Meningeal B Cell Clusters Correlate with Submeningeal Pathology in a Natural Model of Multiple Sclerosis.

Multiple sclerosis (MS) is an idiopathic demyelinating disease in which meningeal inflammation correlates with accelerated disease progression. The study of meningeal inflammation in MS has been limited because of constrained access to MS brain/spinal cord specimens and the lack of experimental models recapitulating progressive MS. Unlike induced models, a spontaneously occurring model would offer a unique opportunity to understand MS immunopathogenesis and provide a compelling framework for translational research. We propose granulomatous meningoencephalomyelitis (GME) as a natural model to study neuropathological aspects of MS. GME is an idiopathic, progressive neuroinflammatory disease of young dogs with a female bias. In the GME cases examined in this study, the meninges displayed focal and disseminated leptomeningeal enhancement on magnetic resonance imaging, which correlated with heavy leptomeningeal lymphocytic infiltration. These leptomeningeal infiltrates resembled tertiary lymphoid organs containing large B cell clusters that included few proliferating Ki67+ cells, plasma cells, follicular dendritic/reticular cells, and germinal center B cell-like cells. These B cell collections were confined in a specialized network of collagen fibers associated with the expression of the lympho-organogenic chemokines CXCL13 and CCL21. Although neuroparenchymal perivascular infiltrates contained B cells, they lacked the immune signature of aggregates in the meningeal compartment. Finally, meningeal B cell accumulation correlated significantly with cortical demyelination reflecting neuropathological similarities to MS. Hence, during chronic neuroinflammation, the meningeal microenvironment sustains B cell accumulation that is accompanied by underlying neuroparenchymal injury, indicating GME as a novel, naturally occurring model to study compartmentalized neuroinflammation and the associated pathology thought to contribute to progressive MS.

Ontogeny of inter-alpha inhibitor protein (IAIP) expression in human brain.

Inter-alpha inhibitor proteins (IAIPs) are naturally occurring immunomodulatory molecules found in most tissues. We have reported ontogenic changes in the expression of IAIPs in brain during development in sheep and abundant expression of IAIPs in fetal and neonatal rodent brain in a variety of cellular types and brain regions. Although a few studies identified bikunin, light chain of IAIPs, in adult human brain, the presence of the complete endogenous IAIP protein complex has not been reported in human brain. In this study, we examined the immunohistochemical expression of endogenous IAIPs in human cerebral cortex from early in development through the neonatal period and in adults using well-preserved postmortem brains. We examined total, nuclear, and cytoplasmic staining of endogenous IAIPs and their expression in neurofilament light polypeptide-positive neurons and glial fibrillary acidic protein (GFAP)-positive astrocytes. IAIPs were ubiquitously detected for the first time in cerebral cortical cells at 24-26, 27-28, 29-36, and 37-40 weeks of gestation and in adults. Quantitative analyses revealed that IAIPs were predominately localized in the nucleus in all age groups, but cytoplasmic IAIP expression was more abundant in adult than in the younger ages. Immunoreactivity of IAIPs was expressed in neurons and astrocytes in all age groups. In addition, IAIP co-localization with GFAP-positive astrocytes was more abundant in adults than in the developing brain. We conclude that IAIPs exhibit ubiquitous expression, and co-localize with neurons and astrocytes in the developing and adult human brain suggesting a potential role for IAIPs in development and endogenous neuroprotection.

Gastrointestinal iron excretion and reversal of iron excess in a mouse model of inherited iron excess.

The current paradigm in the field of mammalian iron biology states that body iron levels are determined by dietary iron absorption, not by iron excretion. Iron absorption is a highly regulated process influenced by iron levels and other factors. Iron excretion is believed to occur at a basal rate irrespective of iron levels and is associated with processes such as turnover of intestinal epithelium, blood loss, and exfoliation of dead skin. Here we explore iron excretion in a mouse model of iron excess due to inherited transferrin deficiency. Iron excess in this model is attributed to impaired regulation of iron absorption leading to excessive dietary iron uptake. Pharmacological correction of transferrin deficiency not only normalized iron absorption rates and halted progression of iron excess but also reversed body iron excess. Transferrin treatment did not alter the half-life of 59Fe in mutant mice. 59Fe-based studies indicated that most iron was excreted via the gastrointestinal tract and suggested that iron-loaded mutant mice had increased rates of iron excretion. Direct measurement of urinary iron levels agreed with 59Fe-based predictions that urinary iron levels were increased in untreated mutant mice. Fecal ferritin levels were also increased in mutant mice relative to wild-type mice. Overall, these data suggest that mice have a significant capacity for iron excretion. We propose that further investigation into iron excretion is warranted in this and other models of perturbed iron homeostasis, as pharmacological targeting of iron excretion may represent a novel means of treatment for diseases of iron excess.

