Neural progenitor cells but not astrocytes respond distally to thoracic spinal cord injury in rat models

Traumatic spinal cord injury （SCI） is a detrimental condition that causes loss of sensory and motor function in an individual. Many complex secondary injury cascades occur after SCI and they offer great potential for therapeutic targeting. In this study, we investigated the response of endogenous neural progenitor cells, astrocytes, and microglia to a localized thoracic SCI throughout the neuroaxis. Twenty-five adult female Sprague-Dawley rats underwent mild-contusion thoracic SCI （n = 9）, sham surgery （n = 8）, or no surgery （n = 8）. Spinal cord and brain tissues were fixed and cut at six regions of the neuroaxis. Immunohistochem- istry showed increased reactivity of neural progenitor cell marker nestin in the central canal at all levels of the spinal cord. Increased reactivity of astrocyte-specific marker glial fibrillary acidic protein was found only at the lesion epicenter. The number of activated microglia was significantly increased at the lesion site, and activated microglia extended to the lumbar enlargement. Phagocytic microglia and macrophages were significantly increased only at the lesion site. There were no changes in nestin, glial fibrillary acidic protein, microglia and macrophage response in the third ventricle of rats subjected to mild-contusion thoracic SCI compared to the sham surgery or no surgery. These findings indicate that neural progenitor cells, astrocytes and microglia respond differently to a localized SCI, presumably due to differences in inflammatory signaling. These different cellular responses may have implications in the way that neural progenitor cells can be manipulated for neuroregeneration after SCI. This needs to be further investigated.

Ependymal cell proliferation and apoptosis following acute spinal cord injury in the adult rat

BACKGROUND： Studies have reported that spinal cord injury can induce the reactive proliferation of ependymal cells and secondarily cause the apoptosis of nerve cells. However, there is no generally accepted theory on the apoptotic characteristics of ependymal cells in the injured spinal cord. OBJECTIVE： To observe the reactive proliferation and apoptosis of ependymal cells in adult rats following acute spinal cord injury. DESIGN, TIME AND SETTING： A randomized control study based on neuropathology was performed in the Third Military Medical University of Chinese PLA between 2005 and 2007. MATERIALS： Forty healthy, adult, Wistar rats were included in the present study. METHODS： Moderate spinal cord injury was established in twenty rats using Feeney＇s method, while the remaining 20 rats served as controls and were only treated with laminectomy. All rats were injected intraperitoneally with 1.25 mL of BrdU solution （10 mg BrdU/mL saline） 3 times at 4 hours intervals during the 12 hours prior to sacrifice. MAIN OUTCOME MEASURES： Ependymal cell proliferation and apoptosis in the rat spinal cord were determined by BrdU and nestin immunofluorescence double-labeling, as well as the TUNEL method, at 1, 3, 7, and 14 days after operation. RESULTS： In the moderate spinal cord injury rats, nestin expression was observed in the cytoplasm of ependymal cells. One day immediately following surgery, ependymal cells were BrdU-labeled. The number of BrdU-positive cells increased at 3 days, reached a peak at 7 days, and gradually reduced thereafter. The ependyma developed from a constitutive monolayer cells to a multi-layer cell complex. Some BrdU/Nestin double-positive ependymal cells migrated out from the ependyma. TUNEL-positive cells were also detected in the ependyma in the central region, as well as ischemic regions of the injured spinal cord. In addition, TUNEL-positive cells were visible in the ependyma. No TUNEL-positive ependymal cells were observed in the normal spinal cord. CONCLUSION： Proliferating ependymal cells induced apoptosis in the central and surrounding region following spinal cord injury.

Necrostatin-1 decreases necroptosis and inflammatory markers after intraventricular hemorrhage in mice

Necrostatin-1,an inhibitor of necroptosis,can effectively inhibit necrotic apoptosis in neurological diseases,which results in the inhibition of inflammation,endoplasmic reticulum stress,and reactive oxygen species production and substantial improvement of neurological function.However,the effects of necrostatin-1 on intraventricular hemorrhage(IVH)remain unknown.In this study,we established a mouse model of IVH by injecting autologous blood into the lateral ventricle of the brain.We also injected necrostatin-1 into the lateral ventricle one hour prior to IVH induction.We found that necrostatin-1 effectively reduced the expression levels of the necroptosis markers receptor-interacting protein kinase(RIP)1,RIP3,mixed lineage kinase domain-like protein(MLKL),phosphorylated(p)-RIP3,and p-MLKL and the levels of interleukin-1β,interleukin-6,and tumor necrosis factor-αin the surrounding areas of the lateral ventricle.However,necrostatin-1 did not reduce ependymal ciliary injury or brain water content.These findings suggest that necrostatin-1 can prevent local inflammation and microglial activation induced by IVH but does not greatly improve prognosis.

