Janus Kinase Inhibitor Brepocitinib Rescues Myelin Phagocytosis Under Inflammatory Conditions: In Vitro Evidence from Microglia and Macrophage Cell Lines.

Central nervous system (CNS) injuries induce cell death and consequently the release of myelin and other cellular debris. Microglia as well as hematogenous macrophages actively collaborate to phagocyte them and undergo their degradation. However, myelin accumulation persists in the lesion site long after the injury with detrimental effects on axonal regeneration. This might be due to the presence of inhibitors of phagocytosis in the injury site. As we recently published that some proinflammatory stimuli, like interferon-γ (IFNγ) and lipopolysaccharide (LPS), inhibit myelin phagocytosis in macrophages, we have now studied the signaling pathways involved. A phagocytosis assay in Raw264.7 macrophages and N13 microglia cell lines with labeled myelin was developed with the pHrodo reagent that emits fluorescence in acidic cellular compartments (e.g.lysosomes). Pharmacological inhibition of Janus kinases (Jak) with Brepocitinib restored myelin phagocytosis and rescued the expression of genes related to phagocytosis, like triggering receptor expressed on myeloid cells 2 (TREM2), induced by IFNγ or LPS. In addition, while pharmacological inhibition of the signal transducer and activator of transcription 3 (STAT3) rescued myelin phagocytosis and the expression of phagocytosis related genes in the presence of LPS, it did not have any effect on IFNγ-treated cells. Our results show that Jak pathways participate in the inhibition of myelin phagocytosis by IFNγ and LPS. They also indicate that the resolution of inflammation is important for the clearance of cellular debris by macrophages and subsequent regenerative processes.

Improved Efficacy of Delayed Treatment with Human Bone Marrow-Derived Stromal Cells Evaluated in Rats with Spinal Cord Injury.

The treatment of spinal cord injury (SCI) with uncultivated human bone marrow-derived stromal cells (bmSCs) prepared by negative selection has been proposed to be therapeutically superior to treatment with stem cells that were expanded in vitro. To explore their use in clinical trials, we studied the functional effects of delayed application at 7 days after SCI by testing different doses of bmSCs. Spinal cord contusion injury was induced in adult male Wistar rats at the thoracic level T9. Human bmSCs were prepared by negative selection without expansion in vitro (NeuroCellsTM). Treatment consisted of one 150 µL injection into the cisterna magna containing 0.5 or 2.5 million fresh bmSCs or 2.5 million bmSCs. The recovery of motor functions was evaluated during a surveillance period of six weeks (6 W), during which spinal cords were assessed histologically. Treatment resulted in a significant, dose-dependent therapeutic effect on the recovery of motor performance. The histological analysis revealed a lower degree of axonal degeneration and better survival of neurons and oligodendrocytes in bmSCs treated rats. Our results support delayed intrathecal application of bmSCs prepared by negative selection without expansion in vitro as a treatment of SCI.

Is TGR5 a therapeutic target for the treatment of spinal cord injury?

Bile acids, which are synthesized in liver and colon, facilitate the digestion of dietary lipids. In addition to this metabolic function, they also act as molecular signals with activities in the nervous system. These are mediated primarily by a G-protein-coupled bile acid receptor (known as TGR5). Preceded by a long tradition in Chinese medicine, bile acids are now being investigated as therapeutic options in several neuropathologies. Specifically, one bile acid, tauroursodeoxycholic acid (TUDCA), which passes the blood-brain barrier and shows anti-inflammatory and anti-apoptotic effects, has been tested in animal models of spinal cord injury (SCI). In this review, we discuss the evidence for a therapeutic benefit in these preclinical experiments. At the time of writing, 12 studies with TGR5 agonists have been published that report functional outcomes with rodent models of SCI. Most investigations found cytoprotective effects and benefits regarding the recovery of sensorimotor function in the subacute phase. When TUDCA was applied in a hydrogel into the lesion site, a significant improvement was obtained at 2 weeks after SCI. However, no lasting improvements with TUDCA treatment were found, when animals were assessed in later, chronic stages. A combination of TUDCA with stem cell injection failed to improve the effect of the cellular treatment. We conclude that the evidence does not support the use of TUDCA as a treatment of SCI. Nevertheless, cytoprotective effects suggest that different modes of application or combinatorial therapies might still be explored.

