Microglial Dynamics Research Articles

Measuring Microglial Turnover in the Adult Brain.

Microglia are the main resident immunocompetent cells of the brain with key roles in brain development, homeostasis, and function. Recent reports have started to shed light on the homeostatic mechanisms regulating the composition and turnover of the microglial population under physiological conditions from development to ageing, but our knowledge of the dynamics of microglia is incomplete. Therefore, it appears relevant to provide a standardized approach to quantify the turnover of microglia, with direct application to create a greater understanding of the dynamics of this cell population, and how it may contribute to the pathogenesis and/or progression of neurological disorders. Here we describe a robust immunohistochemical method to analyze microglial proliferation in mouse brain, aiming at providing a shared and universal approach to analyze microglial dynamics across different laboratories.

Microglial Dynamics During Human Brain Development.

Microglial cells are thought to colonize the human cerebrum between the 4th and 24th gestational weeks. Rodent studies have demonstrated that these cells originate from yolk sac progenitors though it is not clear whether this directly pertains to human development. Our understanding of microglial cell dynamics in the developing human brain comes mostly from postmortem studies demonstrating that the beginning of microglial colonization precedes the appearance of the vasculature, the blood–brain barrier, astrogliogenesis, oligodendrogenesis, neurogenesis, migration, and myelination of the various brain areas. Furthermore, migrating microglial populations cluster by morphology and express differential markers within the developing brain and according to developmental age. With the advent of novel technologies such as RNA-sequencing in fresh human tissue, we are beginning to identify the molecular features of the adult microglial signature. However, this is may not extend to the much more dynamic and rapidly changing antenatal microglial population and this is further complicated by the scarcity of tissue resources. In this brief review, we first describe the various historic schools of thought that had debated the origin of microglial cells while examining the evidence supporting the various theories. We then proceed to examine the evidence we have accumulated on microglial dynamics in the developing human brain, present evidence from rodent studies on the functional role of microglia during development and finally identify limitations for the used approaches in human studies and highlight under investigated questions.

Deconvolution of Human Brain Cell Type Transcriptomes Unraveled Microglia-Specific Potential Biomarkers.

Microglial cells form a context-dependent network of brain immunoeffector cells. Despite their indispensable roles, unresolved questions exist around biomarker discovery relevant to their cellular localization, self-renewing potential, and brain developmental dynamics. To resolve the existent gap in the annotation of candidate biomarkers, we conducted a meta-analysis of brain cells using available high-throughput data sets for deciphering microglia-specific expression profiles. We have identified 3,290 significant genes specific to microglia and further selected the top 20 dysregulated genes on the basis of p-value and log2FC. To this list, we added 7 known microglia-specific markers making the candidate list comprising 27 genes for further downstream analyses. Next, we established a connectome of these potential markers with their putative protein partners, which demonstrated strong associations of upregulated genes like Dedicator of cytokinesis 2 (DOCK2) with early/mature microglial markers such as Sphingosine kinase 1 (SPHK1), CD68, and CD45. To elucidate their respective brain anatomical location, we deconvoluted the BrainSpan Atlas expression data. This analysis showed high expression of the majority of candidate genes in microglia-dense regions (Amygdala, Hippocampus, Striatum) in the postnatal brain. Furthermore, to decipher their localized expression across brain ages, we constructed a developmental dynamics map (DDM) comprising extensive gene expression profiles throughout prenatal to postnatal stages, which resulted in the discovery of novel microglia-specific gene signatures. One of the interesting readout from DDM is that all the microglia-dense regions exhibit dynamic regulation of few genes at 37 post conception week (pcw), the transition period between pre- and postnatal stages. To validate these findings and correlate them as potential biomarkers, we analyzed the expression of corresponding proteins in hESC-derived human microglia precursors. The cultured microglial precursors showed expression of Pentraxin 3 (PTX3) and SPHK1 as well as several known markers like CD68, Allograft inflammatory factor 1 (AIF1/IBA1). In summary, this study has furnished critical insights into microglia dynamics across human brain ages and cataloged potential transcriptomic fingerprints that can be further exploited for designing novel neurotherapeutics.

