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Exogenously applied mature naive B220+ /CD19+ /IgM+ /IgD+ B cells are strongly protective in the context of tissue injury. However, the mechanisms by which B cells detect tissue injury and aid repair remain elusive. Here, we show in distinct models of skin and brain injury that MyD88-dependent toll-like receptor (TLR) signaling through TLR2/6 and TLR4 is essential for the protective benefit of B cells in vivo, while B cell-specific deletion of MyD88 abrogated this effect. The B cell response to injury was multi-modal with simultaneous production of both regulatory cytokines, such as IL-10, IL-35, and transforming growth factor beta (TGFβ), and inflammatory cytokines, such as tumor necrosis factor alpha (TNFα), IL-6, and interferon gamma. Cytometry analysis showed that this response was time and environment-dependent in vivo, with 20%-30% of applied B cells adopting an immune modulatory phenotype with high co-expression of anti- and pro-inflammatory cytokines after 18-48 h at the injury site. B cell treatment reduced the expression of TNFα and increased IL-10 and TGFβ in infiltrating immune cells and fibroblasts at the injury site. Proteomic analysis further showed that B cells have a complex time-dependent homeostatic effect on the injured microenvironment, reducing the expression of inflammation-associated proteins, and increasing proteins associated with proliferation, tissue remodeling, and protection from oxidative stress. These findings chart and validate a first mechanistic understanding of the effects of B cells as an immunomodulatory cell therapy in the context of tissue injury.

To understand the diabetic wound healing microenvironment, we profiled 174,962 single cells from foot, forearm, and PBMCs using single-cell RNA sequencing (scRNASeq) approach. Our analysis shows enrichment of a unique population of fibroblasts overexpressing MMP1, MMP3, MMP11, HIF1A, CHI3L1, and TNFAIP6 genes and M1 macrophage polarization in the DFU patients with healing wounds. Further, scRNASeq of spatially separated samples from same patient and spatial transcriptomics (ST) revealed preferential localization of these healing associated fibroblasts toward deep wound/ulcer bed as compared to wound edge or non-wounded skin. ST also validated our findings of higher enrichment of M1 macrophages in healers and M2 macrophages in non-healers. Our analysis provides deep insights into the wound healing microenvironment, identifying cell types that could be critical in promoting DFU healing, and may inform novel therapeutic approaches for DFU treatment.

Cathepsin K deficiency in male mice (Ctsk−/−) results in decreased numbers of hippocampal astrocytes and altered neuronal patterning as well as learning and memory deficits. Additionally, cathepsin K carries essential roles in the thyroid gland where it contributes to the liberation of thyroid hormones (TH). Because TH are essential for brain development, in particular for the cerebellum, we investigated whether cathepsin K's function in the thyroid is directly linked to the brain phenotype of Ctsk−/− mice. Serum levels of thyroid stimulating hormone, brain concentrations of free TH, and deiodinase 2 (Dio2) activity in brain parenchyma as well as cerebellar development were comparable in Ctsk−/− and WT animals, suggesting regular thyroid states and TH metabolism. Despite unaltered transcript levels, protein expression of two TH transporters was enhanced in specific brain regions in Ctsk−/− mice, suggesting altered TH supply to these regions. Thyrotropin releasing hormone (Trh) mRNA levels were enhanced threefold in the hippocampus of Ctsk−/− mice. In the striatum of Ctsk−/− mice the mRNA for Dio2 and hairless were approximately 1.3-fold enhanced, while mRNA levels for monocarboxylate transporter 8 and Trh were reduced to 60% and 40%, respectively, pointing to altered striatal physiology. We conclude that the role of cathepsin K in the thyroid gland is not directly associated with its function in the central nervous system (CNS) of mice. Future studies will show whether the brain region-specific alterations in Trh mRNA may eventually result in altered neuroprotection that could explain the neurobehavioral defects of Ctsk−/− mice.

