A Simplified Method for Isolating Highly Purified Neurons, Oligodendrocytes, Astrocytes, and Microglia from the Same Human Fetal Brain Tissue

Elucidation of the underlying pathogenic mechanisms leading to apoptosis of neurons and oligodendrocytes and activation of microglia and astrocytes in different neurodegenerative and neuroinflammatory disorders remains a challenge in neuroscience. In order to overcome the challenge and find out therapeutic remedies, it is important to study live and death processes in each and every cell type of the brain. Here we present a protocol of isolating highly purified microglia, astrocytes, oligodendrocytes, and neurons, all four major cell types of the CNS, from the same human fetal brain tissue. As found in vivo, these primary neurons and oligodendroglia underwent apoptosis and cell death in response to neurodegenerative challenges. On the other hand, astroglia, and microglia, cells that do not die in neurodegenerative brains, became activated after inflammatory challenge. The availability of highly purified human brain cells will increase the possibility of developing therapies for different neurodegenerative disorders. M. Jana and A. Jana have equal contribution to the work.     

The immunomodulatory sphingosine 1-phosphate analog FTY720 reduces lesion size and improves neurological outcome in a mouse model of cerebral ischemia

Cerebral ischemia is accompanied by fulminant cellular and humoral inflammatory changes in the brain which contribute to lesion development after stroke. A tight interplay between the brain and the peripheral immune system leads to a biphasic immune response to stroke consisting of an early activation of peripheral immune cells with massive production of proinflammatory cytokines followed by a systemic immunosuppression within days of cerebral ischemia that is characterized by massive immune cell loss in spleen and thymus. Recent work has documented the importance of T lymphocytes in the early exacerbation of ischemic injury. The lipid signaling mediator sphingosine 1-phosphate-derived stable analog FTY720 (fingolimod) acts as an immunosuppressant and induces lymphopenia by preventing the egress of lymphocytes, especially T cells, from lymph nodes. We found that treatment with FTY720 (1 mg/kg) reduced lesion size and improved neurological function after experimental stroke in mice, decreased the numbers of infiltrating neutrophils, activated microglia/macrophages in the ischemic lesion and reduced immunohistochemical features of apoptotic cell death in the lesion.     

Mitochondrial contributions to tissue damage in stroke

Tissue infarction, involving death of essentially all cells within a part of the brain, is a common pathology resulting from stroke and an important determinant of the long-term consequences of this disorder. The cell death that leads to infarct formation is likely to be the result of multiple interacting pathological processes. A range of factors, including the severity of the ischemic insult and whether this is permanent or reversed, determine which mechanisms predominate. Although evaluating mitochondrial properties in intact brain is difficult, evidence for several potentially deleterious responses to cerebral ischemia or post-ischemic reperfusion have been obtained from investigations using animal models of stroke. Marked changes in ATP and related energy metabolites develop quickly in response to occlusion of a cerebral artery, as expected from limitations in the delivery of oxygen and glucose. However, these alterations are often only partially reversed on reperfusion despite improved substrate delivery. Ischemia-induced decreases in the mitochondrial capacity for respiratory activity probably contribute to the ongoing impairment of energy metabolism during reperfusion and possibly also to the magnitude of changes seen during ischemia. Conditions during reperfusion are likely to be conducive to the induction of the permeability transition in mitochondria. There are as yet no well-characterized techniques to identify this change in the intact brain. However, the protective effects of some agents that block formation of the transition pore are consistent with both the induction of the permeability transition during early recirculation and a role for this in the development of tissue damage. Release of cytochrome c into the cytoplasm of cells has been observed with both permanent and reversed ischemia and could trigger the death of some cells by apoptosis, a process which probably contributes to the expansion of the ischemic lesion. Mitochondria are also likely to contribute to the widely-accepted role of nitric oxide in the development of ischemic damage. These organelles are a probable target for the deleterious effects of this substance and can also act as a source of superoxide for reaction with the nitric oxide to produce the damaging species, peroxynitrite. Further characterization of these mitochondrial responses should help to elucidate the mechanisms of cell death due to cerebral ischemia and possibly point to novel sites for therapeutic interventions in stroke.     

