An integrated, temporal study of the behavioural, electrophysiological and neuropathological consequences of murine prion disease

We have conducted an integrated study of ME7 prion disease by examining the electrophysiological and neuropathological features of hippocampal slices from behaviourally characterised C57Bl/6J mice 12, 14, 16, 18, 20 and 24 weeks after intracerebral micro-injection of ME7 or normal brain homogenate. We describe the pathogenesis of ME7 as a three-stage process. STAGE ONE: PrPSc deposition, synaptic pathology and abnormal synaptic plasticity. STAGE TWO: Onset of behavioural changes, exemplified by an increase in open-field activity, enhancement of the slow AHP and development of vacuolation. Membrane depolarisation is also an early feature, but its exact timing remains to be confirmed. STAGE THREE: Clinical disease, substantial neurodegeneration and further disruption of the action potential profile. We suggest that the mechanisms underlying the electrophysiological changes of Stages one and two may provide novel approaches to treatment of prion disease, and that those seen in Stage three may be relevant to neurodegenerative diseases more generally.     

Loss of Perineuronal Net in ME7 Prion Disease

Microglial activation and behavioral abnormalities occur before neuronal loss in experimental murine prion disease; the behavioral changes coincide with a reduction in synaptic plasticity. Because synaptic plasticity depends on an intact perineuronal net (PN), a specialized extracellular matrix that surrounds parvalbumin (PV)-positive GABAergic (gamma-aminobutyric acid [GABA]) inhibitory interneurons, we investigated the temporal relationships between microglial activation and loss of PN and PV-positive neurons in ME7 murine prion disease. Anesthetized C57Bl/6J mice received bilateral intracerebral microinjections of ME7-infected or normal brain homogenate into the dorsal hippocampus. Microglial activation, PrP accumulation, the number of PV-positive interneurons, and Wisteria floribunda agglutinin-positive neurons (i.e. those with an intact PN) were assessed in the ventral CA1 and subiculum at 4, 8, 12, 16, and 20 weeks postinjection. Hippocampal areas and total neuron numbers in the ventral CA1 and subiculum were also determined. Loss of PN coincided with early microglial activation and with a reduction in synaptic plasticity. No significant loss of PV-positive interneurons was observed. Our findings suggest that the substrate of the earliest synaptic and behavioral abnormalities in murine prion disease may be inflammatory microglia-mediated degradation of the PN.     

Inflammatory response and retinal ganglion cell degeneration following intraocular injection of ME7

Scrapie is a prion disease which occurs naturally in sheep and which can be transmitted experimentally to rodents. After intracerebral injection of ME7 into mouse, an atypical inflammatory response, characterized by T-lymphocytes and activated microglia is present early in the course of the disease. In the present work, we have investigated the relationship between this inflammatory response, astrocytosis and neuronal loss along the visual pathway after intraocular injection (intraocular) of ME7 in C57BL/6J mice. We have demonstrated that microglia activation and T-lymphocyte recruitment accompanies the spread of prion pathology along the visual pathway and in the early stages of the disease is restricted to the subcortical visual pathway. Inflammation was also present in non-visual areas in association with PrPsc deposition at late stages of the disease, possibily indicating that diffusion of the scrapie agent also contributes to the spread of the disease. After intraocular injection of the prion agent, the disease is believed to be transported into the brain via axons of retinal ganglion cells (RGCs). Despite the high levels of infectivity reported to be present in the retina early in the disease after intraocular injection of ME7, retinal pathology has not been extensively investigated. We have studied the RGCs response in whole mount retinas after intraocular injection of ME7. We have shown that RGCs degenerate after intraocular injection of ME7 whereas amacrine cells, retinal interneurones, are more resistant. Our results suggest that two distinct population of neurones, exposed in vivo at the same time to the same agent scrapie strain, show different susceptibility to the toxic effects of PrPsc.     