PROLIFERATION AND MIGRATION OF EPENDYMAL CELLS IN THE BRAIN OF ADULT RATS AFTER PERMANENT FOCAL CEREBRAL ISCHEMIA

Objective Ependymal cells are thought to be the primary source of neural stem cells in the adult central nervous system. The purpose of this study is to examine spatial and temporal profiles of ependymal cell proliferation and migration after focal cerebral ischemia. Methods Eighty male Sprague Dawley rats underwent permanent middle cerebral artery occlusion after injection of 10 μL of 0.2% Dil into the lateral ventricle. Rats were sacrificed and brain sections were acquired for pathological evaluation and laser confocal imaging at day 1,3,7,11,14,21 and 28 after ischemia. Results The density of Dil-labeled cells in the ischemic ipsilateral subventricular zone was significantly higher than that in the control group and these labeled cells dispersed in the ischemic ipsilateral subventricular zone and/or were located in ependyma from day 1 to 11. In the ischemic ipsilateral cortex, some Dil-labeled cells occurred in peri-infarction and infarction of parietal region at day14 and peaked at day 21 when some Dil-labeled cell nodules were found in this region. During postischemic day 14-28, a significant decrease in labeled cell density in the ischemic ipsilateral subventricular zone was coincident with a significant increase in labeled cells density in the cortex (peri-infarction and infarction). Conclusion The results indicate that ependymal cells proliferate and migrate after focal cerebral ischemia in the adult rat brain.

Cerebral furin deficiency causes hydrocephalus in mice

Furin is a pro-protein convertase that moves between the trans-Golgi network and cell surface in the secretory pathway.We have previously reported that cerebral overexpres-sion of furin promotes cognitive functions in mice.Here,by generating the brain-specific furin conditional knockout(ckO)mice,we investigated the role of furin in brain development.We found that furin deficiency caused early death and growth retardation.Magnetic resonance im-aging showed severe hydrocephalus.In the brain of furin cko mice,impaired ciliogenesis and the derangement of microtubule structures appeared along with the down-regulated expres-sion of RAB28,a ciliary vesicle protein.In line with the widespread neuronal loss,ependymal cell layers were damaged.Further proteomics analysis revealed that cell adhesion molecules including astrocyte-enriched ITGB8 and BCAR1 were altered in furin cKO mice;and astrocyte overgrowth was accompanied by the reduced expression of sox9,indicating a disrupted differ-entiation into ependymal cells.Together,whereas alteration of RAB28 expression correlated with the role of vesicle trafficking in ciliogenesis,dysfunctional astrocytes might be involved in ependymal damage contributing to hydrocephalus in furin ckO mice.The structural and mo-lecular alterations provided a clue for further studying the potential mechanisms of furin.

Endogenous neurogenesis in adult mammals after spinal cord injury

During the whole life cycle of mammals,new neurons are constantly regenerated in the subgranular zone of the dentate gyrus and in the subventricular zone of the lateral ventricles.Thanks to emerging methodologies,great progress has been made in the characterization of spinal cord endogenous neural stem cells(ependymal cells) and identification of their role in adult spinal cord development.As recently evidenced,both the intrinsic and extrinsic molecular mechanisms of ependymal cells control the sequential steps of the adult spinal cord neurogenesis.This review introduces the concept of adult endogenous neurogenesis,the reaction of ependymal cells after adult spinal cord injury(SCI),the heterogeneity and markers of ependymal cells,the factors that regulate ependymal cells,and the niches that impact the activation or differentiation of ependymal cells.

