Neural Debris Research Articles

Microglia cannibalism and efferocytosis leads to shorter lifespans of developmental microglia.

The overproduction of cells and subsequent production of debris is a universal principle of neurodevelopment. Here, we show an additional feature of the developing nervous system that causes neural debris-promoted by the sacrificial nature of embryonic microglia that irreversibly become phagocytic after clearing other neural debris. Described as long-lived, microglia colonize the embryonic brain and persist into adulthood. Using transgenic zebrafish to investigate the microglia debris during brain construction, we identified that unlike other neural cell types that die in developmental stages after they have expanded, necroptosis-dependent microglial debris is prevalent when microglia are expanding in the zebrafish brain. Time-lapse imaging of microglia demonstrates that this debris is cannibalized by other microglia. To investigate features that promote microglia death and cannibalism, we used time-lapse imaging and fate-mapping strategies to track the lifespan of individual developmental microglia. These approaches revealed that instead of embryonic microglia being long-lived cells that completely digest their phagocytic debris, once most developmental microglia in zebrafish become phagocytic they eventually die, including ones that are cannibalistic. These results establish a paradox-which we tested by increasing neural debris and manipulating phagocytosis-that once most microglia in the embryo become phagocytic, they die, create debris, and then are cannibalized by other microglia, resulting in more phagocytic microglia that are destined to die.

Microglia cannibalism and efferocytosis leads to shorter lifespans of developmental microglia.

The overproduction of cells and subsequent production of debris is a universal principle of neurodevelopment. Here we show an additional feature of the developing nervous system that causes neural debris - promoted by the sacrificial nature of embryonic microglia that irreversibly become phagocytic after clearing other neural debris. Described as long-lived, microglia colonize the embryonic brain and persist into adulthood. Using transgenic zebrafish to investigate the microglia debris during brain construction, we identified that unlike other neural cell-types that die in developmental stages after they have expanded, necroptosis-dependent microglial debris is prevalent when microglia are expanding in the zebrafish brain. Time-lapse imaging of microglia demonstrates that this debris is cannibalized by other microglia. To investigate features that promote microglia death and cannibalism, we used time-lapse imaging and fate-mapping strategies to track the lifespan of individual developmental microglia. These approaches revealed that instead of embryonic microglia being long-lived cells that completely digest their phagocytic debris, once most developmental microglia in zebrafish become phagocytic they eventually die, including ones that are cannibalistic. These results establish a paradox -- which we tested by increasing neural debris and manipulating phagocytosis -- that once most microglia in the embryo become phagocytic, they die, create debris and then are cannibalized by other microglia, resulting in more phagocytic microglia that are destined to die.

Aging and sex: Impact on microglia phagocytosis.

Microglia dysfunction and activation are important hallmarks of the aging brain and are concomitant with age‐related neurodegeneration and cognitive decline. Age‐associated changes in microglia migration and phagocytic capacity result in maladaptive responses, chronic neuroinflammation, and worsened outcomes in neurodegenerative disorders. Given the sex bias in the incidence, prevalence, and therapy response of most neurological disorders, we have here examined whether the phagocytic activity of aged microglia is different in males and females. With this aim, the phagocytosis activity of male and female cells was compared in an in vitro aged microglia model and in microglia isolated from adult (5‐month‐old) or aged (18‐month‐old) mice. In both models, the phagocytosis of neural debris increased with aging in male and female cells and was higher in aged female microglia than in aged male cells. However, female aged microglia lost its ability to adapt its phagocytic activity to inflammatory conditions. These findings suggest that microglia phagocytosis of neural debris may represent a previously unexplored neuroprotective characteristic of aged microglia that may contribute to the generation of sex differences in the manifestation of neurodegenerative diseases.

Bidirectional Regulation of Sleep and Synapse Pruning after Neural Injury.