Systemic infusions of anti-interleukin-1ß neutralizing antibodies reduce short-term brain injury after cerebral ischemia in the ovine fetus.

Perinatal hypoxic-ischemic reperfusion (I/R)-related brain injury is a leading cause of neurologic morbidity and life-long disability in children. Infants exposed to I/R brain injury develop long-term cognitive and behavioral deficits, placing a large burden on parents and society. Therapeutic strategies are currently not available for infants with I/R brain damage, except for hypothermia, which can only be used in full term infants with hypoxic-ischemic encephalopathy (HIE). Moreover, hypothermia is only partially protective. Pro-inflammatory cytokines are key contributors to the pathogenesis of perinatal I/R brain injury. Interleukin-1ß (IL-1ß) is a critical pro-inflammatory cytokine, which has been shown to predict the severity of HIE in infants. We have previously shown that systemic infusions of mouse anti-ovine IL-1ß monoclonal antibody (mAb) into fetal sheep resulted in anti-IL-1ß mAb penetration into brain, reduced I/R-related increases in IL-1ß expression and blood-brain barrier (BBB) dysfunction in fetal brain. The purpose of the current study was to examine the effects of systemic infusions of anti-IL-1ß mAb on short-term I/R-related parenchymal brain injury in the fetus by examining: 1) histopathological changes, 2) apoptosis and caspase-3 activity, 3) neuronal degeneration 4) reactive gliosis and 5) myelin basic protein (MBP) immunohistochemical staining. The study groups included non-ischemic controls, placebo-treated ischemic, and anti-IL-1ß mAb treated ischemic fetal sheep at 127days of gestation. The systemic intravenous infusions of anti-IL-1ß mAb were administered at fifteen minutes and four hours after in utero brain ischemia. The duration of each infusion was two hours. Parenchymal brain injury was evaluated by determining pathological injury scores, ApopTag® positive cells/mm2, caspase-3 activity, Fluoro-Jade B positive cells/mm2, glial fibrillary acidic protein (GFAP) and MBP staining in the brains of fetal sheep 24h after 30min of ischemia. Treatment with anti-IL-1ß mAb reduced (P<0.05) the global pathological injury scores, number of apoptotic positive cells/mm2, and caspase-3 activity after ischemia in fetal sheep. The regional pathological scores and Fluoro-Jade B positive cells/mm2 did not differ between the placebo- and anti-IL-1ß mAb treated ischemic fetal sheep. The percent of the cortical area stained for GFAP was lower (P<0.05) in the placebo ischemic treated than in the non-ischemic group, but did not differ between the placebo- and anti-IL-1ß mAb treated ischemic groups. MBP immunohistochemical expression did not differ among the groups. In conclusion, infusions of anti-IL-1ß mAb attenuate short-term I/R-related histopathological tissue injury, apoptosis, and reduce I/R-related increases in caspase-3 activity in ovine fetal brain. Therefore, systemic infusions of anti-IL-1ß mAb attenuate short-term I/R-related parenchymal brain injury in the fetus.

Oxytocin- and arginine vasopressin-containing fibers in the cortex of humans, chimpanzees, and rhesus macaques.

Oxytocin (OT) and arginine-vasopressin (AVP) are involved in the regulation of complex social behaviors across a wide range of taxa. Despite this, little is known about the neuroanatomy of the OT and AVP systems in most non-human primates, and less in humans. The effects of OT and AVP on social behavior, including aggression, mating, and parental behavior, may be mediated primarily by the extensive connections of OT- and AVP-producing neurons located in the hypothalamus with the basal forebrain and amygdala, as well as with the hypothalamus itself. However, OT and AVP also influence social cognition, including effects on social recognition, cooperation, communication, and in-group altruism, which suggests connectivity with cortical structures. While OT and AVP V1a receptors have been demonstrated in the cortex of rodents and primates, and intranasal administration of OT and AVP has been shown to modulate cortical activity, there is to date little evidence that OT-and AVP-containing neurons project into the cortex. Here, we demonstrate the existence of OT- and AVP-containing fibers in cortical regions relevant to social cognition using immunohistochemistry in humans, chimpanzees, and rhesus macaques. OT-immunoreactive fibers were found in the straight gyrus of the orbitofrontal cortex as well as the anterior cingulate gyrus in human and chimpanzee brains, while no OT-immunoreactive fibers were found in macaque cortex. AVP-immunoreactive fibers were observed in the anterior cingulate gyrus in all species, as well as in the insular cortex in humans, and in a more restricted distribution in chimpanzees. This is the first report of OT and AVP fibers in the cortex in human and non-human primates. Our findings provide a potential mechanism by which OT and AVP might exert effects on brain regions far from their production site in the hypothalamus, as well as potential species differences in the behavioral functions of these target regions.