Cx3cr1-deficiency exacerbates alpha-synuclein-A53T induced neuroinflammation and neurodegeneration in a mouse model of Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder characterized by the degeneration of dopaminergic neurons of the substantia nigra and the accumulation of protein aggregates, called Lewy bodies, where the most abundant is alpha-synuclein (α-SYN). Mutations of the gene that codes for α-SYN (SNCA), such as the A53T mutation, and duplications of the gene generate cases of PD with autosomal dominant inheritance. As a result of the association of inflammation with the neurodegeneration of PD, we analyzed whether overexpression of wild-type α-SYN (α-SYNWT ) or mutated α-SYN (α-SYNA53T ) are involved in the neuronal dopaminergic loss and inflammation process, along with the role of the chemokine fractalkine (CX3CL1) and its receptor (CX3CR1). We generated in vivo murine models overexpressing human α-SYNWT or α-SYNA53T in wild type (Cx3cr1+/+ ) or deficient (Cx3cr1-/- ) mice for CX3CR1 using unilateral intracerebral injection of adeno-associated viral vectors. No changes in CX3CL1 levels were observed by immunofluorescence or analysis by qRT-PCR in this model. Interestingly, the expression α-SYNWT induced dopaminergic neuronal death to a similar degree in both genotypes. However, the expression of α-SYNA53T produced an exacerbated neurodegeneration, enhanced in the Cx3cr1-/- mice. This neurodegeneration was accompanied by an increase in neuroinflammation and microgliosis as well as the production of pro-inflammatory markers, which were exacerbated in Cx3cr1-/- mice overexpressing α-SYNA53T . Furthermore, we observed that in primary microglia CX3CR1 was a critical factor in the modulation of microglial dynamics in response to α-SYNWT or α-SYNA53T . Altogether, our study reveals that CX3CR1 plays an essential role in neuroinflammation induced by α-SYNA53T .

Comparative dynamics of microglial and glioma cell motility at the infiltrative margin of brain tumours.

Microglia are a major cellular component of gliomas, and abundant in the centre of the tumour and at the infiltrative margins. While glioma is a notoriously infiltrative disease, the dynamics of microglia and glioma migratory patterns have not been well characterized. To investigate the migratory behaviour of microglia and glioma cells at the infiltrative edge, we performed two-colour time-lapse fluorescence microscopy of brain slices generated from a platelet-derived growth factor-B (PDGFB)-driven rat model of glioma, in which glioma cells and microglia were each labelled with one of two different fluorescent markers. We used mathematical techniques to analyse glioma cells and microglia motility with both single cell tracking and particle image velocimetry (PIV). Our results show microglia motility is strongly correlated with the presence of glioma, while the correlation of the speeds of glioma cells and microglia was variable and weak. Additionally, we showed that microglia and glioma cells exhibit different types of diffusive migratory behaviour. Microglia movement fit a simple random walk, while glioma cell movement fits a super diffusion pattern. These results show that glioma cells stimulate microglia motility at the infiltrative margins, creating a correlation between the spatial distribution of glioma cells and the pattern of microglia motility.

Abstract TP110: Temporally Characteristic Phenotypes of Microglia and Blood-Derived Macrophage on Neuroinflammatory Responses in Cerebral Ischemia Model Using CX3CR1::EGFP Transgenic Mice

Ischemic stroke is a devastating disease, second only to cardiac ischemia as a cause of death worldwide. As the blood supply is insufficient to the brain tissue followed by occlusion of the cerebral artery, molecular cues generated by cerebral ischemia activate the components of innate immunity, promote inflammatory signaling and contribute to tissue damage. Microglia known as an immune cell in the central nervous system (CNS) has functions similar to those of macrophages in the periphery. At the functional point of view, resident microglia (M0) can be polarized by molecular cues: M1 phase microglia and M2 phase microglia. Moreover, blood-brain barrier (BBB) causes the extravasation of blood-derived macrophage. However, the interaction between the microglia and blood-derived macrophage is still unknown. Methods: we used CX3CR1::EGFP transgenic mice to visualize the microglia and blood-derived monocytes dynamics on neuroinflammatory responses. Also, we used the two-photon microscope technique on neuroinflammatory responses by transient middle cerebral artery occlusion (tMCAO) intravial system, time-dependently. Results: Our preliminary data showed that it is morphologically possible to distinguish activated microglia and infiltrated monocytes from the resident microglia specifically. In addition, infiltrated blood-derived monocytes and resident microglia showed different polarization patterns by specific markers (Iba1, CD86, CD206, and CD45) in time-dependent manners (n=3 / 6h, 1, 3, 5, 7 days) after tMCAO. Furthermore, we performed the cytokine/chemokine array to evaluate the level of specific cytokine/chemokine on ischemic stroke using the serum and brain tissue. In results, the levels of serum and brain tissue of specific cytokine/chemokine showed different levels in all experimental time courses. Conclusion: Taken together, the overall data demonstrated that dynamics of microglia and blood-derived macrophage using specific type of transgenic mice could be a novel strategy for regulating the M2 anti-inflammatory microglia phenotype rather than M1 pro-inflammatory microglia and finding the optimal time point of drug treatment attenuating ischemic stroke.