Specific Imaging of Inflammation with the 18kDa Translocator Protein Ligand DPA-714 in Animal Models of Epilepsy and Stroke

Inflammation is a pathophysiological hallmark of many diseases of the brain. Specific imaging of cells and molecules that contribute to cerebral inflammation is therefore highly desirable, both for research and in clinical application. The 18 kDa translocator protein (TSPO) has been established as a suitable target for the detection of activated microglia/macrophages. A number of novel TSPO ligands have been developed recently. Here, we evaluated the high affinity TSPO ligand DPA-714 as a marker of brain inflammation in two independent animal models. For the first time, the specificity of radiolabeled DPA-714 for activated microglia/macrophages was studied in a rat model of epilepsy (induced using Kainic acid) and in a mouse model of stroke (transient middle cerebral artery occlusion, tMCAO) using high-resolution autoradiography and immunohistochemistry. Additionally, cold-compound blocking experiments were performed and changes in blood-brain barrier (BBB) permeability were determined. Target-to-background ratios of 2 and 3 were achieved in lesioned vs. unaffected brain tissue in the epilepsy and tMCAO models, respectively. In both models, ligand uptake into the lesion corresponded well with the extent of Ox42- or Iba1-immunoreactive activated microglia/macrophages. In the epilepsy model, ligand uptake was almost completely blocked by pre-injection of DPA-714 and FEDAA1106, another high-affinity TSPO ligand. Ligand uptake was independent of the degree of BBB opening and lesion size in the stroke model. We provide further strong evidence that DPA-714 is a specific ligand to image activated microglia/macrophages in experimental models of brain inflammation.     

Assessing Cell Cycle Progression of Neural Stem and Progenitor Cells in the Mouse Developing Brain after Genotoxic Stress

Neurons of the cerebral cortex are generated during brain development from different types of neural stem and progenitor cells (NSPC), which form a pseudostratified epithelium lining the lateral ventricles of the embryonic brain. Genotoxic stresses, such as ionizing radiation, have highly deleterious effects on the developing brain related to the high sensitivity of NSPC. Elucidation of the cellular and molecular mechanisms involved depends on the characterization of the DNA damage response of these particular types of cells, which requires an accurate method to determine NSPC progression through the cell cycle in the damaged tissue. Here is shown a method based on successive intraperitoneal injections of EdU and BrdU in pregnant mice and further detection of these two thymidine analogues in coronal sections of the embryonic brain. EdU and BrdU are both incorporated in DNA of replicating cells during S phase and are detected by two different techniques (azide or a specific antibody, respectively), which facilitate their simultaneous detection. EdU and BrdU staining are then determined for each NSPC nucleus in function of its distance from the ventricular margin in a standard region of the dorsal telencephalon. Thus this dual labeling technique allows distinguishing cells that progressed through the cell cycle from those that have activated a cell cycle checkpoint leading to cell cycle arrest in response to DNA damage.An example of experiment is presented, in which EdU was injected before irradiation and BrdU immediately after and analyzes performed within the 4 hr following irradiation. This protocol provides an accurate analysis of the acute DNA damage response of NSPC in function of the phase of the cell cycle at which they have been irradiated. This method is easily transposable to many other systems in order to determine the impact of a particular treatment on cell cycle progression in living tissues.     

Stereological and Flow Cytometry Characterization of Leukocyte Subpopulations in Models of Transient or Permanent Cerebral Ischemia

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after stroke. These myeloid cell subpopulations can display different phenotypes and functions and need to be distinguished and characterized to study their regulation and contribution to tissue damage. This protocol provides two different methodologies for brain immune cell characterization: a precise stereological approach and a flow cytometric analysis. The stereological approach is based on the optical fractionator method, which calculates the total number of cells in an area of interest (infarcted brain) estimated by a systematic random sampling. The second characterization approach provides a simple way to isolate brain leukocyte suspensions and to characterize them by flow cytometry, allowing for the characterization of microglia, infiltrated monocytes and neutrophils of the ischemic tissue. In addition, it also details a cerebral ischemia model in mice that exclusively affects brain cortex, generating highly reproducible infarcts with a low rate of mortality, and the procedure for histological brain processing to characterize infarct volume by the Cavalieri method.     

Brain plasticity and microglia: is transsynaptic glial activation in the thalamus after limb denervation linked to cortical plasticity and central sensitisation?