Tauroursodeoxycholic Acid Reduces Neuroinflammation but Does Not Support Long Term Functional Recovery of Rats with Spinal Cord Injury.

The bile acid tauroursodeoxycholic acid (TUDCA) reduces cell death under oxidative stress and inflammation. Implants of bone marrow-derived stromal cells (bmSC) are currently under investigation in clinical trials of spinal cord injury (SCI). Since cell death of injected bmSC limits the efficacy of this treatment, the cytoprotective effect of TUDCA may enhance its benefit. We therefore studied the therapeutic effect of TUDCA and its use as a combinatorial treatment with human bmSC in a rat model of SCI. A spinal cord contusion injury was induced at thoracic level T9. Treatment consisted of i.p. injections of TUDCA alone or in combination with one injection of human bmSC into the cisterna magna. The recovery of motor functions was assessed during a surveillance period of six weeks. Biochemical and histological analysis of spinal cord tissue confirmed the anti-inflammatory activity of TUDCA. Treatment improved the recovery of autonomic bladder control and had a positive effect on motor functions in the subacute phase, however, benefits were only transient, such that no significant differences between vehicle and TUDCA-treated animals were observed 1-6 weeks after the lesion. Combinatorial treatment with TUDCA and bmSC failed to have an additional effect compared to treatment with bmSC only. Our data do not support the use of TUDCA as a treatment of SCI.

Bile acids attenuate PKM2 pathway activation in proinflammatory microglia.

Glycolysis is the metabolic pathway that converts glucose into pyruvate. Central nervous system (CNS) pathologies, such as spinal cord injury (SCI) and ischemia, are accompanied by an increase of the glycolytic pathway in the damaged areas as part of the inflammatory response. Pyruvate kinase is a key glycolytic enzyme that converts phosphoenolpyruvate and ADP to pyruvate and ATP. The protein has two isoforms, PKM1 and PKM2, originated from the same gene. As a homodimer, PKM2 loses the pyruvate kinase activity and acts as a transcription factor that regulates the expression of target genes involved in glycolysis and inflammation. After SCI, resident microglia and hematogenous macrophages are key inducers of the inflammatory response with deleterious effects. Activation of the bile acid receptor TGR5 inhibits the pro-inflammatory NFκB pathway in microglia and macrophages. In the present study we have investigated whether bile acids affect the expression of glycolytic enzymes and their regulation by PKM2. Bacterial lipopolysaccharide (LPS) induced the expression of PKM1, PKM2 and its target genes in primary cultures of microglial and Raw264.7 macrophage cells. SCI caused an increase of PKM2 immunoreactivity in macrophages after SCI. Pretreatment with tauroursodeoxycholic acid (TUDCA) or taurolithocholic acid (TLCA) reduced the expression of PKM2 and its target genes in cell cultures. Similarly, after SCI, TUDCA treatment reduced the expression of PKM2 in the lesion center. These results confirm the importance of PKM2 in the inflammatory response in CNS pathologies and indicate a new mechanism of bile acids as regulators of PKM2 pathway.

Taurolithocholic acid but not tauroursodeoxycholic acid rescues phagocytosis activity of bone marrow-derived macrophages under inflammatory stress.

Spinal cord injury (SCI) causes cell death and consequently the breakdown of axons and myelin. The accumulation of myelin debris at the lesion site induces inflammation and blocks axonal regeneration. Hematogenous macrophages contribute to the removal of myelin debris. In this study, we asked how the inflammatory state of macrophages affects their ability to phagocytose myelin. Bone marrow-derived macrophages (BMDM) and Raw264.7 cells were stimulated with lipopolysaccharides (LPS) or interferon gamma (IFNγ), which induce inflammatory stress, and the endocytosis of myelin was examined. We found that activation of the TLR4-NFκB pathway reduced myelin uptake by BMDM, while IFNγ-Jak/STAT1 signaling did not. Since bile acids regulate lipid metabolism and in some cases reduce inflammation, our second objective was to investigate whether myelin clearance could be improved with taurolithocholic acid (TLCA), tauroursodeoxycholic acid or hyodeoxycholic acid. In BMDM only TLCA rescued myelin phagocytosis, when this activity was suppressed by LPS. Inhibition of protein kinase A blocked the effect of TLCA, while an agonist of the farnesoid X receptor did not rescue phagocytosis, implicating TGR5-PKA signaling in the effect of TLCA. To shed light on the mechanism, we measured whether TLCA affected the expression of CD36, triggering receptor on myeloid cells-2 (TREM2), and Gas6, which are known to be involved in phagocytosis and affected by inflammatory stimuli. Concomitant with an increase in expression of tumour necrosis factor alpha, LPS reduced expression of TREM2 and Gas6 in BMDM, and TLCA significantly diminished this downregulation. These findings suggest that activation of bile acid receptors may be used to improve myelin clearance in neuropathologies.

