Parenchymal Fraction Research Articles

The role of the blood‐brain barrier in brain insulin signaling

AbstractBackgroundDefects in central response to insulin is suspected to play a role in AD. However, to act on the brain, pancreas‐secreted insulin must interact first with the blood‐brain barrier (BBB). The insulin receptor (INSR) consists in 2 insulin‐binding extracellular α chains, either short (INSRα‐A) or long (INSRα‐B) isoforms, as well as 2 intracellular β‐chains (INSRβ) containing auto‐phosphorylation sites. The aim of the present study was to better understand the role of INSR located on the BBB in AD.MethodWe used microvessel‐enriched brain samples from individuals classified as Controls, MCI or AD and from 3xTg‐AD mice from different ages. In situ cerebral perfusion was used to directly assess the interaction between circulating insulin and the BBB.ResultsWe first show that INSR in human and mouse brains are concentrated in blood microvessels, with INSRα‐B being almost absent from the parenchymal fractions. Lower levels of INSRα‐B were observed in microvessel extracts from the parietal cortex of AD persons, associated with cognitive decline. The resulting shift toward a higher INSRα‐A:INSRα‐B ratio is consistent with insulin resistance situated on the BBB. Intracarotid infusion of insulin induced a rapid phosphorylation of INSRβ retrieved from cerebrovascular extracts, but not from parenchymal extracts of the mouse brain. In the 3xTg‐AD mouse model of AD neuropathology, lower levels of cerebrovascular INSRα‐B were observed whereas activation of INSRβ was blunted upon intracarotid administration of insulin. Finally, we provide some insights on a possible association between BACE1 activation and the reduction of cerebrovascular INSR.ConclusionOverall, data gathered from postmortem AD brains and an animal model of AD neuropathology suggest that brain insulin resistance in AD may occur at the level of INSR located on microvessels. These results also indicate that INSR and its downstream signaling within endothelial cells of the BBB are systemically accessible drug targets in AD.

Targeting insulin-like growth factor-1 receptor (IGF1R) for brain delivery of biologics.

The blood-brain barrier (BBB) prevents the majority of drugs from crossing into the brain and reaching neurons. To overcome this challenge, safe and non-invasive technologies targeting receptor-mediated pathways have been developed. In this study, three single-domain antibodies (sdAbs; IGF1R3, IGF1R4, and IGF1R5) targeting the extracellular domain of the human insulin-like growth factor-1 receptor (IGF1R), generated by llama immunization, showed enhanced transmigration across the rat BBB model (SV-ARBEC) in vitro. The rate of brain uptake of these sdAbs fused to mouse Fc (sdAb-mFc) in vivo was estimated using the fluorescent in situ brain perfusion (ISBP) technique followed by optical brain imaging and distribution volume evaluation. Compared to the brains perfused with the negative control A20.1-mFc, the brains perfused with anti-IGF1R sdAbs showed a significant increase of the total fluorescence intensity (~2-fold, p<.01) and the distribution volume (~4-fold, p<.01). The concentration curve for IGF1R4-mFc demonstrated a linear accumulation plateauing at approximately 400µg (~1µM), suggesting a saturable mechanism of transport. Capillary depletion and mass spectrometry analyses of brain parenchyma post-ISBP confirmed the IGF1R4-mFc brain uptake with ~25% of the total amount being accumulated in the parenchymal fraction in contrast to undetectable levels of A20.1-mFc after a 5-min perfusion protocol. Systemic administration of IGF1R4-mFc fused with the non-BBB crossing analgesic peptide galanin (2 and 5mg/kg) induced a dose-dependent suppression of thermal hyperalgesia in the Hargreaves pain model. In conclusion, novel anti-IGF1R sdAbs showed receptor-mediated brain uptake with pharmacologically effective parenchymal delivery of non-permeable neuroactive peptides.