Human polyomavirus receptor distribution in brain parenchyma contrasts with receptor distribution in kidney and choroid plexus.

The human polyomavirus, JCPyV, is the causative agent of progressive multifocal leukoencephalopathy, a rare demyelinating disease that occurs in the setting of prolonged immunosuppression. After initial asymptomatic infection, the virus establishes lifelong persistence in the kidney and possibly other extraneural sites. In rare instances, the virus traffics to the central nervous system, where oligodendrocytes, astrocytes, and glial precursors are susceptible to lytic infection, resulting in progressive multifocal leukoencephalopathy. The mechanisms by which the virus traffics to the central nervous system from peripheral sites remain unknown. Lactoseries tetrasaccharide c (LSTc), a pentasaccharide containing a terminal α2,6-linked sialic acid, is the major attachment receptor for polyomavirus. In addition to LSTc, type 2 serotonin receptors are required for facilitating virus entry into susceptible cells. We studied the distribution of virus receptors in kidney and brain using lectins, antibodies, and labeled virus. The distribution of LSTc, serotonin receptors, and virus binding sites overlapped in kidney and in the choroid plexus. In brain parenchyma, serotonin receptors were expressed on oligodendrocytes and astrocytes, but these cells were negative for LSTc and did not bind virus. LSTc was instead found on microglia and vascular endothelium, to which virus bound abundantly. Receptor distribution was not changed in the brains of patients with progressive multifocal leukoencephalopathy. Virus infection of oligodendrocytes and astrocytes during disease progression is LSTc independent.

Targeting choroid plexus epithelia and ventricular ependyma for drug delivery to the central nervous system.

BACKGROUND: Because the choroid plexus (CP) is uniquely suited to control the composition of cerebrospinal fluid (CSF), there may be therapeutic benefits to increasing the levels of biologically active proteins in CSF to modulate central nervous system (CNS) functions. To this end, we sought to identify peptides capable of ligand-mediated targeting to CP epithelial cells reasoning that they could be exploited to deliver drugs, biotherapeutics and genes to the CNS. METHODS: A peptide library displayed on M13 bacteriophage was screened for ligands capable of internalizing into CP epithelial cells by incubating phage with CP explants for 2 hours at 37C and recovering particles with targeting capacity. RESULTS: Three peptides, identified after four rounds of screening, were analyzed for specific and dose dependent binding and internalization. Binding was deemed specific because internalization was prevented by co-incubation with cognate synthetic peptides. Furthermore, after i.c.v. injection into rat brains, each peptide was found to target phage to epithelial cells in CP and to ependyma lining the ventricles. CONCLUSION: These data demonstrate that ligand-mediated targeting can be used as a strategy for drug delivery to the central nervous system and opens the possibility of using the choroid plexus as a portal of entry into the brain.

Co-localization and regulation of basic fibroblast growth factor and arginine vasopressin in neuroendocrine cells of the rat and human brain.

BACKGROUND: Adult rat hypothalamo-pituitary axis and choroid plexus are rich in basic fibroblast growth factor (FGF2) which likely has a role in fluid homeostasis. Towards this end, we characterized the distribution and modulation of FGF2 in the human and rat central nervous system. To ascertain a functional link between arginine vasopressin (AVP) and FGF2, a rat model of chronic dehydration was used to test the hypothesis that FGF2 expression, like that of AVP, is altered by perturbed fluid balance. METHODS: Immunohistochemistry and confocal microscopy were used to examine the distribution of FGF2 and AVP neuropeptides in the normal human brain. In order to assess effects of chronic dehydration, Sprague-Dawley rats were water deprived for 3 days. AVP neuropeptide expression and changes in FGF2 distribution in the brain, neural lobe of the pituitary and kidney were assessed by immunohistochemistry, and western blotting (FGF2 isoforms). RESULTS: In human hypothalamus, FGF2 and AVP were co-localized in the cytoplasm of supraoptic and paraventricular magnocellular neurons and axonal processes. Immunoreactive FGF2 was associated with small granular structures distributed throughout neuronal cytoplasm. Neurohypophysial FGF2 immunostaining was found in axonal processes, pituicytes and Herring bodies. Following chronic dehydration in rats, there was substantially-enhanced FGF2 staining in basement membranes underlying blood vessels, pituicytes and other glia. This accompanied remodeling of extracellular matrix. Western blot data revealed that dehydration increased expression of the hypothalamic FGF2 isoforms of ca. 18, 23 and 24 kDa. In lateral ventricle choroid plexus of dehydrated rats, FGF2 expression was augmented in the epithelium (Ab773 as immunomarker) but reduced interstitially (Ab106 immunostaining). CONCLUSIONS: Dehydration altered FGF2 expression patterns in AVP-containing magnocellular neurons and neurohypophysis, as well as in choroid plexus epithelium. This supports the involvement of centrally-synthesized FGF2, putatively coupled to that of AVP, in homeostatic mechanisms that regulate fluid balance.