Microglial dynamics during brain development.

Microglial dynamics during brain development.

The THIK and Thin of Microglia Dynamics

Madry etal. (2018) show that the two-pore potassium channel THIK-1 is tonically active in microglia and promotes microglial ramification and surveillance of the brain parenchyma. Interestingly, THIK-1 is not essential to damage-induced outgrowth of microglial processes but is critical to microglial IL-1β release.

Microglial dynamics after axotomy-induced retinal ganglion cell death

BackgroundMicroglial cells (MCs) are the sentries of the central nervous system. In health, they are known as surveying MCs because they examine the tissue to maintain the homeostasis. In disease, they activate and, among other functions, become phagocytic to clean the cellular debris. In this work, we have studied the behavior of rat retinal MCs in two models of unilateral complete intraorbital optic nerve axotomy which elicit a different time course of retinal ganglion cell (RGC) loss.MethodsAlbino Sprague-Dawley rats were divided into these groups: (a) intact (no surgery), (b) fluorogold (FG) tracing from the superior colliculi, and (c) FG tracing + crush or transection of the left optic nerve. The retinas were dissected from 2 days to 2 months after the lesions (n = 4–12 group/lesion and time point) and then were subjected to Brn3a and Iba1 double immunodetection. In each intact retina, the total number of Brn3a+RGCs and Iba+MCs was quantified. In each traced retina (b and c groups), FG-traced RGCs and phagocytic microglial cells (PMCs, FG+Iba+) were also quantified. Topographical distribution was assessed by neighbor maps.ResultsIn intact retinas, surveying MCs are homogenously distributed in the ganglion cell layer and the inner plexiform layer. Independently of the axotomy model, RGC death occurs in two phases, one quick and one protracted, and there is a lineal and topographical correlation between the appearance of PMCs and the loss of traced RGCs. Furthermore, the clearance of FG+RGCs by PMCs occurs 3 days after the actual loss of Brn3a expression that marks RGC death. In addition, almost 50% of MCs from the inner plexiform layer migrate to the ganglion cell layer during the quick phase of RGC loss, returning to the inner plexiform layer during the slow degeneration phase. Finally, in contrast to what happens in mice, in rats, there is no microglial phagocytosis in the contralateral uninjured retina.ConclusionsAxotomy-induced RGC death occurs earlier than RGC clearance and there is an inverse correlation between RGC loss and PMC appearance, both numerically and topographically, suggesting that phagocytosis occurs as a direct response to RGC death rather than to axonal damage.

Developmental alcohol exposure impairs synaptic plasticity without overtly altering microglial function in mouse visual cortex

Fetal alcohol spectrum disorder (FASD), caused by gestational ethanol (EtOH) exposure, is one of the most common causes of non-heritable and life-long mental disability worldwide, with no standard treatment or therapy available. While EtOH exposure can alter the function of both neurons and glia, it is still unclear how EtOH influences brain development to cause deficits in sensory and cognitive processing later in life. Microglia play an important role in shaping synaptic function and plasticity during neural circuit development and have been shown to mount an acute immunological response to EtOH exposure in certain brain regions. Therefore, we hypothesized that microglial roles in the healthy brain could be permanently altered by early EtOH exposure leading to deficits in experience-dependent plasticity. We used a mouse model of human third trimester high binge EtOH exposure, administering EtOH twice daily by subcutaneous injections from postnatal day 4 through postnatal day 9 (P4-:P9). Using a monocular deprivation model to assess ocular dominance plasticity, we found an EtOH-induced deficit in this type of visually driven experience-dependent plasticity. However, using a combination of immunohistochemistry, confocal microscopy, and in vivo two-photon microscopy to assay microglial morphology and dynamics, as well as fluorescence activated cell sorting (FACS) and RNA-seq to examine the microglial transcriptome, we found no evidence of microglial dysfunction in early adolescence. We also found no evidence of microglial activation in visual cortex acutely after early ethanol exposure, possibly because we also did not observe EtOH-induced neuronal cell death in this brain region. We conclude that early EtOH exposure caused a deficit in experience-dependent synaptic plasticity in the visual cortex that was independent of changes in microglial phenotype or function. This demonstrates that neural plasticity can remain impaired by developmental ethanol exposure even in a brain region where microglia do not acutely assume nor maintain an activated phenotype.