An Improved Method for Physical Separation of Cerebral Vasculature and Parenchyma Enables Detection of Blood-Brain-Barrier Dysfunction

The neurovascular niche is crucial for constant blood supply and blood-brain barrier (BBB) function and is altered in a number of different neurological conditions, making this an intensely active field of research. Brain vasculature is unique for its tight association of endothelial cells with astrocytic endfeet processes. Separation of the vascular compartment by centrifugation-based methods confirmed enrichment of astrocytic endfeet processes, making it possible to study the entire vascular niche with such methods. Several centrifugation-based separation protocols are found in the literature; however, with some constraints which limit their applicability and the scope of the studies. Here, we describe and validate a protocol for physically separating the neurovascular niche from the parenchyma, which is optimized for smaller tissue quantities. Using endothelial, neuronal, and astrocyte markers, we show that quantitative Western blot-based target detection can be performed of both the vessel-enriched and parenchymal fractions using as little as a single mouse brain hemisphere. Validation of our protocol in rodent stroke models by detecting changes in tight junction protein expression, serum albumin signals and astrocyte activation, i.e., increased glial fibrillary acidic protein expression, between the ipsilateral and the lesion-free contralateral hemisphere demonstrates this protocol as a new way of detecting BBB breakdown and astrogliosis, respectively.

APOE4 GENOTYPE-DEPENDENT DEFICITS IN DHA CONTAINING PHOSPHOLIPIDS AND DHA TRANSPORTERS IN THE CEREBROVASCULATURE OF ALZHEIMER'S DISEASE PATIENTS

The contribution of apolipoprotein E (APOE) E4 to the cerebrovascular dysfunction in Alzheimer’s disease (AD) is characterized by the presence of severe cerebral amyloid angiopathy, increased blood-brain-barrier (BBB) breakdown, reduced cerebral vascularization, and basement membrane thinning in E4 carriers compared to non-carriers. It has also been proposed that the diminished capacity of the apoE4 protein to transport essential polyunsaturated fatty acids (PUFAs) that are required for the structural and functional maintenance and vascular integrity of the brain also contribute to AD pathogenesis. However, it remains to be determined if there are changes in the profiles of phospholipids (PL) and in the expression of lipid transporters within the brain vasculature in relation to the E4 allele and AD diagnosis. We performed liquid chromatography/mass spectrometry based lipidomic analysis of the cerebrovascular and parenchymal fractions from autopsied human brain tissue of pathologically confirmed AD cases and controls stratified by APOE genotype. In order to determine if there were changes in the expression of lipid transporters in relation to the APOE E4 allele, we performed antibody based examination of the major facilitator superfamily domain containing 2A (mfsd2a) protein in the cerebrovasculature from these subjects. Total phosphatidylcholine (PC) was significantly lower in the cerebrovascular fractions of AD patients compared to controls. While docosahexaenoic acid (DHA) containing PL species were lower in heterozygous E4 AD patients compared to E4 controls in both the cerebrovascular and parenchymal fractions, an ether PC species containing arachidonic acid (AA) was elevated within the cerebrovasculature of E4 carriers relative to non-carriers and was highest among E4 AD specimens compared to E4 controls. We also observed an APOE E4 dependent difference in mfsd2a expression. Among AD patients, E4 homozygotes had lower expression of mfsd2a than E4 heterozygotes and non-carriers. These findings demonstrate that deficiencies in DHA within the brains of APOE E4 carriers may, in part, be due to lower expression of mfsd2a. Thus, targeting this transport mechanism may improve the bioavailability of DHA to the brain of APOE E4 individuals providing a novel approach to the treatment of AD.

Resolution of central nervous system astrocytic and endothelial sources of CCL2 gene expression during evolving neuroinflammation.

BackgroundThe chemokine CCL2 is a critical mediator of neuroinflammation in diseases such as multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). CCL2 drives mononuclear cell infiltration into the central nervous system (CNS), alters expression and distribution of microvascular endothelial tight junction proteins, and disrupts the blood–brain and blood-spinal cord barriers. Immunohistochemistry has consistently revealed astrocytes to be a source of this chemokine during neuroinflammation, while providing less uniform evidence that CNS endothelial cells may also express CCL2. Moreover, the relative contributions of these cell types to the CNS pool of CCL2 during MS/EAE are unclear and the aim of this study was to investigate this further.MethodsCCL2 gene expression was determined by qRT-PCR in different populations of CNS cells at different times following EAE induced by immunization with MOG35–55 peptide and adjuvants, or after injection with adjuvants alone. CNS cells types were isolated by two different protocols: bulk isolation to yield crude microvascular and parenchymal fractions (containing astrocytes, other glia, and neurons), or laser capture microdissection (LCM) to acquire more precisely microvascular endothelial cells, astrocytes or other parenchymal cells.ResultsBoth CNS microvessel and parenchymal populations prepared by crude bulk isolation showed up-regulation of CCL2 mRNA following MOG immunization or injection of adjuvants alone. More exact dissection by LCM revealed microvascular endothelial cells and astrocytes to be the specific sources of CCL2 gene induction following MOG immunization, while only astrocytes showed elevated CCL2 mRNA in response to just adjuvants. Astrocytes displayed the greatest degree of stimulation of CCL2 gene expression following EAE induction.ConclusionsHigh-precision LCM affirmed both microvascular endothelial cells and astrocytes as the major CNS sources of CCL2 gene expression during EAE. Given the high accessibility of the CNS microvascular endothelium, endothelial-derived CCL2 could prove a viable target for therapeutic intervention in neuroinflammatory disease.