Ovariectomy Influences Cognition and Markers of Alzheimer's Disease.

Alzheimer's disease (AD) is one of the most devastating and costly diseases, and prevalence of AD increases with age. Furthermore, females are twice as likely to suffer from AD compared to males. The cessation of reproductive steroid hormone production during menopause is hypothesized to cause this difference. Two rodent AD models, APP21 and APP+PS1, and wild type (WT) rats underwent an ovariectomy or sham surgery. Changes in learning and memory, brain histology, amyloid-ß (Aß) deposition, levels of mRNAs involved in Aß production and clearance, and synaptic and cognitive function were determined. Barnes maze results showed that regardless of ovariectomy status, APP+PS1 rats learned slower and had poor memory retention. Ovariectomy caused learning impairment only in the APP21 rats. High levels of Aß42 and very low levels of Aß40 were observed in the brain cortices of APP+PS1 rats indicating limited endogenous PS1. The APP+PS1 rats had 43-fold greater formic acid soluble Aß42 than Aß40 at 17 months. Furthermore, levels of formic acid soluble Aß42 increased 57-fold in ovariectomized APP+PS1 rats between 12 and 17 months of age. The mRNA encoding Grin1 significantly decreased due to ovariectomy whereas levels of Bace1, Chat, and Prkcb all decreased with age. The expression levels of mRNAs involved in Aß degradation and AßPP cleavage (Neprilysin, Ide, Adam9, and Psenen) were found to be highly correlated with each other as well as hippocampal Aß deposition. Taken together, these results indicate that both ovariectomy and genotype influence AD markers in a complex manner.

Activation of suprachiasmatic nuclei and primary visual cortex depends upon time of day.

The human suprachiasmatic nucleus (SCN), the master biological clock, is a small (approximately 2 mm(3)) and deep structure located in the anterior hypothalamus. Previous methods do not allow in vivo study of the human SCN in a non-invasive manner. Therefore, we explored blood oxygen level-dependent (BOLD)-functional magnetic resonance imaging (fMRI) with OFF-ON-OFF block-designed visual stimuli to record the activities in the 'SCN and peri SCN in the anterior hypothalamus' (SCN+) and the primary visual area V1 using a 3T Siemens scanner and six normal subjects. We found that: (i) the BOLD-fMRI response to light and the mean of percentage activation in the SCN+ at midday was significantly less than that at night; and (ii) the number of activated voxels in most of the visual area V1 at midday was significantly higher than that at night. We conclude that BOLD-fMRI responses to light in the SCN+ and the V1 areas vary with time of day. This conclusion is consistent with: (i) the previously measured phase-response curve to light [J. Physiol., 549.3 (2003) 945] for the SCN activity at critical intensity threshold; and (ii) the interaction of the melanopsin-based signals with the rod-cone signals at the 'giant' retinal ganglion cells [Nature, 433 (2005) 749] for the V1 activity.

Dorsomedial SCN neuronal subpopulations subserve different functions in human dementia.

The suprachiasmatic nuclei (SCN) are necessary and sufficient for the maintenance of circadian rhythms in primate and other mammalian species. The human dorsomedial SCN contains populations of non-species-specific vasopressin and species-specific neurotensin neurons. We made time-series recordings of core body temperature and locomotor activity in 19 elderly, male, end-stage dementia patients and 8 normal elderly controls. Following the death of the dementia patients, neuropathological diagnostic information and tissue samples from the hypothalamus were obtained. Hypothalamic tissue was also obtained from eight normal control cases that had not had activity or core temperature recordings previously. Core temperature was analysed for parametric, circadian features, and activity was analysed for non-parametric and parametric circadian features. These indices were then correlated with the degree of degeneration seen in the SCN (glia/neuron ratio) and neuronal counts from the dorsomedial SCN (vasopressin, neurotensin). Specific loss of SCN neurotensin neurons was associated with loss of activity and temperature amplitude without increase in activity fragmentation. Loss of SCN vasopressin neurons was associated with increased activity fragmentation but not loss of amplitude. Evidence for a circadian rhythm of vasopressinergic activity was seen in the dementia cases but no evidence was seen for a circadian rhythm in neurotensinergic activity. These results provide evidence that the SCN is necessary for the maintenance of the circadian rhythm in humans, information on the role of neuronal subpopulations in subserving this function and the utility of dementia in elaborating brain-behaviour relationships in the human.

Enteric Pathologic Manifestations of Alpha-Synucleinopathies.