The microglial fractalkine receptor is not required for activity-dependent plasticity in the mouse visual system.

Microglia have recently been implicated as key regulators of activity-dependent plasticity, where they contribute to the removal of inappropriate or excess synapses. However, the molecular mechanisms that mediate this microglial function are still not well understood. Although multiple studies have implicated fractalkine signaling as a mediator of microglia-neuron communications during synaptic plasticity, it is unclear whether this is a universal signaling mechanism or whether its role is limited to specific brain regions and stages of the lifespan. Here, we examined whether fractalkine signaling mediates microglial contributions to activity-dependent plasticity in the developing and adolescent visual system. Using genetic ablation of fractalkine's cognate receptor, CX3 CR1, and both ex vivo characterization and in vivo imaging in mice, we examined whether fractalkine signaling is required for microglial dynamics and modulation of synapses, as well as activity-dependent plasticity in the visual system. We did not find a role for fractalkine signaling in mediating microglial properties during visual plasticity. Ablation of CX3 CR1 had no effect on microglial density, distribution, morphology, or motility, in either adolescent or young adult mice across brain regions that include the visual cortex. Ablation of CX3 CR1 also had no effect on baseline synaptic turnover or contact dynamics between microglia and neurons. Finally, we found that fractalkine signaling is not required for either early or late forms of activity-dependent visual system plasticity. These findings suggest that fractalkine is not a universal regulator of synaptic plasticity, but rather has heterogeneous roles in specific brain regions and life stages.

Microglial depletion and activation: A [11C]PBR28 PET study in nonhuman primates

BackgroundThe 18-kDa translocator protein (TSPO) is an important target for assessing neuroimmune function in brain with positron-emission tomography (PET) imaging. The goal of this work was to assess two [11C]PBR28 imaging paradigms for measuring dynamic microglia changes in Macaca mulatta.MethodsDynamic [11C]PBR28 PET imaging data with arterial blood sampling were acquired to quantify TSPO levels as [11C]PBR28 VT. Scans were acquired at three timepoints: baseline, immediately post-drug, and prolonged post-drug.ResultsIn one animal, a colony-stimulating factor 1 receptor kinase inhibitor, previously shown to deplete brain microglia, reduced [11C]PBR28 VT in brain by 46 ± 3% from baseline, which recovered after 12 days to 7 ± 5% from baseline. In a different animal, acute lipopolysaccharide administration, shown to activate brain microglia, increased [11C]PBR28 VT in brain by 39 ± 9% from baseline, which recovered after 14 days to −11 ± 3% from baseline.ConclusionsThese studies provide preliminary evidence of complementary paradigms to assess microglia dynamics via in vivo TSPO imaging.

Microfetti: a fate-mapping system for microglia

A multicolor reporter mouse model enables monitoring of the fate and dynamics of microglia in health and disease.

A new fate mapping system reveals context-dependent random or clonal expansion of microglia

Microglia constitute a highly specialized network of tissue-resident immune cells that is important for the control of tissue homeostasis and the resolution of diseases of the CNS. Little is known about how their spatial distribution is established and maintained in vivo. Here we establish a new multicolor fluorescence fate mapping system to monitor microglial dynamics during steady state and disease. Our findings suggest that microglia establish a dense network with regional differences, and the high regional turnover rates found challenge the universal concept of microglial longevity. Microglial self-renewal under steady state conditions constitutes a stochastic process. During pathology this randomness shifts to selected clonal microglial expansion. In the resolution phase, excess disease-associated microglia are removed by a dual mechanism of cell egress and apoptosis to re-establish the stable microglial network. This study unravels the dynamic yet discrete self-organization of mature microglia in the healthy and diseased CNS.