Chronic wounds affect 12-15% of patients with diabetes and are associated with a drastic decrease in their quality of life. Here, we demonstrate that purified mature naive B220+ /CD19+ /IgM+ /IgD+ B cells improve healing of acute and diabetic murine wounds after a single topical application. B cell treatment significantly accelerated acute wound closure by 2-3 days in wild-type mice and 5-6 days in obese diabetic mice. The treatment led to full closure in 43% of chronic diabetic wounds, as compared to only 5% in saline-treated controls. Applying equivalent numbers of T cells or disrupted B cells failed to reproduce these effects, indicating that live B cells mediated pro-healing responses. Topically applied B cell treatment was associated with significantly reduced scar size, increased collagen deposition and maturation, enhanced angiogenesis, and increased nerve growth into and under the healing wound. β-III tubulin+ nerve endings in scars of wounds treated acutely with B cells showed increased relative expression of growth-associated protein 43. The improved healing associated with B cell treatment was supported by significantly increased fibroblast proliferation and decreased apoptosis in the wound bed and edges, altered kinetics of neutrophil infiltration, as well as an increase in TGF-β and a significant reduction in MMP2 expression in wound granulation tissue. Our findings indicate that the timeline and efficacy of wound healing can be experimentally manipulated through the direct application of mature, naive B cells, which effectively modify the balance of mature immune cell populations within the wound microenvironment and accelerate the healing process.

Among the cellular processes that follow injury to the central nervous system, glial scar formation is thought to be one of the major factors that prevent regeneration. In regeneration-competent organisms, glial scar formation has been a matter of controversy. We addressed this issue by examining the glial population after spinal cord injury in a model of regeneration competency, the knifefish Apteronotus leptorhynchus. Analysis of spinal cord sections immunostained against the glial markers glial fibrillary acidic protein, vimentin, or chondroitin sulfate proteoglycan failed to produce any evidence for the formation of a glial scar in the area of the lesion at post-injury survival times ranging from 5 to 185 days. This result was independent of the lesion paradigm applied-amputation of the caudal part of the spinal cord or hemisection lesioning-and similar after examination of transverse and longitudinal sections. We hypothesize that the well-developed network of radial glia in both the intact and the injured spinal cord provides a support system for regeneration of tissue lost to injury. This glial network is likely also involved in the generation of new cells, as indicated by the large subset of glial fibrillary acidic protein-labeled glia that express the stem cell marker Sox2.

The treatment of skin with a low-power continuous-wave (CW) near-infrared (NIR) laser prior to vaccination is an emerging strategy to augment the immune response to intradermal vaccine, potentially substituting for chemical adjuvant, which has been linked to adverse effects of vaccines. This approach proved to be low cost, simple, small, and readily translatable compared with the previously explored pulsed-wave medical lasers. However, little is known on the mode of laser–tissue interaction eliciting the adjuvant effect. In this study, we sought to identify the pathways leading to the immunological events by examining the alteration of responses resulting from genetic ablation of innate subsets including mast cells and specific dendritic cell populations in an established model of intradermal vaccination and analyzing functional changes of skin microcirculation upon the CW NIR laser treatment in mice. We found that a CW NIR laser transiently stimulates mast cells via generation of reactive oxygen species, establishes an immunostimulatory milieu in the exposed tissue, and provides migration cues for dermal CD103+ dendritic cells without inducing prolonged inflammation, ultimately augmenting the adaptive immune response. These results indicate that use of an NIR laser with distinct wavelength and power is a safe and effective tool to reproducibly modulate innate programs in skin. These mechanistic findings would accelerate the clinical translation of this technology and warrant further explorations into the broader application of NIR lasers to the treatment of immune-related skin diseases.