BACKGROUND: Gastrointestinal (GI) symptoms are common in Parkinson disease (PD), often preceding neurological manifestations; however, early diagnostic utility of GI biopsies remains controversial. Studies suggest aberrant deposition of alpha-synuclein (α-syn) follows step-wise progression in central nervous system though histologic interpretation of normal and aberrant staining patterns have shown variable results. This study examines whether GI α-syn mRNA expression combined with standard α-syn immunohistochemical staining enhance the role of GI biopsy in PD. MATERIALS AND METHODS: Four groups were examined, including pediatric (21) and adult control patients (18), PD clinic patients (17), and pathologically confirmed PD cases from hospital archives (16). Enteric nervous system α-syn staining was evaluated by immunohistochemistry in 33 PD and 39 controls. α-Syn mRNA levels were compared between patient groups using quantitative polymerase chain reaction and stomach and colon levels in PD. RESULTS: PD patients had Lewy bodies (LB) and diffuse neuronal α-syn staining. GI tissues from elderly controls, children, and young adults exhibited diffuse positivity. LB were limited to PD. Myenteric plexus immunoreactivity varied in different regions. Widespread staining was noted within stomach and colon. Immunoreactivity was present within esophagus, appendix, and small bowel. α-Syn mRNA expression was highest in PD; however, levels varied between proximal and distal GI tract. CONCLUSIONS: α-Syn is normally present within young and elderly enteric nervous system; furthermore, while α-syn mRNA is always detectable, levels are highest and most variable in PD. This suggests that enteric α-syn may be altered in neurodegenerative disease. The presence of LB in the GI tract, not solely α-syn expression, may prove useful, distinguishing neurodegenerative disease patients from normal controls.

High-mobility group box-1 translocation and release after hypoxic ischemic brain injury in neonatal rats.

Inflammation contributes to neonatal brain injury. Pro-inflammatory cytokines represent key inflammatory meditators in neonatal hypoxic-ischemic (HI) brain injury. The high mobility group box-1 (HMGB1) protein is a nuclear protein with pro-inflammatory cytokine properties when it is translocated from the nucleus and released extracellularly after stroke in adult rodents. We have previously shown that HMGB1 is translocated from the nucleus to cytosolic compartment after ischemic brain injury in fetal sheep. In the current study, we utilized the Rice-Vannucci model to investigate the time course of HMGB1 translocation and release after HI injury in neonatal rats. HMGB1 was located in cellular nuclei of brains from sham control rats. Nuclear to cytoplasmic translocation of HMGB1 was detected in the ipsilateral-HI hemisphere as early as zero h after HI, and released extracellularly as early as 6 h after HI. Immunohistochemical double staining detected HMGB1 translocation mainly in neurons along with release from apoptotic cells after HI. Serum HMGB1 increased at 3 h and decreased by 24 h after HI. In addition, rat brains exposed to hypoxic injury alone also exhibited time dependent HMGB1 translocation at 3, 12 and 48 h after hypoxia. Consequently, HMGB1 responds similarly after HI injury in the brains of neonatal and adult subjects. We conclude that HMGB1 is sensitive early indicator of neonatal HI and hypoxic brain injury.

Neuroprotective effects of inter-alpha inhibitor proteins after hypoxic-ischemic brain injury in neonatal rats.

Hypoxic-ischemic (HI) brain injury is one of the most common neurological problems occurring in the perinatal period. Hypothermia is the only approved intervention for neonatal HI encephalopathy. However, this treatment is only partially protective, has a narrow therapeutic time window after birth and only can be used to treat full-term infants. Consequently, additional therapies are critically needed. Inflammation is an important contributing factor to the evolution of HI brain injury in neonates. Inter-alpha Inhibitor Proteins (IAIPs) are immunomodulatory proteins with anti-inflammatory properties. We have previously shown that IAIPs reduce neuronal cell death and improve behavioral outcomes when given after carotid artery ligation, but before hypoxia in male neonatal rats. The objective of the current study was to investigate the neuroprotective effects of treatment with IAIPs given immediately or 6 h after HI in both male and female neonatal rats. HI was induced with the Rice-Vannucci method in postnatal (P) day 7 rats. After ligation of the right common carotid artery, P7 rats were exposed to 90 min of hypoxia (8% oxygen). Human plasma-derived IAIPs or placebo (phosphate buffered saline) was given at zero, 24, and 48 h after HI. Brains were perfused, weighed and fixed 72 h after HI at P10. In a second, delayed treatment group, the same procedure was followed except that IAIPs or placebo were given at 6, 24 and 48 h after HI. Separate sham-operated, placebo-treated groups were exposed to identical protocols but were not exposed to carotid artery ligation and remained in room air. Rat sex was recorded. The effects of IAIPs on HI brain injury were examined using histopathological scoring and immunohistochemical analyses of the brain and by using infarct volume measurements on frozen tissue of the entire brain hemispheres ipsilateral and contralateral to HI injury. IAIPs given immediately after HI improved (P < 0.050) histopathological brain injury across and within the cingulate, caudate/putamen, thalamus, hippocampus and parietal cortex in males, but not in females. In contrast, IAIPs given immediately after HI reduced (P < 0.050) infarct volumes of the hemispheres ipsilateral to HI injury in similarly both the males and females. Treatment with IAIPs also resulted in higher (P < 0.050) brain weights compared with the placebo-treated HI group, reduced (P < 0.050) neuronal and non-neuronal cell death in the cortex and total hemisphere, and also increased the total area of oligodendrocytes determined by CNPase in the ipsilateral hemisphere and corpus callosum (P < 0.050) of male, but not female subjects exposed to HI. Delayed treatment with IAIPs 6 h after HI did not improve histopathological brain injury in males or females, but resulted in higher (P < 0.050) brain weights compared with the placebo-treated HI males. Therefore, treatment with IAIPs immediately after HI improved brain weights and reduced neuropathological brain injury and cell death in male rats, and reduced infarct volume in both male and female neonatal rats. We conclude that IAIPs exert neuroprotective effects after exposure to HI in neonatal rats and may exhibit some sex-related differential effects.