Transcriptomic signatures of peroxisome proliferator-activated receptor α (PPARα) in different mouse liver models identify novel aspects of its biology.

BackgroundThe peroxisome proliferator-activated receptor alpha (PPARα) is a ligand-activated transcription factor that regulates lipid catabolism and inflammation and is hepatocarcinogenic in rodents. It is presumed that the functions of PPARα in liver depend on cross-talk between parenchymal (hepatocytes) and non-parenchymal (Kupffer and endothelial cells) fractions as well as inter-organ interactions. In order to determine how cellular composition and inter-organ interactions influence gene expression upon pharmacological activation of PPARα, we performed a meta-analysis of transcriptomics data obtained from mouse hepatocytes (containing only the parenchymal fraction), mouse liver slices (containing both fractions), and mouse livers exposed to a PPARα agonist. The aim was to obtain a comprehensive view of common and model-specific PPARα-dependent genes and biological processes to understand the impact of cross-talk between parenchymal and non-parenchymal fractions as well as the effect of inter-organ interactions on the hepatic PPARα transcriptome.To this end we analyzed microarray data of experiments performed in mouse primary hepatocytes treated with the PPARα agonist Wy14643 for 6 or 24 h (in vitro), mouse precision cut liver slices treated with Wy14643 for 24 h (ex vivo), and livers of wild type and Ppara knockout mice treated with Wy14643 for 6 h or 5 days (in vivo).ResultsIn all models, activation of PPARα significantly altered processes related to various aspects of lipid metabolism. In ex vivo and in vivo models, PPARα activation significantly regulated processes involved in inflammation; these processes were unaffected in hepatocytes. Only in vivo models showed significant regulation of genes involved in coagulation, carcinogenesis, as well as vesicular trafficking and extracellular matrix.ConclusionsPPARα-dependent regulation of genes/processes involved in lipid metabolism is mostly independent of the presence of non-parenchymal cells or systemic factors, as it was observed in all liver models. PPARα-dependent regulation of inflammatory genes requires the presence of non-parenchymal cells, as it was observed only ex vivo and in vivo. However, the full spectrum of PPARα biology at the level of lipid metabolism, immunity, carcinogenesis, as well as novel aspects of PPARα signaling such as coagulation, vesicular trafficking and the extracellular matrix, seems to require systemic factors, as it was observed exclusively in vivo.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1106) contains supplementary material, which is available to authorized users.

Default network activity is a sensitive and specific biomarker of memory in multiple sclerosis

Background: Patients with multiple sclerosis (MS) suffer memory impairment but the link between MS-related neuroanatomical changes (brain atrophy) and memory is relatively weak. Objective: The purpose of this study was to use functional magnetic resonance imaging (fMRI) to investigate task-induced default network (DN) deactivation as a neurophysiologic biomarker of memory functioning in MS. Methods: Twenty-eight MS patients underwent high-resolution MRIs to measure brain atrophy (third ventricle width, cerebral gray matter, cerebral white matter, parenchymal fraction, and thalamic, caudate, hippocampal, and amygdala volumes), and fMRI blood oxygen level dependent (BOLD) signal to measure DN deactivation during sustained attention relative to rest. Neuropsychological assessment of episodic memory was performed on a separate day. We used hierarchical regression to predict memory, with age, education, and depression in step one, brain atrophy within step two, DN activity within step three, and the interaction between brain atrophy and DN activity in step four. Results: Brain atrophy predicted worse memory but DN activity independently predicted memory over-and-above measurements of brain atrophy (R 2=0.108), with greater DN activity (lesser deactivation) linked to better memory. A significant brain atrophy by DN activity interaction indicated a stronger relationship between memory and DN activity among patients with more advanced disease, at which point higher DN activity protects patients from disease/atrophy-related memory impairment. To establish specificity, we showed no relationship between DN activity and non-memory cognition, and no relationship between non-DN brain activity and memory. Conclusion: Maintenance of DN activity during sustained attention was supported as a sensitive and specific neurophysiologic biomarker of episodic memory functioning in MS, even when controlling for neuroanatomical changes (brain atrophy).