Microglia are a subset of tissue-macrophages that are ubiquitously distributed throughout the entire CNS. In health, they remain largely dormant until activated by a pathological stimulus. The availability of more sensitive detection techniques has allowed the early measurement of the cell responses of microglia in areas with few signs of active pathology. Subtle neuronal injury can induce microglial activation in retrograde and anterograde projection areas remote from the primary lesion focus. There is also evidence that in cases of long-standing abnormal neuronal activity, such as in patients after limb amputation with chronic pain and phantom sensations, glial activation may occur transsynaptically in the thalamus. Such neuronally driven glial responses may be related to the emergence central sensitisation in chronic pain states or plasticity phenomena in the cerebral cortex. It is suggested, that such persistent low-level microglial activation is not adequately described by the traditional concept of phagocyte-mediated tissue damage that largely evolved from studies of acute brain lesion models or acute human brain pathology. Due to the presence of signal molecules that can act on neurons and microglia alike, the communication between neurons and microglia is likely to be bi-directional. Persistent subtle microglial activity may modulate basal synaptic transmission and thus neuronal functioning either directly or through the interaction with astrocytes. The activation of microglia leads to the emergence of microstructural as well as functional compartments in which neurokines, interleukins and other signalling molecules introduce a qualitatively different, more open mode of cell-cell communication that is normally absent from the healthy adult brain. This 'neo-compartmentalisation', however, occurs along predictable neuronal pathways within which these glial changes are themselves under the modulatory influence of neurons or other glial cells and are subject to the evolving state of the pathology. Depending on the disease state, yet relatively independent of the specific disease cause, fluctuations in the modulatory influence by non-neuronal cells may form the cellular basis for the variability of brain plasticity phenomena, i.e. the plasticity of plasticity.     

Pinocytosis as a select marker of ramified microglia in vivo and in vitro

Based on previous observations in tissue culture, we investigated pinocytotic activity as a potential cell marker for brain microglia. This functional activity was assessed in three different preparations derived from rat: primary cultures of mixed cerebral cortical cells, tissue slabs of whole cerebrum, and cultures of isolated or enriched microglial cells. Each preparation was incubated with the fluorescent dye lucifer yellow as a soluble tracer and then processed for light microscopy. Under the conditions utilized, ramified microglia specifically exhibited differentially high pinocytotic labeling in all cases; the dye was mainly localized within the cell somata, where it was sequestered in pinocytotic vesicles. In each preparation, the identity of the labeled cell population was confirmed as microglia through immunohistochemical staining with the monoclonal antibody (MAb) OX-42, a specific microglial marker. Therefore, pinocytotic labeling is proposed as a select cell marker for microglia, which may be extremely useful in the identification and study of ramified microglial cells.     

Human NK cells kill resting but not activated microglia via NKG2D- and NKp46-mediated recognition

Microglia are resident macrophage-like APCs of the CNS. To avoid escalation of inflammatory processes and bystander damage within the CNS, microglia-driven inflammatory responses need to be tightly regulated and both spatially and temporally restricted. Following traumatic, infectious, and autoimmune-mediated brain injury, NK cells have been found in the CNS, but the functional significance of NK cell recruitment and their mechanisms of action during brain inflammation are not well understood. In this study, we investigated whether and by which mechanisms human NK cells might edit resting and activated human microglial cells via killing in vitro. IL-2-activated NK cells efficiently killed both resting allogeneic and autologous microglia in a cell-contact-dependent manner. Activated NK cells rapidly formed synapses with human microglial cells in which perforin had been polarized to the cellular interface. Ab-mediated NKG2D and NKp46 blockade completely prevented the killing of human microglia by activated NK cells. Up-regulation of MHC class I surface expression by TLR4 stimulation protected microglia from NK cell-mediated killing, whereas MHC class I blockade enhanced cytotoxic NK cell activity. These data suggest that brain-infiltrating NK cells might restrict innate and adaptive immune responses within the human CNS via elimination of resting microglia.     

Phosphoinositide 3-kinase-gamma expression is upregulated in brain microglia and contributes to ischemia-induced microglial activation in acute experimental stroke

Microglia, the resident microphages of the CNS, are rapidly activated after ischemic stroke. Inhibition of microglial activation may protect the brain by attenuating blood-brain barrier damage and neuronal apoptosis after ischemic stroke. However, the mechanisms by which microglia is activated following cerebral ischemia is not well defined. In this study, we investigated the expression of PI3Kγ in normal and ischemic brains and found that PI3Kγ mRNA and protein are constitutively expressed in normal brain microvessels, but significantly upregulated in postischemic brain primarily in activated microglia following cerebral ischemia. In vitro, the expression of PI3Kγ mRNA and protein was verified in mouse brain endothelial and microglial cell lines. Importantly, absence of PI3Kγ blocked the early microglia activation (at 4 h) and subsequent expansion (at 24-72 h) in PI3Kγ knockout mice. The results suggest that PI3Kγ is an ischemia-responsive gene in brain microglia and contributes to ischemia-induced microglial activation and expansion.     