The knifefish Apteronotus leptorhynchus exhibits indeterminate growth throughout adulthood. This phenomenon extends to the spinal cord, presumably through the continuous addition of new neurons and glial cells. However, little is known about the developmental dynamics of cells added during adult growth. The present work characterizes the structural and functional development of the adult spinal cord in this model organism through a comprehensive quantitative analysis of the spatial and temporal dynamics of new cells at various developmental stages. This analysis, based on a novel statistical mapping approach, revealed within the adult spinal cord a wide distribution of both mitotically active and quiescent Sox2-expressing stem/progenitor cells (SPCs). While such cells are particularly concentrated within the ependymal layer near the central canal, the majority of them reside in the parenchyma, resembling the distribution of SPCs observed in the mammalian spinal cord. The active SPCs in the adult knifefish spinal cord give rise to transit amplifying progenitor cells that undergo a few additional mitotic divisions before developing into Hu C/D+ neurons and S100+ glial cells. There is no evidence of long-distance migration of the newborn cells. The persistence of cell proliferation and differentiation, combined with low levels of apoptosis, leads to a continuous addition of cells to the existing tissue. Newly generated neurons have functional and behavioral relevance, as indicated by the integration of axons of new electromotor neurons into the electric organ of these weakly electric fish. This results in a gradual increase in the amplitude of the electric organ discharge during adult development. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1269-1307, 2017.

Adult neurogenesis has been described in dozens of brain regions in teleost fish, with the largest number of new neurons being generated in the cerebellum. Here, we characterized the cerebellar neural stem/progenitor cells (NSPCs) in the brown ghost knifefish (Apteronotus leptorhynchus), an established model system of adult neurogenesis. The majority of the new cerebellar cells arise from neurogenic niches located medially, at the interface of the dorsal/ventral molecular layers and the granular layer. NSPCs within these niches give rise to transit-amplifying progenitors which populate the molecular layer, where they continue to proliferate during their migration toward target areas in the granular layer. At any given time, the majority of proliferating cells are located in the molecular layer. Immunohistochemical staining revealed that the stem cell markers Sox2, Meis1/2/3, Islet1, and, to a lesser extent, Pax6, are widely expressed in all regions of the adult cerebellum. A large subpopulation of these NSPCs coexpress S100, GFAP, and/or vimentin, indicating astrocytic identity. This is further supported by the specific effect of the gliotoxin l-methionine sulfoximine, which leads to a targeted decrease in the number of GFAP+ cells that coexpress Sox2 or the proliferation marker PCNA. Pulse-chase analysis of the label size associated with new cells after administration of 5-bromo-2′-deoxyuridine demonstrated that, on average, two additional cell divisions occur after completion of the initial mitotic cycle. Overall numbers of NSPCs in the cerebellum niches increase consistently over time, presumably in parallel with the continuous growth of the brain. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 39–65, 2015

Neural stem/progenitor cells in the neurogenic niches of the adult brain are widely assumed to give rise predominantly to neurons, rather than glia. Here, we performed a quantitative analysis of the resident neural progenitors and their progeny in the adult pacemaker nucleus (Pn) of the weakly electric fish Apteronotus leptorhynchus. Approximately 15% of all cells in this brainstem nucleus are radial glia-like neural stem/progenitor cells. They are distributed uniformly within the tissue and are characterized by the expression of Sox2 and Meis 1/2/3. Approximately 2-3% of them are mitotically active, as indicated by expression of proliferating cell nuclear antigen. Labeling of proliferating cells with a single pulse of BrdU, followed by chases of up to 100 days, revealed that new cells are generated uniformly throughout the nucleus and do not undergo substantial migration. New cells differentiate into S100+ astrocytes and Hu C/D+ small interneurons at a ratio of 4:1, reflecting the proportions of the total glia and neurons in this brain region. The continuous addition of new cells leads to a diffuse growth of the Pn, which doubles in volume and total cell number over the first 2 years following sexual maturation of the fish. However, the number of pacemaker and relay cells, which constitute the oscillatory neural network, remains constant throughout adult life. We hypothesize that the dominance of gliogenesis is an adaptation to the high-frequency firing of the oscillatory neurons in this nucleus.