Direct comparison of microglial dynamics and inflammatory profile in photothrombotic and arterial occlusion evoked stroke

Many focal cerebral ischemia models utilize the middle cerebral artery occlusion (MCAO) evoked by coagulation to induce ischemic damage in the cortex and mimic the pathology observed in human patients. A second, increasingly popular model, the photothrombotic stroke, uses a laser beam to irradiate the MCA after administration of a photosensitizing dye. This widely used procedure is slowly replacing the MCAO model because of the easiness of the surgical protocol and the reproducibility of the damage. However, the photochemical reaction also results in wider microvascular injury. In this study, we have evaluated the impact of these two types of stroke in the cell survival and evolution of stroke, focusing on microglial cells, the first responders to cell injury. Two groups of heterozygote Cx3CR1-GFP reporter mice (to follow microglia) were subject to stroke injury either with coagulator-mediated occlusion or photothrombotic MCA damage. Microglial cells’ dynamics of activation and phagocytosis together with astrocytic response and leukocyte infiltration were characterized at 1, 3 and 7days after damage. Photothrombotic stroke delayed microglial and astrocytic invasion of the ischemic core and accumulation of phagocytic microglia. It also elicited higher levels of inflammatory cytokines/chemokines and increased infiltration from the periphery. In addition, only the neurons in the MCAO stroke showed phenotype plasticity by downregulating the transcription factor NeuN. These data provide a better understanding of the exact temporal and spatial dynamics of the inflammatory response in these two animal models of stroke and identify more relevant targets for human therapy.

Neurogenesis-Promoting Natural Product α-Asarone Modulates Morphological Dynamics of Activated Microglia.

α-Asarone is an active constituent of Acori Tatarinowii, one of the widely used traditional Chinese Medicine to treat cognitive defect, and recently is shown to promote neurogenesis. Here, we demonstrated that low level (3 μM) of α-asarone attenuated LPS-induced BV2 cell bipolar elongated morphological change, with no significant effect on the LPS-induced pro-inflammatory cytokine expressions. In addition, time-lapse analysis also revealed that α-asarone modulated LPS-induced BV2 morphological dynamics. Consistently a significant reduction in the LPS-induced Monocyte Chemoattractant Protein (MCP-1) mRNA and protein levels was also detected along with the morphological change. Mechanistic study showed that the attenuation effect to the LPS-resulted morphological modulation was also detected in the presence of MCP-1 antibodies or a CCR2 antagonist. This result has also been confirmed in primary cultured microglia. The in vivo investigation provided further evidence that α-asarone reduced the proportion of activated microglia, and reduced microglial tip number and maintained the velocity. Our study thus reveals α-asarone effectively modulates microglial morphological dynamics, and implies this effect of α-asarone may functionally relate to its influence on neurogenesis.

Neuroadhesive L1 coating attenuates acute microglial attachment to neural electrodes as revealed by live two-photon microscopy.

Implantable neural electrode technologies for chronic neural recordings can restore functional control to paralysis and limb loss victims through brain-machine interfaces. These probes, however, have high failure rates partly due to the biological responses to the probe which generate an inflammatory scar and subsequent neuronal cell death. L1 is a neuronal specific cell adhesion molecule and has been shown to minimize glial scar formation and promote electrode-neuron integration when covalently attached to the surface of neural probes. In this work, the acute microglial response to L1-coated neural probes was evaluated invivo by implanting coated devices into the cortex of mice with fluorescently labeled microglia, and tracking microglial dynamics with multi-photon microscopy for the ensuing 6h in order to understand L1's cellular mechanisms of action. Microglia became activated immediately after implantation, extending processes towards both L1-coated and uncoated control probes at similar velocities. After the processes made contact with the probes, microglial processes expanded to cover 47.7% of the control probes' surfaces. For L1-coated probes, however, there was a statistically significant 83% reduction in microglial surface coverage. This effect was sustained through the experiment. At 6h post-implant, the radius of microglia activation was reduced for the L1 probes by 20%, shifting from 130.0 to 103.5μm with the coating. Microglia as far as 270μm from the implant site displayed significantly lower morphological characteristics of activation for the L1 group. These results suggest that the L1 surface treatment works in an acute setting by microglial mediated mechanisms.