Hypothesis: are neoplastic macrophages/microglia present in glioblastoma multiforme?

Most malignant brain tumours contain various numbers of cells with characteristics of activated or dysmorphic macrophages/microglia. These cells are generally considered part of the tumour stroma and are often described as TAM (tumour-associated macrophages). These types of cells are thought to either enhance or inhibit brain tumour progression. Recent evidence indicates that neoplastic cells with macrophage characteristics are found in numerous metastatic cancers of non-CNS (central nervous system) origin. Evidence is presented here suggesting that subpopulations of cells within human gliomas, specifically GBM (glioblastoma multiforme), are neoplastic macrophages/microglia. These cells are thought to arise following mitochondrial damage in fusion hybrids between neoplastic stem cells and macrophages/microglia.     

Endogenous neurogenesis following ischaemic brain injury: Insights for therapeutic strategies

Ischaemic stroke is among the most common yet most intractable types of central nervous system (CNS) injury in the adult human population. In the acute stages of disease, neurons in the ischaemic lesion rapidly die and other neuronal populations in the ischaemic penumbra are vulnerable to secondary injury. Multiple parallel approaches are being investigated to develop neuroprotective, reparative and regenerative strategies for the treatment of stroke. Accumulating evidence indicates that cerebral ischaemia initiates an endogenous regenerative response within the adult brain that potentiates adult neurogenesis from populations of neural stem and progenitor cells. A major research focus has been to understand the cellular and molecular mechanisms that underlie the potentiation of adult neurogenesis and to appreciate how interventions designed to modulate these processes could enhance neural regeneration in the post-ischaemic brain. In this review, we highlight recent advances over the last 5 years that help unravel the cellular and molecular mechanisms that potentiate endogenous neurogenesis following cerebral ischaemia and are dissecting the functional importance of this regenerative mechanism following brain injury.This article is part of a Directed Issue entitled: Regenerative Medicine: the challenge of translation.     

The type 1 interleukin‐1 receptor is essential for multiple aspects of brain inflammation

Interleukin‐1 (IL‐1) is induced immediately after brain imjury and elevated levels of IL‐1 have been strongly implicated in the neurodegeneration that accompanies stroke, Alzheimer's disease and Multiple Sclerosis. Antagonizing IL‐1 reduces cell death; however, the basis for this protection has not been elucidated. Here we analyzed the response to penetrating brain injury in mice lacking the type 1 interleukin receptor (IL‐1R1) to determine which cellular and molecular mediators of tissue damage require IL‐1 signaling. At the cellular level fewer amoeboid microglia/macrophages appeared adjacent to the injured brain tissue in IL‐1R1 null mice, and those microglia present at early postinjury intervals retained their resting morphology. Astrogliosis also was mildly abrogated. At the molecular level, cyclooxygenase 2 and IL‐6 expression were depressed and delayed. Interestingly, basal levels of cyclooxygenase 2, IL‐1 and IL‐6 were significantly lower in the IL‐1R1 null mice. Additionally, stimulation of VCAM‐1 mRNA was depressed in the IL‐1R1 null mice, and correspondingly, there was reduced migration of peripheral macrophages into the IL‐1R1 null brain after injury. This observation correlated with a reduced number of cyclooxygenase 2+ amoeboid phagocytes adjacent to the injury. By contrast, the production of nerve growth factor was only mildly affected. Since antagonizing IL‐1 protects neural cells in experimental models of stroke and multiple sclerosis, our data suggest that cell preservation is achieved by abrogating microglial/macrophage activation and the subsequent self‐propagating cycle of inflammation. Acknowledgements: Supported by NMSS Award #RG 3837.     