Isolation of CD133+ Liver Stem Cells for Clonal Expansion

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In addition, liver stem cells are hypothesized to be the precursors for a subset of liver cancer, Hepatocellular carcinoma. One of the primary challenges to stem cell work in any solid organ like the liver is the isolation of a rare population of cells for detailed analysis. For example, the vast majority of cells in the liver are hepatocytes (parenchymal fraction), which are significantly larger than non-parenchymal cells. By enriching the specific cellular compartments of the liver (i.e. parenchymal and non-parenchymal fractions), and selecting for CD45 negative cells, we are able to enrich the starting population of stem cells by over 600-fold.The proceduresdetailed in this report allow for a relatively rare population of cells from a solid organ to be sorted efficiently. This process can be utilized to isolateliver stem cells from normal murine liver as well as chronic liver injury models, which demonstrate increased liver stem cell proliferation. This method has clear advantages over standard immunohistochemistry of frozen or formalin fixed liver as functional studies using live cells can be performed after initial co-localization experiments. To accomplish the procedure outlined in this report, a working relationship with a research based flow-cytometry core is strongly encouraged as the details of FACS isolation are highly dependent on specialized instrumentation and a strong working knowledge of basic flow-cytometry procedures. The specific goal of this process is to isolate a population of liver stem cells that can be clonally expanded in vitro.

Isolation of CD133+ Liver Stem Cells for Clonal Expansion

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In addition, liver stem cells are hypothesized to be the precursors for a subset of liver cancer, Hepatocellular carcinoma. One of the primary challenges to stem cell work in any solid organ like the liver is the isolation of a rare population of cells for detailed analysis. For example, the vast majority of cells in the liver are hepatocytes (parenchymal fraction), which are significantly larger than non-parenchymal cells. By enriching the specific cellular compartments of the liver (i.e. parenchymal and non-parenchymal fractions), and selecting for CD45 negative cells, we are able to enrich the starting population of stem cells by over 600-fold.The proceduresdetailed in this report allow for a relatively rare population of cells from a solid organ to be sorted efficiently. This process can be utilized to isolateliver stem cells from normal murine liver as well as chronic liver injury models, which demonstrate increased liver stem cell proliferation. This method has clear advantages over standard immunohistochemistry of frozen or formalin fixed liver as functional studies using live cells can be performed after initial co-localization experiments. To accomplish the procedure outlined in this report, a working relationship with a research based flow-cytometry core is strongly encouraged as the details of FACS isolation are highly dependent on specialized instrumentation and a strong working knowledge of basic flow-cytometry procedures. The specific goal of this process is to isolate a population of liver stem cells that can be clonally expanded in vitro.

Plasma levels of 24S-hydroxycholesterol reflect brain volumes in patients without objective cognitive impairment but not in those with Alzheimer's disease

Objectives: Cholesterol has been linked to Alzheimer's disease (AD) and plasma 24S-hydroxycholesterol (24OHC) has been suggested as a surrogate marker for brain cholesterol metabolism. This study investigates the relation of 24OHC as well as markers of extracerebral cholesterol homeostasis (lanosterol, lathosterol, cholesterol, LDL-C, HDL-C and 27-hydroxycholesterol) with brain volumes in memory clinic patients. Methods: 96 patients (33 with subjective cognitive impairment—SCI; 36 with mild cognitive impairment—MCI; 27 with AD) referred to the Memory Clinic at Karolinska University Hospital, Sweden. Plasma assessments were done by isotope dilution-mass spectrometry. MRI measurements were done using custom-made software BMAP (imaging laboratory, Karolinska Institutet), running on HERMES platform. Results: Ratios of 24-hydroxycholesterol, 27-hydroxycholesterol, lanosterol and lathosterol to cholesterol (R_24OHC, R_27OHC, R_lanosterol and R_lathosterol) were significantly lower in patients with AD. In the whole population, after controlling for age, sex, APOE genotype and statins, R_24OHC was positively related to gray matter (GM) fraction. However, when groups were considered separately, the relation to GM volume, GM and parenchymal fractions was significant in the SCI group only ( p < 0.05). There was a significant positive association between cholesterol and white matter (WM) volume, WM and parenchymal fractions in patients with AD. Conclusions: Plasma R_24OHC was lower in patients with AD, but R_24OHC was significantly related to brain volumes in the control group only. One reason may be the previously demonstrated abnormal expression of cholesterol 24S-hydroxylase in astrocytes in AD, which may limit the usefulness of this plasma marker in this specific disease. The findings on cholesterol agree with previous reports of decreasing plasma cholesterol levels in AD patients, suggesting a CNS-mediated effect on extracerebral cholesterol homeostasis.