Synaptic changes characterize early behavioural signs in the ME7 model of murine prion disease

Prion diseases are fatal, chronic neurodegenerative diseases of mammals, characterized by amyloid deposition, astrogliosis, microglial activation, tissue vacuolation and neuronal loss. In the ME7 model of prion disease in the C57BL/6 J mouse, we have shown previously that these animals display behavioural changes that indicate the onset of neuronal dysfunction. The current study examines the neuropathological correlates of these early behavioural changes. After injection of ME7-infected homogenate into the dorsal hippocampus, we found statistically significant impairment of burrowing, nesting and glucose consumption, and increased open field activity at 13 weeks. At this time, microglia activation and PrPSc deposition was visible selectively throughout the limbic system, including the hippocampus, entorhinal cortex, medial and lateral septum, mamillary bodies, dorsal thalamus and, to a lesser degree, in regions of the brainstem. No increase in apoptosis or neuronal cell loss was detectable at this time, while in animals at 19 weeks postinjection there was 40% neuronal loss from CA1. There was a statistically significant reduction in synaptophysin staining in the stratum radiatum of the CA1 at 13 weeks indicating loss of presynaptic terminals. Damage to the dorsal hippocampus is known to disrupt burrowing and nesting behaviour. We have demonstrated a neuropathological correlate of an early behavioural deficit in prion disease and suggest that this should allow insights into the first steps of the neuropathogenesis of prion diseases.     

Optical imaging detects apoptosis in the brain and peripheral organs of prion-infected mice

Activation of the caspase family of cysteine proteases is proposed to be an important cell death mechanism in transmissible spongiform encephalopathies or prion diseases. We determined the extent of caspase activation in the brain and peripheral organs of mice that showed clinical signs after intracerebral inoculation with mouse-adapted prions by in vivo administration of a red fluorescent pan-caspase inhibitor, sulforhodamine B-Val-Ala-Asp(OMe)-fluoromethylketone. Fluorescence reflectance imaging identified a significant increase in active caspases in brains of prion-infected, but not uninfected, mice that correlated with increases in procaspase-3 and cleaved caspase-3, a central effector caspase, assessed by Western immunoblot analysis. Fluorescence was found in brain regions in which neuronal loss occurs; immunohistochemical analysis indicated that fluorescence was localized within and adjacent to deposits of abnormal disease-associated conformers of the prion protein (PrP Sc). Fluorescence was also significantly increased in the kidney, lung, and ileum of prion-infected mice. This premortem labeling of caspase activation in the brain, and importantly in peripheral organs, could be exploited as a biomarker for longitudinal monitoring of prion disease progression and the impact of therapy in vivo in addition to, or independently of, PrP and spongiform changes.     

MCP-1 and murine prion disease: separation of early behavioural dysfunction from overt clinical disease

Prion diseases are chronic, fatal neurodegenerative conditions of the CNS. We have investigated the role of monocyte chemoattractant protein-1 (MCP-1) in the ME7 model of murine prion disease. MCP-1 expression increased in the CNS throughout disease progression and was positively correlated with microglial activation. We subsequently compared the inflammatory response, pathology and behavioural changes in wild-type (wt) mice and MCP-1 knockout mice (MCP-1-/-) inoculated with ME7. Late-stage clinical signs were delayed by 4 weeks in MCP-1-/- mice, and survival time increased by 2-3 weeks. By contrast, early changes in affective behaviours and locomotor activity were not delayed in onset. There was also no difference in microglial activation or neuronal death in the hippocampus and thalamus of wt mice and MCP-1-/- mice. These results highlight an important dissociation between prolonged survival, early behavioural dysfunction and hippocampal/thalamic pathology when considering therapeutic intervention for human prion diseases and other chronic neurodegenerative conditions.     

Aberrant Alterations of Mitochondrial Factors Drp1 and Opa1 in the Brains of Scrapie Experiment Rodents

The abnormal mitochondrial dynamics has been reported in the brains of some neurodegenerative diseases, such as Alzheimer’s disease (AD) and Parkinson’s disease (PD), but limitedly described in prion disease. Dynamin-related protein 1 (Drpl) and optic atrophy protein 1 (Opa1) are two essential elements for mitochondria fission and fusion. To evaluate possible changes of mitochondria dynamics during prion infection, the situations of brain Drp1 and Opa1 of scrapie strains 139A, ME7, and S15 mice, as well as 263K-infected hamsters, were analyzed. Significant decreases of brain Drp1 were observed in scrapie-infected rodents at terminal stage by Western blots and immunohistochemical assays, while the levels of Opa1 also showed declined tendency in the brains of scrapie-infected rodents. Immunofluorescent assays illustrated well localization of Drp1 or Opa1 within NeuN-positive cells. Moreover, the S-nitrosylated forms of Drp1significantly increased in the brain tissues of 139A- and ME7-infected mice at terminal stage. Dynamic analysis of Drp1 and SNO-Dpr1 in the brains collected at different time points within the incubation period of 139A-infected mice demonstrated that the whole Drp1 decreased at all tested samples, whereas the SNO-Drp1 remarkably increased in the sample of 90-day post-infection (dpi), reached to the peak in that of 120 dpi and dropped down but still maintained at higher level at the end of disease. The levels of apoptotic factors cleaved caspase 9, caspase 3, and Bax were also markedly increased in the brain tissues of the mice infected with agents 139A and ME7. Our data indicate a disorder of mitochondria dynamics in the brains of prion infection, largely depending on the abnormal alteration of brain Drp1.     