Cerebral cortical arteriolar angiopathy, vascular beta-amyloid, smooth muscle actin, Braak stage, and APOE genotype.

BACKGROUND AND PURPOSE: We examined the associations among the vascular beta-amyloid levels, smooth muscle actin, wall thickness, and lumen diameter to achieve greater understanding of the arteriolar changes that accompany Alzheimer disease (AD). METHODS: Post-mortem pathology brain specimens from 76 patients with AD and 19 non-AD age control subjects were studied. We analyzed arterioles of the frontal cortex (Brodmann area 10) by immunohistochemistry and morphometry, and derived measures of vascular beta-amyloid level, smooth muscle actin (SMA) volume, and arteriolar wall thickness and lumen diameter. APOE genotype was determined for each case. RESULTS: Overall, there was a striking reciprocal relationship between arteriolar beta-amyloid volume and smooth muscle actin (P<0.0001). In addition, there was a strong positive association between progressively accumulating vascular beta-amyloid and augmentations in both wall thickness (P<0.0001) and lumen width (P<0.0001). In comparison with non-AD control subjects, smooth muscle actin was decreased in patients clinically diagnosed with AD and was reduced >10-fold in cases with AD pathology (Braak I to VI) compared with those lacking AD neuropathology. Significantly altered composition and structure of cortical vessels in pre-Braak stages corroborated our hypothesis that arterioles are devastated early in the AD pathological process. Smooth muscle actin, arteriolar wall thickness, and luminal diameter did not vary with Braak stage severity (P>0.05), indicating that substantial arteriolar damage may precede at least some of the interstitial plaques and neuronal tangles. Moreover, the structural and biochemical arteriolar abnormalities did not vary as a function of APOE genotype (P>0.05). CONCLUSIONS: We postulate that in elderly patients, the continually progressing beta-amyloid-associated angiopathy, at the arteriolar level, harms the contractile apparatus and cerebral blood flow autoregulation, thereby making the downstream capillaries vulnerable to damage. Collectively, our observations lend further support to the idea that microvascular damage has a role, perhaps relatively early, in the onset of major AD pathology.

Hippocampal RAGE immunoreactivity in early and advanced Alzheimer's disease.

Microvascular accumulation and neuronal overproduction of amyloid-beta peptide (Abeta) are pathologic features of Alzheimer's disease (AD). In this study, we examined the receptor for advanced glycation endproducts (RAGE), a multi-ligand receptor found in both neurons and cerebral microvascular endothelia that binds Abeta. RAGE expression was assessed in aged controls (n = 6), patients with early AD-like pathology (n = 6), and severe, Braak V-VI AD (n = 6). Human hippocampi were stained with a specific polyclonal antibody directed against RAGE (Research Diagnostics, Flanders, NJ). Immunoreactivity was localized in both neurons and cerebral endothelial cells. Quantitative image-analyses were performed on grayscale images to assess the total surface area of endothelial RAGE immunoreaction product in cross sections of cerebral microvessels (5-20 microm). Confocal images were acquired for confirmation of RAGE immunoreactivity in both microvessels and neurons by coupling RAGE with CD-31 and neurofilament, respectively. A significant increase in endothelial RAGE immunoreactivity was found in severe Braak V-VI AD patients when compared to aged controls (p < 0.001), and when compared to patients with early AD pathology (p = 0.0125). In addition, a significant increase in endothelial RAGE immunoreactivity was witnessed when comparing aged controls having no reported AD pathology with patients having early AD-like pathology (p = 0.038). Our data suggest that microvascular RAGE levels increase in conjunction with the onset of AD, and continue to increase linearly as a function of AD pathologic severity (p < 0.0001).