Fibroblast growth factor-2 levels are elevated in the cerebrospinal fluid of multiple sclerosis patients

In recent years a role has been recognized for fibroblast growth factor (FGF)-2 in the pathogenesis of demyelination and the failure of remyelination in experimental models of multiple sclerosis (MS). FGF-2 levels were determined using a sensitive immunoassay in the cerebrospinal fluid (CSF) of 20 patients with clinically isolated syndrome (CIS), 40 patients with relapsing-remitting (R-R) MS, and 30 patients with secondary progressive (SP) MS, correlated with MRI measures. Control CSF samples were obtained from 20 subjects who underwent lumbar puncture for diagnostic purposes and for whom all instrumental and laboratory analyses excluded systemic and nervous system diseases. FGF-2 levels in the CSF of MS and CIS patients were significantly higher than controls (P<0.001 and P<0.05, respectively). The highest levels were detected in R-R MS patients during relapse and in SP MS patients with an increase of 1 point in EDSS scores in the last 6 months. A significant correlation was found in SP MS patients with lesional load (R=0.43, P<0.01) but not with parenchymal fractions as measures of brain atrophy. A slight increase in serum FGF-2 levels was also found in R-R MS patients during relapse with gadolinium enhancing lesions and in SP patients with disability progression. These findings support the implication of FGF-2 in the pathogenesis of MS and concur with recent reports of the involvement of FGF receptor signalling in the disruption of myelin production in differentiated oligodendrocytes and in the loss of adult oligodendrocytes and myelin in vivo due to FGF-2.

Characterisation of non parenchymal cell fractions with hepatic progenitor cells from surgical resected human liver tissue

Characterisation of non parenchymal cell fractions with hepatic progenitor cells from surgical resected human liver tissue

Cerebral volume changes in multiple sclerosis patients treated with high-dose intravenous methylprednisolone.

Multiple sderosis (MS) patients develop varying degrees of cerebral atrophy, which may already begin at disease onset The purpose of this study is to examine the effect of steroid treatment on cerebral volume in MS patients. Thirty-five MS patients participating in a clinical trial of oral interferon beta, which induded monthly MRI, were included in this study. They suffered from an acute relapse and were treated with intravenous methylprednisolone (IV-MP); 13 of the patients were treated with oral prednisolone tapering after IV-MP. The last MRI scan before and the first (and second for oral tapering patients) scan after IV-MP treatment were used for measuring parenchymal fraction (PF) and ventricular fraction (VF). Changes in PF and VF were analysed using Student's t test. For the total population no significant changes in PF or VF were found. However, the subgroup of patients receiving oral tapering after IV-MP showed changes, compatible with atrophy in both PF and VF, that were significant immediately after IV-MP treatment and still persisted (though not statistically significant anymore) after a mean interval of 30 days. The magnitude of these changes was about the same as the annual change in cerebral volume as reported in natural history studies. Our data indicate that short courses of intravenous steroids (restricted to three or five days) have no major impact, whereas prolonged treatment with oral tapering does significantly affect brain volume. These findings are important for longitudinal studies and clinical trials in which brain volume is used as an outcome measure.

Longitudinal brain volume measurement in multiple sclerosis: rate of brain atrophy is independent of the disease subtype.