Changes in the localization of brain prion proteins during scrapie infection

Prion proteins (PrP) were localized in the brains of normal and scrapie-infected hamsters by immunohistochemistry and Western blotting. PrP monoclonal antibodies and monospecific anti-PrP peptide sera, which react with both the cellular (PrPC) and scrapie (PrPSc) isoforms of the prion protein, were used to locate PrP in tissue sections. In normal hamsters, PrPC was located primarily in nerve cell bodies throughout the CNS; whereas, in the terminal stages of scrapie, PrP immunoreactivity was shifted to the neuropil and was absent from most nerve cell bodies. Prion proteins were not uniformly dispersed throughout the gray matter of scrapie-infected hamster brains; rather, they were concentrated in those regions that exhibited spongiform degeneration and reactive astrogliosis. Since earlier studies showed that the level of PrPC remains constant during scrapie infection as measured in whole brain homogenates and no antibodies are presently available that can distinguish PrPC from PrPSc, we analyzed individual brain regions by Western blotting. Analysis of proteinase K-digested homogenates of dissected brain regions showed that most of the regional changes in PrP immunoreactivity that are seen during scrapie infection are due to the accumulation of PrPSc. These observations indicate that the tissue pathology of scrapie can be directly correlated with the accumulation of PrPSc in the neuropil, and they suggest that the synthesis and distribution of the prion protein has a central role in the pathogenesis of this disorder.     

Absence of detectable IL-1beta production in murine prion disease: a model of chronic neurodegeneration

Murine prion disease is accompanied by a modified inflammatory response characterized by early but prolonged microglial activation and T-lymphocyte recruitment. In this model, we look at the profile of cytokine production, particularly IL-1beta. Mice inoculated with prion-infected brain homogenate show typical signs of prion disease. We were unable to detect any IL-1beta using immunohistochemistry, with various fixation protocols, or ELISA between 8 and 24 wk post-inoculation. Also, there was no increase in mRNA for IL-1beta, IL-6, IFNgamma, and iNOS as measured by quantitative RT-PCR. Using the same procedures and examining tissues at the same time, IL-1beta immunostaining was detected in infiltrating inflammatory cells in mouse brains injected with LPS or in a delayed-type hypersensitivity response in the brain. Soluble IL-1beta was also increased, as measured by ELISA, and there was an increase in mRNA species for IL-1beta, IL-6, TNFalpha but not IFNgamma or iNOS in these brains. These data reveal that chronic neurodegeneration seen in prion disease does not induce production of a range of proinflammatory mediators despite showing marked microglial activation and raise the question as to whether IL-1beta would exacerbate the neurodegeneration as it does in acute neurodegeneration following head injury and stroke.     

Endoplasmic reticulum stress features are prominent in Alzheimer disease but not in prion diseases in vivo

Prion diseases and Alzheimer disease (AD) share a variety of clinical and neuropathologic features (e.g. progressive dementia, accumulation of abnormally folded proteins in diseased tissue, and pronounced neuronal loss) as well as pathogenic mechanisms like generation of oxidative stress molecules and complement activation. Recently, it was suggested that neuronal death in AD may have its origin in the endoplasmic reticulum (ER). Cellular stress conditions can interfere with protein folding and subsequently cause accumulation of unfolded or misfolded proteins in the ER lumen. The ER responds to this by the activation of adaptive pathways, which are termed unfolded protein response (UPR). The UPR transducer PERK, which launches the most immediate response to ER stress (i.e. the transient attenuation of mRNA translation), and the downstream effector of PERK, eIF2alpha, were shown to be activated in AD. We demonstrate that neither in sporadic nor in infectiously acquired or inherited human prion diseases can the activated forms of PERK and eIF2alpha be detected, except when concomitant neurofibrillary pathology is present; whereas the distribution of phosphorylated PERK correlates with abnormally phosphorylated tau in AD. In brains of scrapie-affected mice and mice infected with sporadic or variant Creutzfeldt-Jakob disease, activated PERK is only very faintly expressed. The lack of prominent activation of the PERK-eIF2alpha pathway in prion diseases suggests that, in contrast to AD, ER stress does not play a crucial role in neuronal death in prion disorders.     