Multiplicity of cerebrospinal fluid functions: New challenges in health and disease.

UNLABELLED: This review integrates eight aspects of cerebrospinal fluid (CSF) circulatory dynamics: formation rate, pressure, flow, volume, turnover rate, composition, recycling and reabsorption. Novel ways to modulate CSF formation emanate from recent analyses of choroid plexus transcription factors (E2F5), ion transporters (NaHCO3 cotransport), transport enzymes (isoforms of carbonic anhydrase), aquaporin 1 regulation, and plasticity of receptors for fluid-regulating neuropeptides. A greater appreciation of CSF pressure (CSFP) is being generated by fresh insights on peptidergic regulatory servomechanisms, the role of dysfunctional ependyma and circumventricular organs in causing congenital hydrocephalus, and the clinical use of algorithms to delineate CSFP waveforms for diagnostic and prognostic utility. Increasing attention focuses on CSF flow: how it impacts cerebral metabolism and hemodynamics, neural stem cell progression in the subventricular zone, and catabolite/peptide clearance from the CNS. The pathophysiological significance of changes in CSF volume is assessed from the respective viewpoints of hemodynamics (choroid plexus blood flow and pulsatility), hydrodynamics (choroidal hypo- and hypersecretion) and neuroendocrine factors (i.e., coordinated regulation by atrial natriuretic peptide, arginine vasopressin and basic fibroblast growth factor). In aging, normal pressure hydrocephalus and Alzheimer's disease, the expanding CSF space reduces the CSF turnover rate, thus compromising the CSF sink action to clear harmful metabolites (e.g., amyloid) from the CNS. Dwindling CSF dynamics greatly harms the interstitial environment of neurons. Accordingly the altered CSF composition in neurodegenerative diseases and senescence, because of adverse effects on neural processes and cognition, needs more effective clinical management. CSF recycling between subarachnoid space, brain and ventricles promotes interstitial fluid (ISF) convection with both trophic and excretory benefits. Finally, CSF reabsorption via multiple pathways (olfactory and spinal arachnoidal bulk flow) is likely complemented by fluid clearance across capillary walls (aquaporin 4) and arachnoid villi when CSFP and fluid retention are markedly elevated. A model is presented that links CSF and ISF homeostasis to coordinated fluxes of water and solutes at both the blood-CSF and blood-brain transport interfaces. OUTLINE: 1 Overview2 CSF formation2.1 Transcription factors2.2 Ion transporters2.3 Enzymes that modulate transport2.4 Aquaporins or water channels2.5 Receptors for neuropeptides3 CSF pressure3.1 Servomechanism regulatory hypothesis3.2 Ontogeny of CSF pressure generation3.3 Congenital hydrocephalus and periventricular regions3.4 Brain response to elevated CSF pressure3.5 Advances in measuring CSF waveforms4 CSF flow4.1 CSF flow and brain metabolism4.2 Flow effects on fetal germinal matrix4.3 Decreasing CSF flow in aging CNS4.4 Refinement of non-invasive flow measurements5 CSF volume5.1 Hemodynamic factors5.2 Hydrodynamic factors5.3 Neuroendocrine factors6 CSF turnover rate6.1 Adverse effect of ventriculomegaly6.2 Attenuated CSF sink action7 CSF composition7.1 Kidney-like action of CP-CSF system7.2 Altered CSF biochemistry in aging and disease7.3 Importance of clearance transport7.4 Therapeutic manipulation of composition8 CSF recycling in relation to ISF dynamics8.1 CSF exchange with brain interstitium8.2 Components of ISF movement in brain8.3 Compromised ISF/CSF dynamics and amyloid retention9 CSF reabsorption9.1 Arachnoidal outflow resistance9.2 Arachnoid villi vs. olfactory drainage routes9.3 Fluid reabsorption along spinal nerves9.4 Reabsorption across capillary aquaporin channels10 Developing translationally effective models for restoring CSF balance11 Conclusion.

Choroid plexus genes for CSF production and brain homeostasis are altered in Alzheimer's disease.