In multiple sclerosis (MS), brain atrophy depicted by magnetic resonance imaging reflects overall tissue loss, including axonal loss. To determine the course of atrophy by studying the rate of development of brain atrophy in patients who have different subtypes of MS. Eighty-three patients with MS (42 with relapsing-remitting, 21 with secondary progressive, and 20 with primary progressive) were studied longitudinally, with an interval of 2 to 4 years. Magnetic resonance imaging included T1- and T2-weighted images to obtain 2 brain volume measurements: (1) the parenchymal fraction as a marker of global brain atrophy and (2) the ventricular fraction as a marker of central atrophy. The annualized rate of global and central brain atrophy was compared between those with different subtypes of MS and related to clinical characteristics, including sex, age, disease duration, and disability. There was a significant decrease of the parenchymal fraction (-0.7% per year; SEM, 0.11% per year) and a significant increase of ventricular fraction (3.7% per year; SEM, 0.54% per year) in the total group. Significant tissue loss was also seen in all 3 subtypes of MS; the decrease in parenchymal fraction was not different between subtypes, whereas the increase in ventricular fraction tended to be larger in patients with secondary progressive MS compared with patients with primary progressive MS. Marginal associations were found between clinical determinants and the rate of brain atrophy. Annualized increase in the ventricular fraction was correlated with age (r = -0.26) and duration of symptoms (r = -0.22): younger patients (mainly patients with relapsing-remitting MS who have a limited disability) displayed a larger increase in ventricular fraction compared with older patients. The rate of development of brain atrophy is largely independent of the course of the disease and other clinical characteristics. The relentless loss of tissue occurring in MS is not restricted to later (progressive) phases of the disease, thereby stressing the need for early neuroprotective treatment in MS.

Brain atrophy in multiple sclerosis: impact of lesions and of damage of whole brain tissue.

In multiple sclerosis (MS), brain atrophy measurement on magnetic resonance imaging (MRI) reflects overall tissue loss, especially demyelination and axonal loss. We studied which factor contributes most to the development of brain atrophy extent and severity of lesions or damage of whole brain issue (WBT). Eighty-six patients with MS [32 primary progressive (PP), 32 secondary progressive (SP)] and 22 relapsing-remitting (RR) were studied MRI included T1- and T2-weighted imaging to obtain hypointense T1 lesion volume (T1LV) and two brain volume measurements: 1) the parenchymal fraction (PF; whole brain parenchymal volume/intracranial volume) as a marker of overall brain volume, and 2) the ventricular fraction (VF; ventricular volume/intracranial volume) as a marker of central atrophy. From magnetization transfer ratio (MTR) histograms, the relative peak height (rHp) was derived as an index of damage of WBT (a lower peak height reflects damage of WBT). Multiple linear regression analysis revealed that damage of WBT explains most of the variance of PF (standardized coeffcient beta = 0.59, p < 0.001 for WBT and beta = -0.19, p < 0.05 for T1LV). These findings are independent of disease phase; even in RR patients, damage of WBT plays a dominant role in explaining the variance in overall brain volume. By contrast the variance in VF is explained by both T1LV and damage of WBT (standardized coefficient beta = 0.43, p < 0.001 for T1LV and beta =-0.38, p < 0.001 for WBT). This study shows that overall brain volume (PF) is best explained by damage of WBT, supporting the significance of nonfocal pathology in MS in producing tissue loss. Central atrophy (VP) is determined by both lesion volume and damage of WBT. Our results underline the importance of nonfocal pathology even in the early (RR) phase of the disease.

Optimizing the association between disability and biological markers in MS.

Axonal damage is an important feature of MS pathology and the likely substrate of development of progressive disability. Brain volume measurement on MRI can be used as an overall marker of tissue damage and axonal loss. The authors studied the relation of brain volume measurements with the MS Functional Composite (MSFC) in an attempt to improve the clinico-radiologic association. In 137 patients with MS (80 relapsing-remitting [RR], 36 secondary progressive [SP], and 21 primary progressive [PP]) and 12 healthy controls, a brain MRI scan was obtained. Patients also underwent MSFC and Expanded Disability Status Scale (EDSS) assessments. MRI analysis included determination of hypointense T1- and hyperintense T2-weighted lesion load, and two brain volume measurements: 1) the parenchymal fraction (PF): whole brain parenchyma/intracranial volume; and 2) the ventricular fraction (VF): ventricular volume/whole brain parenchyma. The median PF was smaller and the median VF larger in the patient group (0.81 for PF and 0.029 for VF) than in the control group (0.87 for PF, p < 0.001; and 0.013 for VF, p < 0.01). For the patient population, moderate correlations were found between brain volume measurements and MSFC (0.36 for PF and -0.40 for VF). Patients with short disease duration showed a correlation of MSFC with both brain and lesion volume measurements on MRI, whereas patients with long disease duration only showed a correlation with brain volume measurements. Brain volume measurements are correlated with disability as assessed by the MSFC. Although in the early phase of the disease the amount of focal demyelination is important, the residual brain volume seems to be more relevant in determining disability in later phases of the disease.