Rapid detection of Creutzfeldt-Jakob disease and scrapie prion proteins

Creutzfeldt-Jakob disease (CJD) and Gerstmann-Str?ussler syndrome (GSS) of humans as well as scrapie of animals are caused by prions. The scrapie prion protein isoform (PrPSc) is the only macromolecule identified to date which is a component of the infectious prion particle. PrPSc is converted to PrP 27-30 by limited proteolysis while the cellular isoform, designated PrPC, is completely digested under the same conditions. ELISA studies demonstrated that native PrP 27-30 bound to plastic surfaces resisted proteolysis and exhibited little or no immunoreactivity but after denaturation with guanidinium thiocyanate (GdnSCN), immunoreactivity was greatly enhanced. PrPSc bound to nitrocellulose also exhibited enhanced immunoreactivity after denaturation. PrPSc was readily detected in brain extracts from scrapie-infected hamsters, mice, and sheep by dot-blot immunoassays using limited proteolysis followed by GdnSCN denaturation. The high sensitivity and specificity of the immunoassay allowed detection of regional differences in PrPSc in sheep brain. CJD prion protein isoform (PrPCJD) was also detected in the brains of all 10 patients tested with neuropathologically confirmed CJD and in 1 patient with GSS. Enhanced immunoreactivity of PrPSc or PrPCJD after denaturation cannot only be used for immunodiagnosis of prion diseases but may also form the basis of new assays in experimental studies directed at the chemical structure of the prion particle.     

Vascular endothelial growth factor (VEGF) modulates vascular permeability and inflammation in rat brain.

Vascular endothelial growth factor (VEGF) is an angiogenic growth factor that also induces vascular permeability and macrophage migration. VEGF expression is weak in normal adult brain, but is strongly upregulated in glioma cells and reactive astrocytes, suggesting that chronic overexpression of VEGF in the brain contributes to blood-brain barrier (BBB) breakdown. We examined the effects of chronic VEGF overexposure on the integrity of the BBB using the following approaches: 1) continuous intracerebral infusion of VEGF via miniosmotic pump; and 2) intracerebral injection of an adenoviral vector encoding the VEGF165 gene (AdCMV.VEGF). After 6 days both treatments produced approximately 10-fold breakdown of the BBB (as measured by transport of 14C-aminoisobutyric acid (AIB) from blood into brain) compared with the respective controls (albumin infusion or AdCMV.beta gal virus). BBB disruption in AdCMV.VEGF-treated brains was accompanied by a severe inflammatory response not observed in brains receiving AdCMV.beta gal or VEGF protein infusion, indicating that neither VEGF nor viral particles alone were responsible for the inflammatory response. However, injection of AdCMV.beta gal followed by VEGF infusion to the same site also elicited inflammation. Chronic overexposure of normal brain to VEGF also increased intercellular adhesion molecule-1 (ICAM-1) and major histocompatibility complex (MHC) class I and II expression. Although VEGF itself is not inflammatory, VEGF may modulate immune responses in the central nervous system (CNS) by opening the BBB, altering the immunoprivileged status of the brain, and allowing contact between normally sequestered CNS antigens and blood-borne immune mediators.     

Elevated intracranial IL-18 in humans and mice after traumatic brain injury and evidence of neuroprotective effects of IL-18-binding protein after experimental closed head injury.