In contrast to mammals, in teleost fish radial glia persist beyond early development. This persistence parallels the enormous potential of teleosts to continuously generate a large number of new neurons in dozens of specific proliferation zones in the adult brain. In the present study, we characterized in the teleost fish Apteronotus leptorhynchus the immunological properties of radial glia in the corpus cerebelli—a cerebellar subdivision with particularly high proliferative activity—and examined their possible function in the guidance of migrating young neurons. Radial glia stained immunopositive for glial fibrillary acidic protein (GFAP) and vimentin, and in most cases the two intermediate filament proteins co-localized. GFAP immunolabeling combined with immunohistochemistry against the mitotic marker 5-bromo-2'-deoxyuridine (BrdU) revealed an abundance of elongated BrdU-labeled nuclei closely apposed to, or localized within, GFAP-immunoreactive radial glia. The association of BrdU-labeled nuclei and GFAP-immunoreactive radial glial fibers was particularly pronounced 2 days after BrdU administration, when the migratory activity of the young cells is highest. When the new cells reach the granular layer, they start expressing the neuronal marker protein Hu C/D, but continue their close association with radial glial fibers. These results suggest the role of radial glia in the guidance of migrating adult-born neurons in the teleostean cerebellum. This function appears to be mediated both by somal translocation and by a glial-guided mode of locomotion.

Teleost fish are distinguished by their ability to constitutively generate new neurons in the adult central nervous system ('adult neurogenesis'), and to regenerate whole neurons after injury ('neuronal regeneration'). In the brain, new neurons are produced in large numbers in several dozens of proliferation zones. In the spinal cord, proliferating cells are present in the ependymal layer and throughout the parenchyma. In the retina, new cells arise from the ciliary marginal zone and from Muller glia. Experimental evidence has suggested that both radial glia and non-glial cells can function as adult stem cells. The proliferative activity of these cells can be regulated by molecular factors, such as fibroblast growth factor and Notch, as well as by social and behavioral experience. The young cells may either reside near the respective proliferation zone, or migrate to specific target areas. Approximately half of the newly generated cells persist for the rest of the fish's life, and many of them differentiate into neurons. After injury, a massive surge of apoptotic cell death occurs at the lesion site within a few hours. Apoptosis is followed by a marked increase in cell proliferation and neurogenesis, leading to repair of the tissue. The structural regeneration is paralleled by partial or complete recovery of function. Recent investigations have led to the identification of several dozens of molecular factors that are potentially involved in the process of regeneration.

Temperature manipulation has been shown to significantly affect recovery after spinal cord injury in various mammalian model systems. Little has been known thus far about the impact of temperature on structural and functional recovery after central nervous system lesions in regeneration-competent, poikilotherm organisms. In the present study, we addressed this aspect using an established model of adult spinal cord regeneration, the weakly electric teleost fish Apteronotus leptorhynchus. We observed an overall beneficial effect of increased temperature on both structural and behavioral recovery after amputation of the caudal spinal cord. Fish kept at 30 degrees C recovered the amplitude of the electric organ discharge at more than twice the rate observed in fish kept at 22 degrees C, within the first 20 days post-injury. This improved recovery was supported by increased cell proliferation and decreased apoptosis levels in fish kept at 30 degrees C. The high temperature appeared to have a direct inhibitory effect on apoptosis and to lead to a compression of the duration of the wave of post-lesion apoptosis. The latter effect was presumably induced through the acceleration of the metabolic rate, a phenomenon also supported by the observation that re-growth of the tail was significantly increased in fish kept at 30 degrees C.

This study describes a method for the determination of relative age in a tropical teleost, the brown ghost knifefish Apteronotus leptorhynchus. This method is based on identification of the maximum number of scale circuli, which is thought to be associated with the oldest scales, and thus to be the most indicative of the age of a given fish. Relative age can be inferred by relating differences in maximum circulus counts to the average rate of circulus addition, which was estimated at 34 circuli per year in adult fish through oxytetracycline marking. This method shows high inter-investigator reliability and has a limited effect on fish because of the low number of scales required in order to determine the maximum number of circuli with a sufficiently high confidence level. Analysis of the frequency distribution of the circulus counts revealed periodic patterns that are similar among fish, presumably reflecting the environmental life history of the individuals. Regression analysis and comparison of addition rates showed that scale circulus counts and otolith ring counts are equivalent approaches for age estimation, but scale analysis is superior because of its limited invasiveness and the lower demand in terms of technical skills and expensive instrumentation.