Retinoic acid increases phagocytosis of myelin by macrophages.

Traumatic injuries of the central nervous system (CNS) are followed by the accumulation of cellular debris including proteins and lipids from myelinated fiber tracts. Insufficient phagocytic clearance of myelin debris influences the pathological process because it induces inflammation and blocks axonal regeneration. We investigated whether ligands of nuclear receptor families retinoic acid receptors (RARs), retinoid X receptors, peroxisome proliferator-activated receptors, lipid X receptors, and farnesoid X receptors increase myelin phagocytosis by murine bone marrow-derived macrophages and Raw264.7 cells. Using in vitro assays with 3,3'-dioctadecyloxacarbocyanine perchlorate- and pHrodo-labeled myelin we found that the transcriptional activator all-trans retinoic acid (RA)enhanced endocytosis of myelin involving the induction of tissue transglutaminase-2. The RAR-dependent increase of phagocytosis was not associated with changes in gene expression of receptors FcγR1, FcγR2b, FcγR3, TREM2, DAP12, CR3, or MerTK. The combination of RA and myelin exposure significantly reduced the expression of M1 marker genes inducible nitric oxide synthase and interleukin-1ß and increased expression of transmembrane proteins CD36 and ABC-A1, which are involved in lipid transport and metabolism. The present results suggest an additional mechanism for therapeutic applications of RA after CNS trauma. It remains to be studied whether endogenous RA-signaling regulates phagocytosis in vivo.

Expression patterns of chloride transporters in the auditory brainstem of developing chicken.

GABAergic transmission changes from depolarization to hyperpolarization in most vertebrate brain regions during development. By contrast, in the auditory brainstem of chicken a depolarizing effect of GABA persists after hatching. Since auditory brainstem neurons that receive GABAergic input have a Cl- reversal potential above resting membrane potential, a specifically tuned activity of Cl- transporters is likely. We here present a developmental study of the expression patterns of several members of the SLC12 family (NKCC1, NKCC2, KCC1, KCC2, KCC4, CCC6, CCC9) and of AE3 at developmental ages E7, E10, E12, E15, E17, and P1 with quantitative RT-PCR. NKCC2 and CCC9 were not detected in auditory brainstem (positive control: kidney). KCC1, CCC6 and AE3 were expressed, but not regulated, while NKCC1, KCC2 and KCC4 were regulated. The expression of the latter transporters increased, with KCC2 exhibiting the strongest expression at all time points. Biochemical analysis of the protein expression of NKCC1, KCC2 and KCC4 corroborated the findings on the mRNA level. All three transporters showed a localization at the outer rim of the cells, with NKCC1 and KCC2 expressed in neurons, and KCC4 predominantly in glia. The comparison of the published chloride reversal potential and expression of transporter proteins suggest strong differences in the efficiency of the three transporters. Further, the strong KCC2 expression could reflect a role in the structural development of auditory brainstem synapses that might lead to changes in the physiological properties.

Treatment of rats with spinal cord injury using human bone marrow-derived stromal cells prepared by negative selection.

