compressive myelopathy patients

We investigated the serum levels of the phosphorylated form of the high molecular weight neurofilament subunit (pNF-H) in patients with cervical compressive myelopathy. pNF-H is becoming increasingly recognized as a biomarker for axonal injury, however, it remains unclear whether serum pNF-H is elevated in chronic spinal cord compression. We examined 26 patients who underwent surgery for cervical compressive myelopathy. Peripheral blood samples were obtained both preoperatively and 1 week after surgery to evaluate the serum pNF-H levels using an enzyme-linked immunosorbent assay. A history of recent aggravation of myelopathy was also investigated. Of the 26 myelopathy patients, the preoperative serum pNF-H level was negative in 20 patients and moderately elevated in six. Patients who were positive for pNF-H were more likely to have had a recent aggravation of myelopathy compared with the pNF-H negative patients (83 versus 25%; p=0.02). All patients who were positive for pNF-H before surgery remained positive after surgery. Two patients who became positive after surgery demonstrated a neurologic deterioration associated with the surgery. In conclusion, the serum pNF-H level was negative in the majority of patients with cervical compressive myelopathy. Our results suggest that an elevated serum level of pNF-H is associated with an acute worsening of myelopathy and that a positive conversion of pNF-H after surgery is a marker of perioperative neural damage.

Elevated levels of phosphorylated neurofilament heavy subunit in the cerebrospinal fluid of patients with lumbar spinal stenosis: preliminary findings.

BACKGROUND CONTEXT The phosphorylated neurofilament heavy subunit (pNfH) is an axon fiber structural protein that is released into the cerebrospinal fluid (CSF) after nerve damage. Although the previous studies have reported elevated CSF levels of pNfH in various neurological diseases, including amyotrophic lateral sclerosis, these levels have not been examined in patients with spinal stenosis. PURPOSE The purpose of this study was to investigate the CSF levels of pNfH in patients with lumbar spinal stenosis (LSS) and to examine the relationship between CSF levels of pNfH and the severity of LSS. STUDY DESIGN This is a prospective observational study. PATIENT SAMPLE We included consecutive patients with LSS who were undergoing myelography for preoperative evaluation. The CSF samples from patients with idiopathic scoliosis were used as the controls. OUTCOME MEASURES Physiological measures: CSF levels of pNfH were measured using an enzyme-linked immunosorbent assay. The Zurich Claudication Questionnaire (ZCQ) and the Numerical Rating Scale (NRS) for sciatic pain were used to assess the clinical severity of LSS, and patients were grouped into tertiles according to their symptom severity and pain grading. Axial magnetic resonance imaging was used to evaluate the morphological severity of LSS, and patients were classified into three groups based on their morphological grading (using the CSF/rootlet ratio). METHODS Analysis of variance was used to examine the relationship between the CSF levels of pNfH and the severity of LSS. RESULTS Thirty-three patients with LSS were included (13 men and 20 women and mean age 73.2 [range 58-88] years). Most patients (n=32) were positive for pNfH in their CSF (mean 1,344 [149-9,250] pg/mL), whereas all control subjects were negative for pNfH in their CSF. Regarding the association with clinical severity, patients in the third tertiles of ZCQ and NRS tended to have higher levels of pNfH compared with the other groups. There was no association between the CSF level of pNfH and the morphological severity of LSS. CONCLUSIONS This study detected elevated pNfH levels in the CSF of patients with LSS. Patients with severe clinical symptoms were more likely to exhibit high levels of pNfH. Our results indicate the potential usefulness of pNfH as a biomarker for compressive spinal disorders.

Mental State Can Influence the Degree of Postoperative Axial Neck Pain Following Cervical Laminoplasty

Study design: A retrospective cohort study. Objective: To investigate factors influencing the incidence of moderate to severe postoperative axial neck pain following cervical laminoplasty. Methods: We reviewed 125 patients with cervical myelopathy who underwent double-door laminoplasty. The primary outcomes were the Numerical Rating Scale score (NRS score, 0-10) for neck pain, the Short Form 36 (SF-36) Health Survey score (Physical and Mental Component Summary scores [PCS and MCS, respectively]), and satisfaction. Imaging parameters on plain radiographs and magnetic resonance imaging were also evaluated. Patients with moderate to severe postoperative neck pain (NRS ≥ 5) were compared with those with no or mild neck pain (NRS ≤ 4). Results: One hundred and three patients (82%) with complete data were eligible for inclusion. There were 67 men and 36 women, with a mean age of 65 years (32-89 years). Twenty-five patients (23%) had moderate to severe postoperative axial pain (NRS ≥ 5) and were compared with the other 78 patients (NRS ≤ 4), which revealed several predictive factors, including female sex, the presence of preoperative neck pain, low postoperative PCS, low preoperative and postoperative MCS, and satisfaction with the treatment. Multivariable logistic regression analysis revealed that the postoperative MCS (P = .002) was a risk factor for postoperative neck pain, although the preoperative MCS did not reach statistical significance (P = .06). Conclusions: Patients with a low mental state, possibly before surgery, are at a high risk for postoperative axial neck pain. None of the imaging parameters were statistically different.

Applications and Limitations of pNF-H, a Novel Biomarker for Spinal Cord Injury: Strategy for the Evaluation of Therapeutic Outcomes

The development of proper outcome measurements is as important as developing novel therapies. One of the characteristic issues in spinal cord injury (SCI) is the difficulty in severity evaluation at an early time point. In general, the assessment of tissue damage can be achieved by measurement of tissue-specific proteins released into peripheral blood. Phosphorylated neurofilament NF-H (pNF-H) is a cytoskeletal structural protein in neurons. In addition to its physiological function, pNF-H has been reported as a biomarker for axonal damage because of its stability within blood. In both rodent SCI models and human SCI patients, higher pNF-H values in blood correlate with more severe neural tissue damage. It would be possible to determine the severity of SCI by measuring blood pNF-H at either 24 or 96 h after injury. This better prognostic evaluation may reduce the number of required patients in clinical studies and facilitate the development of novel therapies for SCI.