Proinflammatory cytokines are important mediators of neuroinflammation after traumatic brain injury. The role of interleukin (IL)-18, a new member of the IL-1 family, in brain trauma has not been reported to date. The authors investigated the posttraumatic release of IL-18 in murine brains following experimental closed head injury (CHI) and in CSF of CHI patients. In the mouse model, intracerebral IL-18 was induced within 24 hours by ether anesthesia and sham operation. Significantly elevated levels of IL-18 were detected at 7 days after CHI and in human CSF up to 10 days after trauma. Published data imply that IL-18 may play a pathophysiological role in inflammatory CNS diseases; therefore its inhibition may ameliorate outcome after CHI. To evaluate the functional aspects of IL-18 in the injured brain, mice were injected systemically with IL-18-binding protein (IL-18BP), a specific inhibitor of IL-18, 1 hour after trauma. IL-18BP-treated mice showed a significantly improved neurological recovery by 7 days, accompanied by attenuated intracerebral IL-18 levels. This demonstrates that inhibition of IL-18 is associated with improved recovery. However, brain edema at 24 hours was not influenced by IL-18BP, suggesting that inflammatory mediators other than IL-18 induce the early detrimental effects of intracerebral inflammation.     

Endotoxin induces a delayed loss of TH-IR neurons in substantia nigra and motor behavioral deficits

We have previously reported that a single injection of endotoxin, lipopolysaccharide (LPS, 5mg/kg, i.p.), causes a delayed and progressive loss of TH-IR neurons in the substantia nigra (SN) in C57BL/six male mice. In this study, we determined sex differences and behavioral deficits accompanying the loss of TH-IR neurons in response to peripheral LPS injection. A single injection of LPS (5mg/kg, i.p.) failed to produce any loss of TH-IR neurons in the SN of female mice over a 12-month period. To determine if multiple-injections were required, female mice received five injections of LPS (5mg/kg, i.p.) at either weekly or monthly intervals. Behavioral motor ability and TH-IR neuronal loss were determined after the first injection of LPS. We found significant differences in both behavioral activities and neuronal loss between these two injection paradigms. Between 7 and 20 months after the first injection of LPS, progressive behavioral changes, measured by rotor-rod and open-field activities, and neuronal loss in SN were observed in monthly injected, but not in weekly injected mice. In addition, reduced rotor-rod ability in monthly injected mice were restored following treatment of l-dopa/carbidopa (30 mg/3mg/kg), i.p.). Approximately 40 and 50% loss of TH-IR neurons at 9 and 20 months, respectively, was observed after exposure to LPS, suggesting that the behavioral deficit is related to loss of dopamine function in the nigra-striatal pathway. More intense immuno-staining of alpha-synuclein and inflammatory markers were detected in brain sections exposed to LPS. In conclusion, these results show that multi-LPS monthly injections can induce a delayed and progressive loss of TH-IR neurons and motor deficits which resemble the progressive nature of Parkinson's disease. Further, the present study reveals a clear sex difference: female mice are more resistant to LPS than male mice. Repeated monthly LPS injections are required to cause both motor behavioral deficits and DA neuronal loss in female mice.     

Neuronal apoptosis in immunodeficient mice infected with the challenge virus standard strain of rabies virus by intracerebral inoculation

The challenge virus standard-11 strain (CVS) of fixed rabies virus produces neuronal apoptosis in widespread areas of the brain of mice after intracerebral inoculation. The role of the adaptive immune response in producing neuronal apoptosis in this model was evaluated by comparing the infections in adult C57BL/6J mice with nude mice (T cell deficient) and Rag1 mice (T and B cell deficient). Both strains of immunodeficient mice showed very similar clinical disease and neuropathological findings, including marked neuronal apoptosis. The adaptive immune response is unlikely of fundamental importance in producing neuronal apoptosis in the brains of mice in this model.     

Intrinsic physiological and morphological properties of principal cells of the hippocampus and neocortex in hamsters infected with scrapie.

Scrapie is a transmissible spongiform encephalopathy, or "prion disease." We investigated the effects of intracerebral Sc237 scrapie inoculation in hamsters on the physiology and morphology of principal cells from neocortical and hippocampal slices. Scrapie inoculation resulted in increased branching of basal dendrites of hippocampal CA1 pyramidal cells (Sholl analysis), reduced amplitudes of medium and late afterhyperpolarizations (AHPs) in CA1 pyramidal cells and layer V neocortical cells, loss of frequency potentiation of depolarizing afterpotentials (DAPs), and double action potentials in synaptically evoked CA1 pyramidal cell responses. Postsynaptic double action potentials could also be evoked in normal hamster CA1 pyramidal cells by acute pharmacological block of AHPs, suggesting that the depressed AHPs in scrapie-infected hamsters caused the action potential doublets. Both the AHP and the DAP potentiations depend on increased intracellular calcium, which suggests that the underlying deficit, in hamsters infected with Sc237 scrapie, may lie in calcium entry and/or homeostasis. Fast IPSPs, passive membrane properties, and density of dendritic spines remained unchanged. These last two results differ markedly from recent studies on mice infected with ME7 scrapie, indicating diversity of pathophysiology in this group of diseases, perhaps associated with the progressive and substantial neuronal loss found in the ME7, and not the Sc237, model.     