Lineage tracing reveals the origin of Nestin-positive cells are heterogeneous and rarely from ependymal cells after spinal cord injury

Nestin is expressed extensively in neural stem/progenitor cells during neural development, but its expression is mainly restricted to the ependymal cells in the adult spinal cord. After spinal cord injury(SCI), Nestin expression is reactivated and Nestinpositive(Nestin;) cells aggregate at the injury site. However, the derivation of Nestin;cells is not clearly defined. Here, we found that Nestin expression was substantially increased in the lesion edge and lesion core after SCI. Using a tamoxifen inducible CreER(T2)-loxP system, we verified that ependymal cells contribute few Nestin;cells either to the lesion core or the lesion edge after SCI. In the lesion edge, GFAP+astrocytes were the main cell type that expressed Nestin;they then formed an astrocyte scar.In the lesion core, Nestin;cells expressed αSMA or Desmin, indicating that they might be derived from pericytes. Our results reveal that Nestin;cells in the lesion core and edge came from various cell types and rarely from ependymal cells after complete transected SCI, which may provide new insights into SCI repair.

Single-cell RNA sequencing reveals Nestin+active neural stem cells outside the central canal after spinal cord injury

Neural stem cells(NSCs)in the spinal cord hold great potential for repair after spinal cord injury(SCI).The ependyma in the central canal(CC)region has been considered as the NSCs source in the spinal cord.However,the ependyma function as NSCs after SCI is still under debate.We used Nestin as a marker to isolate potential NSCs and their immediate progeny,and characterized the cells before and after SCI by single-cell RNA-sequencing(scRNA-seq).We identified two subgroups of NSCs:the subgroup located within the CC cannot prime to active NSCs after SCI,while the subgroup located outside the CC were activated and exhibited the active NSCs properties after SCI.We demonstrated the comprehensive dynamic transcriptome of NSCs from quiescent to active NSCs after SCI.This study reveals that Nestin+cells outside CC were NSCs that activated upon SCI and may thus serve as endogenous NSCs for regenerative treatment of SCI in the future.

Microtubule-bundling protein Spef1 enables mammalian ciliary central apparatus formation

Cilia are cellular protrusions containing nine microtubule(MT)doublets and function to propel cell movement or extracellular liquid flow through beating or sense environmental stimuli through signal transductions.Cilia require the central pair(CP)apparatus,consisting of two CP MTs covered with projections of CP proteins,for planar strokes.How the CP MTs of such‘9+2’cilia are constructed,however,remains unknown.Here we identify Spef1,an evolutionarily conserved microtubule-bundling protein,as a core CP MT regulator in mammalian cilia.Spef1 was selectively expressed in mammalian cells with 9+2 cilia and specifically localized along the CP.Its depletion in multiciliated mouse ependymal cells by RNAi completely abolished the CP MTs and markedly attenuated ciliary localizations of CP proteins such as Hydin and Spag6,resulting in rotational beat of the ependymal cilia.Spef1,which binds to MTs through its N-terminal calponin.homologous domain,formed homodimers through its C-terminal coiled coil region to bundle and stabilize MTs.Disruption of either the MT-binding or the dimerization activity abolished the ability of exogenous Spef1 to restore the structure and functions of the CP apparatus.We propose that Spefl bundles and stabilizes central MTs to enable the assembly and functions of the CP apparatus.

β-Catenin Deletion in Regional Neural Progenitors Leads to Congenital Hydrocephalus in Mice

Congenital hydrocephalus is a major neurological disorder with high rates of morbidity and mortality;however,the underlying cellular and molecular mechanisms remain largely unknown.Reproducible animal models mirroring both embryonic and postnatal hydrocephalus are also limited.Here,we describe a new mouse model of congenital hydrocephalus through knockout ofβ-catenin in Nkx2.1-expressing regional neural progenitors.Progressive ventriculomegaly and an enlarged brain were consistently observed in knockout mice from embryonic day 12.5 through to adulthood.Transcriptome profiling revealed severe dysfunctions in progenitor maintenance in the ventricular zone and therefore in cilium biogenesis afterβ-catenin knockout.Histological analyses also revealed an aberrant neuronal layout in both the ventral and dorsal telencephalon in hydrocephalic mice at both embryonic and postnatal stages.Thus,knockout ofβ-catenin in regional neural progenitors leads to congenital hydrocephalus and provides a reproducible animal model for studying pathological changes and developing therapeutic interventions for this devastating disease.