BACKGROUND: The roles of the choroid plexus (CP) and cerebrospinal fluid (CSF) production have drawn increasing attention in Alzheimer's disease (AD) research. Specifically, studies document markedly decreased CSF production and turnover in moderate-to-severe AD. Moreover, reduced CP function and CSF turnover lead to impaired clearance of toxic metabolites, likely promote neuroinflammation, and may facilitate neuronal death during AD progression. We analyzed CP gene expression in AD compared with control subjects, specifically considering those genes involved with CSF production and CP structural integrity. METHODS: The Brown-Merck Gene Expression Omnibus (GEO) database (CP transcripts) was mined to examine changes in gene expression in AD compared to controls with a focus on assorted genes thought to play a role in CSF production. Specifically, genes coding for ion transporters in CP epithelium (CPE) and associated enzymes like Na-K-ATPase and carbonic anhydrase, aquaporins, mitochondrial transporters/enzymes, blood-cerebrospinal fluid barrier (BCSFB) stability proteins, and pro-inflammatory mediators were selected for investigation. Data were analyzed using t test p-value and fold-change analysis conducted by the GEO2R feature of the GEO database. RESULTS: Significant expression changes for several genes were observed in AD CP. These included disruptions to ion transporters (e.g., the solute carrier gene SLC4A5, p = 0.004) and associated enzyme expressions (e.g., carbonic anhydrase CA4, p = 0.0001), along with decreased expression of genes involved in BCSFB integrity (e.g., claudin CLDN5, p = 0.039) and mitochondrial ATP synthesis (e.g., adenosine triphosphate ATP5L, p = 0.0004). Together all changes point to disrupted solute transport at the blood-CSF interface in AD. Increased expression of pro-inflammatory (e.g., interleukin IL1RL1, p = 0.00001) and potential neurodegenerative genes (e.g., amyloid precursor APBA3, p = 0.002) also implicate disturbed CP function. CONCLUSIONS: Because the altered expression of numerous transcripts in AD-CP help explain decreased CSF production in AD, these findings represent a first step towards identifying novel therapeutic targets in AD.

Memory deficiency, cerebral amyloid angiopathy, and amyloid-ß plaques in APP+PS1 double transgenic rat model of Alzheimer's disease.

Transgenic rat models of Alzheimer's disease were used to examine differences in memory and brain histology. Double transgenic female rats (APP+PS1) over-expressing human amyloid precursor protein (APP) and presenilin 1 (PS1) and single transgenic rats (APP21) over-expressing human APP were compared with wild type Fischer rats (WT). The Barnes maze assessed learning and memory and showed that both APP21 and APP+PS1 rats made significantly more errors than the WT rats during the acquisition phase, signifying slower learning. Additionally, the APP+PS1 rats made significantly more errors following a retention interval, indicating impaired memory compared to both the APP21 and WT rats. Immunohistochemistry using an antibody against amyloid-ß (Aß) showed extensive and mostly diffuse Aß plaques in the hippocampus and dense plaques that contained tau in the cortex of the brains of the APP+PS1 rats. Furthermore, the APP+PS1 rats also showed vascular changes, including cerebral amyloid angiopathy with extensive Aß deposits in cortical and leptomeningeal blood vessel walls and venous collagenosis. In addition to the Aß accumulation observed in arterial, venous, and capillary walls, APP+PS1 rats also displayed enlarged blood vessels and perivascular space. Overall, the brain histopathology and behavioral assessment showed that the APP+PS1 rats demonstrated behavioral characteristics and vascular changes similar to those commonly observed in patients with Alzheimer's disease.

Blood-Cerebrospinal Fluid Barrier Gradients in Mild Cognitive Impairment and Alzheimer's Disease: Relationship to Inflammatory Cytokines and Chemokines.

Background: The pathophysiology underlying altered blood-cerebrospinal fluid barrier (BCSFB) function in Alzheimer's disease (AD) is unknown but may relate to endothelial cell activation and cytokine mediated inflammation. Methods: Cerebrospinal fluid (CSF) and peripheral blood were concurrently collected from cognitively healthy controls (N = 21) and patients with mild cognitive impairment (MCI) (N = 8) or AD (N = 11). The paired serum and CSF samples were assayed for a panel of cytokines, chemokines, and related trophic factors using multiplex ELISAs. Dominance analysis models were conducted to determine the relative importance of the inflammatory factors in relationship to BCSFB permeability, as measured by CSF/serum ratios for urea, creatinine, and albumin. Results: BCSFB disruption to urea, a small molecule distributed by passive diffusion, had a full model coefficient of determination (r2) = 0.35, and large standardized dominance weights (>0.1) for monocyte chemoattractant protein-1, interleukin (IL)-15, IL-1rα, and IL-2 in serum. BCSFB disruption to creatinine, a larger molecule governed by active transport, had a full model r2 = 0.78, and large standardized dominance weights for monocyte inhibitor protein-1b in CSF and tumor necrosis factor-α in serum. BCSFB disruption to albumin, a much larger molecule, had a full model r2 = 0.62, and large standardized dominance weights for IL-17a, interferon-gamma, IL-2, and VEGF in CSF, as well IL-4 in serum. Conclusions: Inflammatory proteins have been widely documented in the AD brain. The results of the current study suggest that changes in BCSFB function resulting in altered permeability and transport are related to expression of specific inflammatory proteins, and that the shifting distribution of these proteins from serum to CSF in AD and MCI is correlated with more severe perturbations in BCSFB function.