Following acute neural injury, severed axons undergo programmed Wallerian degeneration over several following days. While sleep has been linked with synaptic reorganization under other conditions, the role of sleep in responses to neural injuries remains poorly understood. To study the relationship between sleep and neural injury responses, we examined Drosophila melanogaster following the removal of antennae or other sensory tissues. Daytime sleep is elevated after antennal or wing injury, but sleep returns to baseline levels within 24h after injury. Similar increases in sleep are not observed when olfactory receptor neurons are silenced or when other sensory organs are severed, suggesting that increased sleep after injury is not attributed to sensory deprivation, nociception, or generalized inflammatory responses. Neuroprotective disruptions of the E3 ubiquitin ligase highwire and c-Jun N-terminal kinase basket in olfactory receptor neurons weaken the sleep-promoting effects of antennal injury, suggesting that post-injury sleep may be influenced by the clearance of damaged neurons. Finally, we show that pre-synaptic active zones are preferentially removed from severed axons within hours after injury and that depriving recently injured flies of sleep slows the removal of both active zones and damaged axons. These data support a bidirectional interaction between sleep and synapse pruning after antennal injury: locally increasing the need to clear neural debris is associated with increased sleep, which is required for efficient active zone removal after injury.

In Vitro Engulfment Assay to Measure Phagocytic Activity of Astrocytes Using Synaptosomes.

Astrocytes eliminate unnecessary synapses, neural debris, and pathogenic proteins such as amyloid β plaque. Although the emerging evidences suggest that the phagocytic roles of astrocytes are critical in maintaining brain homeostasis during development as well as pathogenic conditions, the efficient assay for measuring phagocytic capacity and kinetics of astrocytes has been lacking. Here we present in vitro engulfment assay using purified astrocytes and synaptosomes. Based on imaging methods, either fluorescent or pH indicator-conjugated synaptosomes can be used in this assay.

The serrulatane diterpenoid natural products RAD288 and RAD289 stimulate properties of olfactory ensheathing cells useful for neural repair therapies

Olfactory ensheathing cells (OECs) are being trialled for cell transplantation therapies for neural repair as they have unique properties which can enhance neuron regeneration. However, improvements in cell viability, proliferation and migration are needed to enhance therapeutic outcomes. Growth factors can enhance cell activity, but they can also induce side effects as they can act on numerous cell types. An alternative approach is to identify natural products (NPs) that more selectively activate specific cell functions. We have examined two pure NPs, 3-acetoxy-7,8-dihydroxyserrulat-14-en-19-oic acid (RAD288) and 3,7,8-trihydroxyserrulat-14-en-19-oic acid (RAD289) isolated from the Australian plant Eremophila microtheca. We determined that RAD288 and RAD289 stimulated the viability and proliferation of OECs in two-dimensional cultures and increased cell viability in three-dimensional spheroids. Both compounds also enhanced OEC-mediated phagocytosis of neural debris. However, only RAD288 stimulated migration of OECs, demonstrating that key structural changes to the compound can dramatically affect the resultant cellular action. In addition, cell-type specific action is highlighted by the result that neither compound stimulated the viability of Schwann cells which are a closely-related glial cell type. Therefore, these small molecules may have high potential for selective activation of specific therapeutically-useful activities of OECs for transplantation therapies to repair the nervous system.

Lipopolysaccharide and Curcumin Co-Stimulation Potentiates Olfactory Ensheathing Cell Phagocytosis Via Enhancing Their Activation.

The gradual deterioration following central nervous system (CNS) injuries or neurodegenerative disorders is usually accompanied by infiltration of degenerated and apoptotic neural tissue debris. A rapid and efficient clearance of these deteriorated cell products is of pivotal importance in creating a permissive environment for regeneration of those damaged neurons. Our recent report revealed that the phagocytic activity of olfactory ensheathing cells (OECs) can make a substantial contribution to neuronal growth in such a hostile environment. However, little is known about how to further increase the ability of OECs in phagocytosing deleterious products. Here, we used an in vitro model of primary cells to investigate the effects of lipopolysaccharide (LPS) and curcumin (CCM) co-stimulation on phagocytic activity of OECs and the possible underlying mechanisms. Our results showed that co-stimulation using LPS and CCM can significantly enhance the activation of OECs, displaying a remarkable up-regulation in chemokine (C-X-C motif) ligand 1, chemokine (C-X-C motif) ligand 2, tumor necrosis factor-α, and Toll-like receptor 4, increased OEC proliferative activity, and improved phagocytic capacity compared with normal and LPS- or CCM-treated OECs. More importantly, this potentiated phagocytosis activity greatly facilitated neuronal growth under hostile culture conditions. Moreover, the up-regulation of transglutaminase-2 and phosphatidylserine receptor in OECs activated by LPS and CCM co-stimulation are likely responsible for mechanisms underlying the observed cellular events, because cystamine (a specific inhibitor of transglutaminase-2) and neutrophil elastase (a cleavage enzyme of phosphatidylserine receptor) can effectively abrogate all the positive effects of OECs, including phagocytic capacity and promotive effects on neuronal growth. This study provides an alternative strategy for the repair of traumatic nerve injury and neurologic diseases with the application of OECs in combination with LPS and CCM.