Burrowing into prion disease.

Mice received intra-hippocampal injections of scrapie-infected brain homogenate. Open field activity increased from around week 12 post-injection. Concomitantly the tendency to displace food from a tube inside the home cage decreased. The food was generally dug out with the feet, rather than carried by mouth, so its displacement was called burrowing. Food restriction was unnecessary for this burrowing to occur. Only later, around 18 weeks, did more general motor impairments develop. As burrowing in scrapie-infected mice decreased when open field activity increased, and preceded later motor impairments, it was not due to motor dysfunction. Burrowing is a simple, sensitive, objective, ethological measure, sensitive to preclinical prion disease. Other potential applications are in transgenic and knockout mice, models of ageing and Alzheimer's disease, and pharmacology, particularly neuroleptics.     

Early unsuspected neuron and axon terminal loss in scra pie-infected mice revealed by morphometry and immunocytochemistry

Neuronal loss is often quoted as an element of the pathology of the transmissible spongiform encephalo-pathies, but few data are published. To determine whether neuronal loss is a salient feature of murine scrapie, and whether there is a relationship with the other hallmark lesions of scrapie we compared the numbers of neurons, severity of vacuolation, axonal bouton density and distribution of prion protein (PrP) in the dorsal lateral geniculate nucleus (dLGN) following intraocular infection of C57BL/FaBtDk mice with ME 7 scrapie. This route of infection limits the initial spread of infection to the retinal efferents, thus directing infectivity and subsequent pathological changes to the dLGN which is a major projection of the optic nerve. Morphometric assessment of neuron number in the dLGN was made on semi-serial sections from five infected and five normal brain injected controls at four 50-day intervals during the incubation period, and on terminally affected mice. The number of neurons decreased from around 20 000 at 50 days to under 1000 in the terminal group. Significant loss was identified in individual mice at 150 days post-infection, coincident with the onset of vacuolation: neuron number was found to have an inverse relationship to the severity of vacuolation. Axonal boutons in the dLGN (demonstrated by synaptophysin immunolabelling) were reduced at 200 days, and virtually absent in terminal mice. The intensity of PrP immunostaining progressively increased from 150 days, and in a separate experiment PrP was detected from 175 days by polyacrylamide gel electrophoresis of brain extracts. These results show that early neuronal loss is a significant feature of experimental scrapie infection, and the possible mechanisms of this degeneration are discussed.     

The Unique Characteristics of Inflammatory Responses in Mouse Brain are Acquired During Postnatal Development

The kinetics of leukocyte recruitment during acute inflammation in adult mouse brain differ from the stereotyped response occurring in non-CNS tissues; neutrophil recruitment is minimal and monocyte recruitment occurs after a 48 h delay. One aspect of the CNS microenvironment which may contribute to restricted leukocyte recruitment is the highly differentiated nature of resident CNS macrophages, the microglia. Thus we studied the inflammatory response to intracerebral injections of endotoxin in neonates in which microglia are less differentiated and resemble more closely macrophages of non-CNS tissues. Mice injected with endotoxin on the day of birth exhibited both neutrophil and monocyte recruitment to the parenchyma, but the response differed from that occurring in non-CNS tissues such as skin. Leukocyte recruitment was very slow, the mononuclear phagocyte response peaking 14 days after endotoxin injection. This sluggish inflammatory response was reminiscent of that previously described in fetal wounds. However, when endotoxin was injected into brains of 7-day-old neonates the inflammatory response resembled that seen in non-CNS tissues; i.e. prolific neutrophil recruitment and a brisk mononuclear phagocyte response. Thus the unusual inflammatory cell kinetics are a property of the mature CNS microenvironment; all signals necessary to support typical leukocyte recruitment are present in the brain by 7 days of age but the brain becomes able to restrict leukocyte immigration during subsequent postnatal development. Developmental changes in the host response to identical inflammatory challenges suggest a window during which the brain may be particularly vulnerable to inflammatory bystander damage.