Although the generation of new neurons in the adult nervous system ('adult neurogenesis') has been studied intensively in recent years, little is known about this phenomenon in non-mammalian vertebrates. Here, we examined the generation, migration, and differentiation of new neurons and glial cells in the Mozambique tilapia (Oreochromis mossambicus), a representative of one of the largest vertebrate taxonomic orders, the perciform fish. The vast majority of new cells in the brain are born in specific proliferation zones of the olfactory bulb; the dorsal and ventral telencephalon; the periventricular nucleus of the posterior tuberculum, optic tectum, and nucleus recessi lateralis of the diencephalon; and the valvula cerebelli, corpus cerebelli, and lobus caudalis of the cerebellum. As shown in the olfactory bulb and the lateral part of the valvula cerebelli, some of the young cells migrate from their site of origin to specific target areas. Labeling of mitotic cells with the thymidine analog 5-bromo-2'-deoxyuridine, combined with immunostaining against the neuron-specific marker protein Hu or against the astroglial marker glial fibrillary acidic protein demonstrated differentiation of the adult-born cells into both neurons and glia. Taken together, the present investigation supports the hypothesis that adult neurogenesis is an evolutionarily conserved vertebrate trait. info:eu-repo/semantics/publishedVersion

Traumatic brain injury and spinal cord injury are devastating conditions that may result in death or long-term disability. A promising strategy for the development of effective cell replacement therapies involves the study of regeneration-competent organisms. Among this group, teleost fish are distinguished by their excellent potential to regenerate nervous tissue and to regain function after injury to the central nervous system. In this chapter, we summarize our current understanding of the cellular processes that mediate this regenerative potential, and we show that several of these processes are shared with the normal development of the intact central nervous system; we describe how the spontaneous self-repair of the teleostean central nervous system leads to functional recovery, at physiological and behavioral levels; we discuss the possible function of molecular factors associated with the degenerative and regenerative processes after injury; and, finally, we speculate on evolutionary aspects of adult neurogenesis and neuronal regeneration, and on how a better understanding of these aspects could catalyze the development of therapeutic strategies to overcome the regenerative limits of the mammalian CNS.

Spinal cord injuries in mammals, including humans, have devastating long-term consequences. Despite substantial research, therapeutic approaches developed in mammalian model systems have had limited success to date. An alternative strategy in the search for treatment of spinal cord lesions is provided by regeneration-competent vertebrates. These organisms, which include fish, urodele amphibians, and certain reptiles, have a spinal cord very similar in structure to that of mammals, but are capable of spontaneous structural and functional recovery after spinal cord injury. The present review aims to provide an overview of the current status of our knowledge of spinal cord regeneration in one of these groups, teleost fish. The findings are discussed from a comparative perspective, with reference to other taxa of regeneration-competent vertebrates, as well as to mammals.

In contrast to mammals, adult teleost fish exhibit a vast potential for central nervous system regeneration after injury. Among other mechanisms, this capacity is mediated by replacement of cells lost to injury by new neurons and glia. Here, we examined the spatio-temporal dynamics of apoptosis, and its relationship to the generation and the differentiation of new cells, during this cell replacement phase. As an experimental paradigm, caudal transection of the spinal cord in the teleost fish Apteronotus leptorhynchus was used. During the cell replacement phase, there was a rather constant percentage of new cells (identified by incorporation of 5-bromo-2′-deoxyuridine into newly synthesized DNA) that underwent apoptosis (identified by anti-active caspase-3 immunolabeling). Many of these cells were also immunopositive for the marker proteins Hu C/D or glial fibrillary acidic protein, indicating that a large portion of cells undergo apoptosis after differentiation into neurons or glia, respectively. The spatial distribution of apoptotic cells was uneven, displaying a radial peak in the mid parenchymal regions and a longitudinal peak at the site of the initial spinal transection. The latter persisted for over 100 days post-injury, indicating possible problems in the integration of new cells at the interface between the old, intact tissue and the regenerated portion of the spinal cord. Taken together, the results of the present study are consistent with the hypothesis that apoptosis plays a role in the development of the new tissue during the cell replacement phase of the regenerating teleostean spinal cord.