Repurposing the NRF2 Activator Dimethyl Fumarate as Therapy Against Synucleinopathy in Parkinson's Disease.

This preclinical study was aimed at determining whether pharmacological targeting of transcription factor NRF2, a master controller of many homeostatic genes, might provide a disease-modifying therapy in the animal model of Parkinson's disease (PD) that best reproduces the main hallmark of this pathology, that is, α-synucleinopathy, and associated events, including nigral dopaminergic cell death, oxidative stress, and neuroinflammation. Pharmacological activation of NRF2 was achieved at the basal ganglia by repurposing dimethyl fumarate (DMF), a drug already in use for the treatment of multiple sclerosis. Daily oral gavage of DMF protected nigral dopaminergic neurons against α-SYN toxicity and decreased astrocytosis and microgliosis after 1, 3, and 8 weeks from stereotaxic delivery to the ventral midbrain of recombinant adeno-associated viral vector expressing human α-synuclein. This protective effect was not observed in Nrf2-knockout mice. In vitro studies indicated that this neuroprotective effect was correlated with altered regulation of autophagy markers SQTSM1/p62 and LC3 in MN9D, BV2, and IMA 2.1 and with a shift in microglial dynamics toward a less pro-inflammatory and a more wound-healing phenotype. In postmortem samples of PD patients, the cytoprotective proteins associated with NRF2 expression, NQO1 and p62, were partly sequestered in Lewy bodies, suggesting impaired neuroprotective capacity of the NRF2 signature. These experiments provide a compelling rationale for targeting NRF2 with DMF as a therapeutic strategy to reinforce endogenous brain defense mechanisms against PD-associated synucleinopathy. DMF is ready for clinical validation in PD. Antioxid. Redox Signal. 25, 61-77.

Immunoimaging with Light Sheet Microscopy: Microglial Dynamics in the Developing Zebrafish Brain

The immune system is integral to the natural development and homeostasis of the brain, and in its response to injury and disease. These processes are highly sensitive to cell position and dynamics, and live imaging is important for quantification of these aspects of the immune response. The zebrafish is well established as a model organism for developmental biology due to its relative transparency and ease of study by microscopy. Recent zebrafish transgenic lines are extending its use to the study of the innate immune system. However, the immune system is characterized in part by the fast, organism-scale motility of its cells, presenting challenges for proper observation of its roles, both in the development of the organism and in immune responses. We apply 1- and 2-photon light sheet microscopy, or selective plane illumination microscopy (SPIM), to achieve rapid, large volume imaging of the developing zebrafish brain and the dynamics of microglia - the main innate immune cell of the brain. The high-speed, low-photodamage imaging by SPIM enables the direct tracking of microglia throughout the entire zebrafish brain in early development. Further, the high spatio-temporal resolution allows observation of the microglial processes during development, as well as of the active state in response to pathological stimuli. In capturing these dynamics, across the full organ, we begin to gain a more complete understanding of neuroimmunology not available with other approaches. This work lays the foundation for the measurement of neuroimmune dynamics and their relationship to developing brain circuitry and vasculature as well as activity and function.

Microglial polarization dynamics in dorsal spinal cord in the early stages following chronic sciatic nerve damage

Peripheral nerve injury can lead to activation of spinal microglia, which can mediate neuroinflammation and contribute to neuropathic pain following nerve injury. Activated microglia may manifest with either pro-inflammatory M1 phenotype or anti-inflammatory M2 phenotype, which may lead to detrimental or beneficial roles in the nervous system. In this study, microglia numbers, morphology and gene profiles were examined in the dorsal spinal cord of rats over 14 days following sciatic nerve chronic constriction injury (CCI). The morphology of some microglia changed from a surveying to an activated state within 1 day of CCI. Neuropathic pain developed within seven to 14 days following injury and microglia numbers were increased, with almost all in the dorsal spinal cord morphologically defined as activated. At day one after CCI, both M1 and M2 microglia-related genes were increased but only M1 microglia-related genes remained elevated at day seven and 14 thereafter. These results indicate that both M1 and M2 microglia were activated in the dorsal spinal cord one day after CCI but the microglia skewed towards M1 phenotype during the following seven and 14 days.