BACKGROUND: Spinal cord injury (SCI) is a highly debilitating pathology without curative treatment. One of the most promising disease modifying strategies consists in the implantation of stem cells to reduce inflammation and promote neural regeneration. In the present study we tested a new human bone marrow-derived stromal cell preparation (bmSC) as a therapy of SCI. METHODS: Spinal cord contusion injury was induced in adult male rats at thoracic level T9/T10 using the Infinite Horizon impactor. One hour after lesion the animals were treated with a sub-occipital injection of human bmSC into the cisterna magna. No immune suppression was used. One dose of bmSC consisted, on average, of 2.3 million non-manipulated cells in 100 µL suspension, which was processed out of fresh human bone marrow from the iliac crest of healthy volunteers. Treatment efficacy was compared with intraperitoneal injections of methylprednisolone (MP) and saline. The recovery of motor functions was assessed during a surveillance period of nine weeks. Adverse events as well as general health, weight and urodynamic functions were monitored daily. After this time, the animals were perfused, and the spinal cord tissue was investigated histologically. RESULTS: Rats treated with bmSC did not reject the human implants and showed no sign of sickness behavior or neuropathic pain. Compared to MP treatment, animals displayed better recovery of their SCI-induced motor deficits. There were no significant differences in the recovery of bladder control between groups. Histological analysis at ten weeks after SCI revealed no differences in tissue sparing and astrogliosis, however, bmSC treatment was accompanied with reduced axonal degeneration in the dorsal ascending fiber tracts, lower Iba1-immunoreactivity (IR) close to the lesion site and reduced apoptosis in the ventral grey matter. Neuroinflammation, as evidenced by CD68-IR, was significantly reduced in the MP-treated group. CONCLUSIONS: Human bmSC that were prepared by negative selection without expansion in culture have neuroprotective properties after SCI. Given the effect size on motor function, implantation in the acute phase was not sufficient to induce spinal cord repair. Due to their immune modulatory properties, allogeneic implants of bmSC can be used in combinatorial therapies of SCI.

Vibratory movements in contests between females of the feather-legged spider (Uloborus plumipes).

Females of the feather-legged spider Uloborus plumipes invade, and compete for, each other's orb webs. In the context of these competitive interactions the question arose how the spiders communicate. Since substrate-borne vibrations are the most important component of the sensory environment of web-building spiders, we investigated vibratory movements that might serve as signals of communication. Three behaviors were found to be associated with female-female contests and to cause propagating vibrations in the spider webs: thread pulling, abdominal trembling, and web shaking. While thread pulling and abdominal trembling were also observed when prey insects were caught in the webs, web shaking occurred only in response to the presence of a competing conspecific. Caused by flexing of the first legs and a vigorous rotary movement of the opisthosoma, web shaking creates a short burst of strong oscillations of the orb web. This behavior always elicited a behavioral reaction by the competitor and may serve as an intraspecific signal in the mutual assessment of competing spiders. We suggest that web shaking communicates resource holding potential in U. plumipes.

Fighting for the web: competition between female feather-legged spiders (Uloborus plumipes).

Most spider species are solitary, and among the few social interactions among them, resource competition between females has received little attention. We discovered that females of the feather-legged spider Uloborus plumipes invade the orb webs of conspecifics and compete for webs. Following observations in the wild, intruder-defender interactions were studied in a terrarium and in controlled laboratory experiments. We found that contests for orb webs occurred spontaneously between adult females. Competitive interactions in U. plumipes were characterized by an escalation of ritualized behaviors. In 27% of the contests the winner was determined by interactions at a distance, which involved behaviors that caused vibratory signaling on the web. The remaining interactions escalated to physical contact, and in 78% of these a fight occurred between the contestants. Using multivariate logistic regression we determined the factors that predicted the outcome of the contests: (i) Web ownership did not give the defender a competitive advantage. (ii) The difference in physical size between the competing spiders was the most important predictor for the outcome of web contests. (iii) Independent of body size, the display of certain behaviors, specifically the ability to reach the hub before the contestant and the frequency of attacks, increased the probability of winning. (iv) Winning or losing a fight did not affect the chances of winning subsequent contests. The interactions reported here provide a promising approach to investigate communication in spiders and to test theoretical models of intraspecific competition.

Cell-free artificial implants of electrospun fibres in a three-dimensional gelatin matrix support sciatic nerve regeneration in vivo.