Anti-inflammatory activity of low molecular weight polysialic acid on human macrophages.

Oligosialic and polysialic acid (oligoSia and polySia) of the glycocalyx of neural and immune cells are linear chains, in which the sialic acid monomers are α2.8-glycosidically linked. Sialic acid-binding immunoglobulin-like lectin-11 (SIGLEC-11) is a primate-lineage specific receptor of human tissue macrophages and microglia that binds to α2.8-linked oligoSia. Here, we show that soluble low molecular weight polySia with an average degree of polymerization 20 (avDP20) interacts with SIGLEC-11 and acts anti-inflammatory on human THP1 macrophages involving the SIGLEC-11 receptor. Soluble polySia avDP20 inhibited the lipopolysaccharide (LPS)-induced gene transcription and protein expression of tumor necrosis factor-α (Tumor Necrosis Factor Superfamily Member 2, TNFSF2). In addition, polySia avDP20 neutralized the LPS-triggered increase in macrophage phagocytosis, but did not affect basal phagocytosis or endocytosis. Moreover, polySia avDP20 prevented the oxidative burst of human macrophages triggered by neural debris or fibrillary amyloid-β1–42. In a human macrophage-neuron co-culture system, polySia avDP20 also reduced loss of neurites triggered by fibrillary amyloid-β1–42. Thus, treatment with polySia avDP20 might be a new anti-inflammatory therapeutic strategy that also prevents the oxidative burst of macrophages.

Combination of small gap suture with nerve fragments in the experimental study of the rat nerve defects

Objective: To study the effect of nerve fragments and small gap suture of epineurium in peripheral nerve repair. Methods: (1) The experimental animals were randomly divided into control group, simple small gap suture group, small gap suture combined neural debris group; (2) observing the nerve anastomosis after two weeks; (3) testing nerve fiber regeneration after eight weeks; (4) testing gastrocnemius wet weight ratio after eight weeks; (5) pathological image analysis: the number of regenerated nerve fibers, the diameter and myelin thickness of regenerated nerve fiber were measured. Results: (1) Scar tissue was formed in traditional suture anastomotic adhesions; in simple small gap suture group, obvious adhesions was not found surrounding the tissue; for combination group of small gap suture with nerve debris, there was no obvious adhesions, and the outer membrane of small gap turned thinner than before. (2) Compared with the normal control group, the wet weight was increased significantly in the small gap suture group; relative to the small gap group, the wet weight was increased significantly in the small gap plus nerve fragments group. (3) Compared with the control group, the total number of myelinated nerve fibers was increased significantly in the small-gap suture group and small gap plusnerve fragments group; the diameter of nerve fibers and myelin thickness were increased significantly in simple small gap group and the small gap plus nerve debris group. (4) Histology showed: Compared with simple small gap suture group, nerve fiber density was increased, arrangement is neat in the small gap plus nerve fragments group. Conclusions: The nerve fragments plus epineurium small gap suture has a significant role in the repair of peripheral nerve injury.