Extracellular vesicles from hypoxia-preconditioned microglia promote angiogenesis and repress apoptosis in stroke mice via the TGF-β/Smad2/3 pathway

Systemic transplantation of oxygen−glucose deprivation (OGD)-preconditioned primary microglia enhances neurological recovery in rodent stroke models, albeit the underlying mechanisms have not been sufficiently addressed. Herein, we analyzed whether or not extracellular vesicles (EVs) derived from such microglia are the biological mediators of these observations and which signaling pathways are involved in the process. Exposing bEnd.3 endothelial cells (ECs) and primary cortical neurons to OGD, the impact of EVs from OGD-preconditioned microglia on angiogenesis and neuronal apoptosis by the tube formation assay and TUNEL staining was assessed. Under these conditions, EV treatment stimulated both angiogenesis and tube formation in ECs and repressed neuronal cell injury. Characterizing microglia EVs by means of Western blot analysis and other techniques revealed these EVs to be rich in TGF-β1. The latter turned out to be a key compound for the therapeutic potential of microglia EVs, affecting the Smad2/3 pathway in both ECs and neurons. EV infusion in stroke mice confirmed the aforementioned in vitro results, demonstrating an activation of the TGF-β/Smad2/3 signaling pathway within the ischemic brain. Furthermore, enriched TGF-β1 in EVs secreted from OGD-preconditioned microglia stimulated M2 polarization of residing microglia within the ischemic cerebral environment, which may contribute to a regulation of an early inflammatory response in postischemic hemispheres. These observations are not only interesting from the mechanistic point of view but have an immediate therapeutic implication as well, since stroke mice treated with such EVs displayed a better functional recovery in the behavioral test analyses. Hence, the present findings suggest a new way of action of EVs derived from OGD-preconditioned microglia by regulating the TGF-β/Smad2/3 pathway in order to promote tissue regeneration and neurological recovery in stroke mice.Subject terms: Cellular neuroscience, Neuroimmunology     

Regeneration of the central nervous system-principles from brain regeneration in adult zebrafish

Poor recovery of neuronal functions is one of the most common healthcare challenges for patients with different types of brain injuries and/or neurodegenerative diseases. Therapeutic interventions face two major challenges: (1) How to generate neurons de novo to replenish the neuronal loss caused by injuries or neurodegeneration (restorative neurogenesis) and (2) How to prevent or limit the secondary tissue damage caused by long-term accumulation of glial cells, including microglia, at injury site (glial scar). In contrast to mammals, zebrafish have extensive regenerative capacity in numerous vital organs, including the brain, thus making them a valuable model to improve the existing therapeutic approaches for human brain repair. In response to injuries to the central nervous system (CNS), zebrafish have developed specific mechanisms to promote the recovery of the lost tissue architecture and functionality of the damaged CNS. These mechanisms include the activation of a restorative neurogenic program in a specific set of glial cells (ependymoglia) and the resolution of both the glial scar and inflammation, thus enabling proper neuronal specification and survival. In this review, we discuss the cellular and molecular mechanisms underlying the regenerative ability in the adult zebrafish brain and conclude with the potential applicability of these mechanisms in repair of the mammalian CNS.     

Effect of early decrease in the lesion size on late brain tissue loss, synaptophysin expression and functionality after a focal brain lesion in rats

The purpose of the present study is to determine the effects of early decrease in the lesion size on late brain tissue loss, synaptogenesis and functionality after a focal brain lesion in rats. The lesion was induced either to the cortex using the photothrombotic ischemic stroke or to the striatum using the malonate poisoning model. The cortical and striatal lesions amounted to 66-80 mm(3) at day 1 post-lesion and were reduced by 50% after the acute administration of dipyridyl (a liposoluble iron chelator) and aminoguanidine (an inhibitor of the inducible nitric oxide synthase), respectively. Loss of histologically intact tissue and synaptophysin expression as an indicator of synaptogenesis were examined at day 35 post-lesion. Both types of lesion resulted in synaptophysin upregulation in contralateral and ipsilateral cortical areas. On the contrary, brain tissue loss was greater after the striatal (-17%) than the cortical lesion (-5%). Synaptophysin expression and tissue loss were not different between drug- and vehicle-treated rats. Moreover, a set of standard neurological tests revealed a difference in deficit between the both types of lesion, yet only in the acute post-lesion stage. However, it did not distinguish between vehicle- and drug-treated rats whatever the lesion location. Our results indicate that late histological endpoints measurements are not recommended to probe the potential neuroprotective properties of a drug administered within the acute post-lesion stage. They also suggest that inhibition of cytotoxic mechanisms involved in lesion growth is of no clinical interest when it cannot lead to a long-term histological protection and/or increased synaptogenesis.     