Surgical repair of larger peripheral nerve lesions requires the use of autologous nerve grafts. At present, clinical alternatives to avoid nerve transplantation consist of empty tubes, which are only suitable for the repair over short distances and have limited success. We developed a cell-free, three-dimensional scaffold for axonal guidance in long-distance nerve repair. Sub-micron scale fibres of biodegradable poly-ε-caprolactone (PCL) and collagen/PCL (c/PCL) blends were incorporated in a gelatin matrix and inserted in collagen tubes. The conduits were tested by replacing 15-mm-long segments of rat sciatic nerves in vivo. Biocompatibility of the implants and nerve regeneration were assessed histologically, with electromyography and with behavioural tests for motor functions. Functional repair was achieved in all animals with autologous transplants, in 12 of 13 rats that received artificial implants with an internal structure and in half of the animals with empty nerve conduits. In rats with implants containing c/PCL fibres, the extent of recovery (compound muscle action potentials, motor functions of the hind limbs) was superior to animals that had received empty implants, but not as good as with autologous nerve transplantation. Schwann cell migration and axonal regeneration were observed in all artificial implants, and muscular atrophy was reduced in comparison with animals that had received no implants. The present design represents a significant step towards cell-free, artificial nerve bridges that can replace autologous nerve transplants in the clinic. Copyright © 2017 John Wiley & Sons, Ltd.

Characterisation of cell-substrate interactions between Schwann cells and three-dimensional fibrin hydrogels containing orientated nanofibre topographical cues.

The generation of complex three-dimensional bioengineered scaffolds that are capable of mimicking the molecular and topographical cues of the extracellular matrix found in native tissues is a field of expanding research. The systematic development of such scaffolds requires the characterisation of cell behaviour in response to the individual components of the scaffold. In the present investigation, we studied cell-substrate interactions between purified populations of Schwann cells and three-dimensional fibrin hydrogel scaffolds, in the presence or absence of multiple layers of highly orientated electrospun polycaprolactone nanofibres. Embedded Schwann cells remained viable within the fibrin hydrogel for up to 7 days (the longest time studied); however, cell behaviour in the hydrogel was somewhat different to that observed on the two-dimensional fibrin substrate: Schwann cells failed to proliferate in the fibrin hydrogel, whereas cell numbers increased steadily on the two-dimensional fibrin substrate. Schwann cells within the fibrin hydrogel developed complex process branching patterns, but, when presented with orientated nanofibres, showed a strong tendency to redistribute themselves onto the nanofibres, where they extended long processes that followed the longitudinal orientation of the nanofibres. The process length along nanofibre-containing fibrin hydrogel reached near-maximal levels (for the present experimental conditions) as early as 1 day after culturing. The ability of this three-dimensional, extracellular matrix-mimicking scaffold to support Schwann cell survival and provide topographical cues for rapid process extension suggest that it may be an appropriate device design for the bridging of experimental lesions of the peripheral nervous system.

Retinoic acid as a survival factor in neuronal development of the grasshopper, Locusta migratoria.

Based on experience with cell cultures of adult insect neurons, we develop a serum-free culture system for embryonic locust neurons. Influences of trophic substances on survival and neurite outgrowth of developing neurons are investigated. For the first time, a positive trophic effect of 9-cis retinoic acid (9-cis RA) was shown in vitro on embryonic neurons of an insect. We observed longer cell survival of 50 % developmental stage neurons in cultures supplemented with 0.3 nM 9-cis RA. Furthermore, an influence on neuron morphology was revealed, as the addition of 9-cis RA to cell culture medium led to an increase in the number of neurites per cell. Although an RA receptor gene, LmRXR (Locusta migratoria retinoid X receptor), was expressed in the central nervous system throughout development, the influence of 9-cis RA on neuronal survival and outgrowth was restricted to 50 % stage embryonic cells.

Three-dimensional configuration of orientated fibers as guidance structures for cell migration and axonal growth.

Peripheral nerve injuries can be surgically repaired by suturing the transected nerve stumps or, in case of larger lesions, by the transplantation of an autologous nerve graft. To avoid donor site morbidity, the development of artificial implants is desired. Clinically, hollow conduits have been used for this purpose but are inferior to the autograft because they lack internal guidance cues for Schwann cells and regenerating axons. In this article, we describe the design of a three-dimensional (3D) scaffold consisting of parallel fibers embedded in a collagen matrix. For this purpose, an electrospinning device was developed to produce and manipulate a 3D array of aligned poly(ɛ-caprolactone) (PCL) microfibers. This fiber array was then incorporated into biodegradable PCL tubes to serve as artificial nerve bridges. Using primary cultures of embryonic chicken dorsal root ganglia, we show that PCL microfibers in the 3D matrix of our composite scaffold guide the direction of Schwann cell migration and axonal growth.