Microglial CD33-Related Siglec-E Inhibits Neurotoxicity by Preventing the Phagocytosis-Associated Oxidative Burst

Sialic acid-binding Ig-like lectins (Siglecs) are members of the Ig superfamily that recognize sialic acid residues of glycoproteins. Siglec-E is a mouse CD33-related Siglec that preferentially binds to sialic acid residues of the cellular glycocalyx. Here, we demonstrate gene transcription and protein expression of Siglec-E by cultured mouse microglia. Siglec-E on microglia inhibited phagocytosis of neural debris and prevented the production of superoxide radicals induced by challenge with neural debris. Soluble extracellular Siglec-E receptor protein bound to the neural glycocalyx. Coculture of mouse microglia and neurons demonstrated a neuroprotective effect of microglial Siglec-E that was dependent on neuronal sialic acid residues. Increased neurotoxicity of microglia after knockdown of Siglece mRNA was neutralized by the reactive oxygen species scavenger Trolox. Data suggest that Siglec-E recognizes the intact neuronal glycocalyx and has neuroprotective function by preventing phagocytosis and the associated oxidative burst.

Lipid-like components released from degenerating dopaminergic neurons trigger the dynamic migration of microglia

In the brain, communication between neural and non-neural cells is crucial for the proper functioning of the central nervous system. Microglia play an important role in the clearance of neural cellular corpses and debris, especially under pathological conditions. It remains, however, unclear how microglia sense the degenerating neurons at a distance in order to migrate to them. In the present study, we explored the interaction between neurons and microglia using an in vitro model of Parkinson’s disease (PD). In primary mesencephalic neuronal cultures, 1-methyl-4-phenylpridinium (MPP+) induced the selective death of dopaminergic (DAergic) neurons in a dose- and time-dependent manner. Transmigration assay showed that the conditioned medium (CM) from mesencephalic cultures treated with MPP+ was enough to trigger the attraction of microglia at an early as well as a late phase of neuronal damage. Microglia preferably reacted with the soluble parts separated by ultracentrifugation over the neural debris-containing pellets. This chemoattractive activity was significantly reduced by the removal of the lipidic components in CM, but not by the removal of proteins, DNA or RNA. These results suggest that as yet-unidentified lipid-like components released from dying DAergic neurons are likely to recruit microglia, and thus have a role in neuronal damage.

Identity of the cells recruited to a lesion in the central nervous system of a decapod crustacean

In a previous study, we analyzed and described the features of the degeneration of the protocerebral tract (PCT) of the crustacean Ucides cordatus, after the extirpation of the eyestalk. In that study, among axons with axoplasmic degeneration, cells with granules resembling blood cells (hemocytes) were seen. Therefore, in the present study, we characterized the circulating hemocytes and compared them with the cells recruited to a lesion, which was produced as in the former study. Using histochemistry, immunohistochemistry, and electron microscopy (transmission and scanning), we confirmed that circulating and recruited cells display a similar morphology. Therefore, in the crab, hemocytes were attracted to the lesion site in the acute stage of degeneration, appearing near local glial cells that showed signs of being responsive. Some of the attracted hemocytes displayed a morphology that was considered to be possibly activated blood cells. Also, the cells that migrated to the injured PCT displayed features, such as the presence of hydrolytic enzymes and an ability to phagocytize neural debris, similar to those of vertebrates. In summary, our results indicate that hemocytes were not only phagocytizing neural debris together with glial cells but also that they may be concerned with creating a favorable environment for regenerating events.

Signal Regulatory Protein-β1: A Microglial Modulator of Phagocytosis in Alzheimer’s Disease

The signal regulatory protein-beta1 (SIRPbeta1) is a DAP12-associated transmembrane receptor expressed in a subset of hematopoietic cells. Recently, it was shown that peritoneal macrophages express SIRPbeta1, which positively regulated phagocytosis. Here, we found that SIRPbeta1 was up-regulated and acted as a phagocytic receptor on microglia in amyloid precursor protein J20 (APP/J20) transgenic mice and in Alzheimer's disease (AD) patients. Interferon (IFN)-gamma and IFN-beta stimulated gene transcription of SIRPbeta1 in cultured microglia. Activation of SIRPbeta1 on cultured microglia by cross-linking antibodies induced reorganization of the cytoskeleton protein beta-actin and suppressed lipopolysaccharide-induced gene transcription of tumor necrosis factor-alpha and nitric oxide synthase-2. Furthermore, activation of SIRPbeta1 increased phagocytosis of microsphere beads, neural debris, and fibrillary amyloid-beta (Abeta). Phagocytosis of neural cell debris and Abeta was impaired after lentiviral knockdown of SIRPbeta1 in primary microglial cells. Thus, SIRPbeta1 is a novel IFN-induced microglial receptor that supports clearance of neural debris and Abeta aggregates by stimulating phagocytosis.