Live imaging of mouse endogenous neural progenitors migrating in response to an induced tumor

Adult neurogenesis is restricted to specific brain regions. Although involved in the continuous supply of interneurons for the olfactory function, the role of neural precursors in brain damage-repair remains an open question. Aiming to in vivo identify endogenous neural precursor cells migrating towards a brain damage site, the monoclonal antibody Nilo2 recognizing cell surface antigens on neuroblasts, was coupled to magnetic glyconanoparticles (mGNPs). The Nilo2-mGNP complexes allowed, by magnetic resonance imaging in living animals, the in vivo identification of endogenous neural precursors at their niche, as well as their migration to a lesion site (induced brain tumor), which was fast (within hours) and orderly. Interestingly, the rapid migration of neuroblasts towards a damage site is a characteristic that might be exploited to precisely localize early damage events in neurodegenerative diseases. In addition, it might facilitate the study of regenerative mechanisms through the activation of endogenous neural cell precursors. A similar approach, combining magnetic glyconanoparticles linked to appropriate antibodies could be applied to flag other small cell subpopulations within the organism, track their migration, localize stem cell niches, cancer stem cells or even track metastatic cells.     

Microglial motility in the rat facial nucleus following peripheral axotomy

Microglial motility was studied in living mammalian brain tissue using infrared gradient contrast microscopy in combination with video contrast enhancement and time lapse video recording. The infrared gradient contrast allows the visualization of living cells up to a depth of 60 μm in brain slices, in regions where cell bodies remain largely uninjured by the tissue preparation and are visible in their natural environment. In contrast to other techniques, including confocal microscopy, this procedure does not require any staining or labeling of cell membranes and thus guarantees the investigation of tissue which has not been altered, apart from during preparation. Microglial cells are activated and increase in number in the facial nucleus following peripheral axotomy. Thus we established the preparation of longitudinal rat brainstem slices containing the axotomized facial nucleus as a source of activated microglial cells. During prolonged video time lapse recordings, two different types of microglial cell motility could be observed. Microglial cells which had accumulated at the surface of the slice remained stationary but showed activity of the cell soma, developing pseudopods of different shape and size which undulated and which were used for phagocytosis of cell debris. Microglial phagocytosis of bacteria could be documented for the first time in situ. In contrast, ameboid microglia which did not display pseudopods but showed migratory capacity, could be observed exclusively in the depth of the tissue. Some of these cells maintained a close contact to neurons and appeared to move along their dendrites, a finding that may be relevant to the role of microglia in “synaptic stripping”, the displacement of synapses following axotomy. This approach provides a valuable opportunity to investigate the interactions between activated microglial cells and the surrounding cellular and extracellular structures in the absence of staining or labeling, thus opening a wide field for the analysis of the cellular mechanisms involved in numerous pathologies of the CNS.     

Intracerebral hemorrhages and syncytium formation induced by endothelial cell infection with a murine leukemia virus.

The mechanisms of endothelial cell damage that lead to cerebral hemorrhage are not completely understood. In this study, a cloned murine retrovirus, TR1.3, that uniformly induced stroke in neonatal BALB/c mice is described. Restriction digest mapping suggests that TR1.3 is part of the Friend murine leukemia virus (FMuLV) family. However, unlike mice exposed to other FMuLVs, mice infected with TR1.3 virus developed tremors and seizures within 8 to 18 days postinoculation. This was uniformly followed by paralysis and death within 1 to 2 days. Postmortem examination of TR1.3-inoculated mice revealed edematous brain tissue with large areas of intracerebral hemorrhage. Histologic analysis revealed prominent small vessel pathology including syncytium formation of endothelial cells. Immunohistochemical analysis of frozen brain sections using double fluorescence staining demonstrated that TR1.3 virus specifically infected small vessel endothelial cells. Although infection of vessel endothelial cells was detected in several organs, only brain endothelial cells displayed viral infection associated with hemorrhage. The primary determinant of TR1.3-induced neuropathogenicity was found to reside within a 3.0-kb fragment containing the 3' end of the pol gene, the env gene, and the U3 region of the long terminal repeat. The restricted tropism and acute pathogenicity of this cloned murine retrovirus provide a model for studying virus-induced stroke and for elucidating the mechanisms involved in syncytium formation by retroviruses in vivo.