Spinal cord injury induced changes of nuclear receptors PPARα and LXRß and modulation with oleic acid/albumin treatment.

In previous studies with animal models of spinal cord injury (SCI) pharmacological activation of peroxisome proliferator activated receptors (PPAR) and liver X receptors (LXR) were used to reduce tissue damage and promote behavioral recovery in animal models. We have studied the endogenous expression of the transcription factors PPARα and LXRß in the chronic stage after SCI in rats. The immunohistochemical investigation revealed a long lasting increase in the level of PPARα in white matter in the vicinity of the lesion site. The source of this signal was identified in a subpopulation of astrocytes outside of the glial scar area. Intrathecal injections of oleic acid/albumin reduced the lesion-induced PPARα immunoreactivity. In addition, ependymal cells displayed a prominent PPARα signal in the non-injured spinal cord, and continued to express the receptor as they proliferated and migrated within the damaged tissue. The nuclear receptor LXRß was detected at similar levels after SCI as in sham operated animals. We found high levels of immunoreactivity in the gray matter, while in the white matter it was present in subpopulations of astrocytes and oligodendrocytes. Macrophages that had accumulated within the center of the lesion contained LXRß in their cell nuclei. Possible endogenous functions of PPARα and LXRß after SCI are discussed, specifically the control of fatty acid and cholesterol metabolism and the regulation of inflammatory reactions.

Expression of retinoid X receptor ß is induced in astrocytes during corpus callosum demyelination.

The experimental activation of retinoid receptors reduces pathological symptoms in animal models of multiple sclerosis. In order to assess the involvement of endogenous retinoid signaling during the process of demyelination we investigated retinoic acid synthesizing enzymes and nuclear receptors using the mouse model of cuprizone toxicity. The initiation of myelin degradation in the corpus callosum was accompanied with a local increase of retinaldehyde dehydrogenase (RALDH) immunoreactivity. On the level of receptors we observed a striking increase in protein expression of the retinoid X receptor (RXR)-ß in the affected corpus callosum. The RXRß immunoreactivity appeared exclusively in astrocytes, where it reached a maximum at five weeks of treatment, following the RALDH response. In the cerebral cortex and basal ganglia of affected mice RXRß was also observed in neurons. Among nuclear receptor antigens RARα showed a cuprizone associated increase in the corpus callosum. Quantitative RT-PCR revealed strong basal expression of RXRß and a significant, over 20-fold upregulation of the peroxisome proliferator-activated receptor-γ during demyelination. The results indicate that compensatory mechanisms during central demyelination may engage nuclear receptor dimers with an RXRß partner.

Functional recovery, serotonergic sprouting, and endogenous progenitor fates in response to delayed environmental enrichment after spinal cord injury.

Environmental enrichment (EE) is a way to induce voluntary locomotor training that positively affects locomotor recovery after acute spinal cord injury (SCI). The beneficial effect on SCI outcome is thought to be based on enhanced plasticity in motor pathways, triggered by locomotor-specific sensory feedback to the spinal cord circuitry for locomotion (central pattern generators [CPGs]). In view of chronic SCI, we tested the hypothesis that EE improves motor outcome after SCI in the rat when started after a clinically relevant delay of 3 weeks. At the CPG level (i.e., the spinal L1-L2 level), where EE-related sensory feedback is processed, two key mechanisms of anatomical plasticity were examined: (1) serotonergic innervation, and (2) survival and differentiation of spinal cord progenitor cells. Delayed EE improved interlimb coordination, which was associated with an increased serotonergic innervation of the ventro-lateral grey matter within the L1-L2 segments. Although spinal cord progenitor cells were found to differentiate into both neurons and glial cells, EE did not affect their survival. These results show that EE induces a substantial improvement of motor outcome after SCI when commenced after a clinically-relevant delay. Increased serotonergic innervation of the lumbar CPG area is therefore suggested to play an important role in the EE-induced recovery of interlimb coordination.