Selective Remodeling: Refining Neural Connectivity at the Neuromuscular Junction

A primer on new research by Fuentes-Medel and colleagues explains the important role of non-neural cells in clearing neural debris, which is continuously produced during the normal remodeling processes that establish and maintain neural connectivity.

Ultrastructural Observations on the Progress of Nerve Degeneration and Regeneration at the Suture Site following Vagal-Hypoglossal Nerve Coaptation in Cats

Nerve degeneration and regeneration have been investigated at the suture site following proximal-to-distal vagal-hypoglossal nerve coaptation (VHC) in cats at different time points (from 3 to 315 days postoperatively; dpo). Massive axonal degeneration and myelin breakdown and removal of degraded neural debris were observed during the first 2 weeks postoperatively. This was followed by active Schwann cell multiplication and inflammatory cell invasion at 14 dpo. Schwann cells appeared mobile, and were guided to the newly developed growth cones, dividing them into axonal sprout clusters. At 18 dpo, the migrating Schwann cells were confined to the preexisting basal lamina scaffolds, forming bands of Büngner. It is suggested that the latter may play a key role in navigating the regenerating axons to their newly acquired target organ at 22 dpo. Remyelination of axons was not observed till 46 dpo. Compared with the rapid axonal reaction in other models of nerve injury, the degeneration process in VHC was protracted and, furthermore, regeneration and remyelination were delayed. The subtle remodeling of the nerve in cross-coaptation may be far greater than previously recognized, and this may have clinical importance since patients undergoing nerve crossover microsurgery exhibit delayed motor rehabilitation, apparently as a direct result of a change in target innervation. Defining the mechanisms underlying the neuroplastic program could thus potentially improve the prognosis of crossover of two different peripheral nerves.

Binary imaging analysis for comprehensive quantitative histomorphometry of peripheral nerve

Quantitative histomorphometry is the current gold standard for objective measurement of nerve architecture and its components. Many methods still in use rely heavily upon manual techniques that are prohibitively time consuming, predisposing to operator fatigue, sampling error, and overall limited reproducibility. More recently, investigators have attempted to combine the speed of automated morphometry with the accuracy of manual and semi-automated methods. Systematic refinements in binary imaging analysis techniques combined with an algorithmic approach allow for more exhaustive characterization of nerve parameters in the surgically relevant injury paradigms of regeneration following crush, transection, and nerve gap injuries. The binary imaging method introduced here uses multiple bitplanes to achieve reproducible, high throughput quantitative assessment of peripheral nerve. Number of myelinated axons, myelinated fiber diameter, myelin thickness, fiber distributions, myelinated fiber density, and neural debris can be quantitatively evaluated with stratification of raw data by nerve component. Results of this semi-automated method are validated by comparing values against those obtained with manual techniques. The use of this approach results in more rapid, accurate, and complete assessment of myelinated axons than manual techniques.

Abstracts

Abstracts

Loss of microglial ramification in microglia-astrocyte cocultures: involvement of adenylate cyclase, calcium, phosphatase, and Gi-protein systems.

Reduction in microglial branching is a common feature in brain pathology and culminates in the transformation into small, rounded, microglia-derived phagocytes in the presence of neural debris. The molecular factors responsible for this transformation are unknown. Here we explored the effect of different classes of intra- and extracellular stimuli in vitro on the morphology of ramified microglia cultured on a confluent astrocyte substrate. These studies showed a strong dose-dependent effect for the Ca(2+) ionophore calcimycine/A21837 (50 microM) and for dibutyryl-cAMP (1 mM), with a loss of microglial ramification. Direct activation of the adenylate cyclase with forskolin (0.1 mM) also led to the disappearance of microglial branching. Okadaic acid (70 nM), the inhibitor of protein phosphatases 1 and 2A (PP1/PP2A), and pertussis toxin (12.5 microg/ml), a G(i)-protein inhibitor, also showed similar effects. No effect was observed for dibutyryl-cGMP or for UTP; addition of ATP had a moderate effect, but only at very high, probably nonphysiological concentrations (100 mM). Extracellular matrix components such as keratatan-sulfate, integrin receptor blockers, the disintegrins kistrin, echistatin, and flavoridin, or the serine protease thrombin all had no effect. Addition of prostaglandin D(2) (PGD(2)), a molecule produced by activated microglial cells, had a transforming effect, but at concentrations two orders of magnitude higher than that of established PGD(2) receptors. In summary, addition of agents causing intracellular elevation of Ca(2+) and cAMP or inhibition of G(i)-proteins and phosphatases to ramified microglia cultured on top of confluent astrocytes leads to a rapid loss of microglial branching. Signaling cascades controlled by these molecules may play an important role in the regulation of this common physiological process in the injured brain.

Axonal regeneration after cold preservation of nerve allografts and immunosuppression with tacrolimus in mice.

The purpose of this study was to combine the immunosuppressive and neuroregenerative effects of tacrolimus (FK506) with cold preservation of peripheral nerve allografts to maximize axonal regeneration across short peripheral nerve gaps. Ninety-six male C3H mice were randomized to six groups, which were composed of animals with isografts (Group 1, positive control), allografts (Group 2, negative control), allografts treated with subtherapeutic doses of FK506 without and with cold preservation (Groups 3 and 4), and allografts treated with therapeutic doses of FK506 without and with cold preservation (Groups 5 and 6). Results were determined using walking-track data and histomorphometric measurements. Three weeks postoperatively, animals treated with therapeutic doses of FK506 after receiving cold-preserved allografts demonstrated accelerated functional recovery relative to all other groups. In addition, histomorphometric parameters in these animals (1,257 +/- 847 total axons, 6.7 +/- 3.3% nerve tissue, 11.8 +/- 6.5% neural debris, 8,844 +/- 4,325 fibers/mm2 nerve density, and 2.53 +/- 0.25 microm fiber width) were the same as or better than in all other groups. The parameters of percent nerve tissue (p < 0.016), nerve density (p < 0.038), and percent neural debris (p < 0.01) were statistically significantly better than those in all other groups, including Group 1 (isograft, positive control). The combination of FK506 treatment with cold preservation of nerve allografts resulted in functional and histomorphometric recovery superior to that with either modality alone.

In vitro model of microglial deramification: ramified microglia transform into amoeboid phagocytes following addition of brain cell membranes to microglia-astrocyte cocultures.

Changes in the morphology of ramified microglia are a common feature in brain pathology and culminate in the appearance of small, rounded, microglia-derived phagocytes in the presence of neural debris. Here, we explored the effect of adding brain cell membranes on the morphology of alphaMbeta2-integrin (CD11b/CD18, CR3) positive microglia cultured on a confluent astrocyte substrate as an in vitro model of deramification. Addition of brain membranes led to a loss of microglial ramification, with full transformation to small, rounded, macrophages at 20-40 microg/ml. Time course studies showed a rapid response, with first effects at 1-3 hours, and full transformation at 24-48 hours. Removal of cell membranes and exchange of the culture medium led to a similarly rapid process of reramification. Comparison of cell membranes from different tissues at 20 microg/ml showed strong transforming effect for the brain, more moderate for kidney and liver, and very weak for spleen and skeletal muscle. Fluorescent labeling of brain membranes revealed uptake by almost all rounded macrophages, by a subpopulation of glial fibrillary acidic protein (GFAP)-positive astrocytes, but not by ramified microglia. Phagocytosis of inert fluorobeads did not lead to a transformation into macrophages but their phagocytosis was inhibited by brain membranes, pointing to a saturable uptake mechanism. In summary, addition of brain cell membranes and their phagocytosis leads to a rapid and reversible loss of ramification. The differences in transforming activity from different tissues and the absence of effect from phagocytosed fluorobeads suggest, however, the need for a second stimulus following the phagocytosis of cell debris.