Evidence for a Sustained Inflammatory Response of the Hepatic Microcirculation after Hemorrhagic Shock

Defined microcirculatory disturbances have been demonstrated during the first hours after hemorrhagic shock involving complex pathophysiological mechanisms. Since clinical complications associated with the inflammatory reaction that accompanies shock and resuscitation become apparent within days, our aim was to establish an experimental model of hemorrhagic shock which simulates this clinical situation. Therefore, a nonlethal rat model was developed allowing for the investigation of the hepatic microcirculation by intravital microscopy up to 4 days after hermorrhagic shock. Anesthetized Sprague-Dawley rats were subjected to hemorrhagic hyptension by blood withdrawal reducing MAP (mean arterial blood pressure) to 40 mm Hg. After 60 min of hypovolemic hypotension, the animals were resuscitated. After 1, 2, 3, or 4 days, sinusoidal perfusion pattern and leukocyte adhesion characteristics were investigated using intravital fluorescence microscopy. In separate, but parallel experiments, the time course of TNFα (tumor necrosis factor α) and ICAM-1 (intercellular adhesion molecule-1) mRNA expressio was examined in enriched parenchymal and non-parenchymal liver cell fractions by Northern blot hybridization. At day 1, sinusoidal narrowing as seen in previous experiments a few hours after shock was not observed but occurred at days 2 and 3 after shock. Permanent adhesion of leukocytes to the sinusoidal endothelium reached a maximum at day 2 after shock and remained significantly increased until day 4 compared with controls. An early TNFα mRNA accumulation was observed in shock animals but not in sham operated controls. In contrast, a sustained expression of ICAM-1 and prolonged accumulation of white blood cells were observed. The data reflect an early transient TNFα mRNA response and a sustained inflammatory reaction associated with shock and resuscitation. The inflammatory pathological alteration of the hepatic microcirculation after hemorrhagic shock are paralleled by a sustained disturbance of the sinusoidal flow patterns.

Late-gestation tracheal occlusion in the fetal lamb causes rapid lung growth with type II cell preservation.

Fetal tracheal occlusion (TO) results in varying degrees of lung growth. This study examines whether gestational age influences lung growth response following TO. Fetal lambs (term = 145 days) underwent TO early (108 days, n = 6) or late (122 days, n = 6) in gestation. Aspirated lung fluid volume (LFV) and intratracheal pressure (ITP) were recorded daily. Two weeks after TO, the fetuses were sacrificed. Lung growth was assessed by lung weight and stereologic volumetry. Type II cellular density was assessed by computer-assisted morphometry using antisurfactant protein B antibody. After early TO, ITP remained below 2 mm Hg for all but one of the first 5 days. In late TO, ITP rose to 4.8 +/- 1.7 mm Hg by Day 1 and remained elevated. LFV remained lower after early than after late TO (P < 0.05) for 8 days. Thereafter, pressure and volume reached similar levels in both TO groups; both were significantly higher than their respective controls (P < 0.05). Parenchymal fraction (1 - air-space fraction) was significantly smaller after late TO (22.8 +/- 1.2%) than after early TO (31.3 +/- 0.5%). Type II density was 38.0 +/- 12.4 x 10(6)/mL after early TO and 84.0 +/- 24.3 x 10(6)/mL in control (P < 0.05); the difference between late TO and control was not significant. Late tracheal occlusion in fetal lambs caused more rapid lung growth than earlier TO, although ultimate lung size was similar in both groups. Late TO also resulted in greater air-space fraction and better preservation of the type II cell population than early TO. Late-gestation tracheal occlusion may therefore be preferable to prolonged occlusion initiated earlier.

Permeability of a neuroprotective compound NS-7 into brain: Comparison between normal and middle cerebral artery-occluded rats

The pharmacokinetics of 4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy) pyrimidine hydrochloride (NS-7), a novel neuroprotective compound, in brains of normal and ischemic rats were investigated. In normal rats, the concentrations of NS-7 in the cerebral cortex and striatum were more than 10-folds higher than those in plasma during 5 min and 12 h after intravenous injection. The time course changes in plasma concentration of NS-7 were fitted to the two-compartment open model, in which elimination half-life (t 1 2 β) was 6.0 h and distribution volume (V 1) was 4.4. The estimated striatal interstitial concentration of NS-7 measured by microdialysis was unexpectedly low and almost constant after intravenous injection. Subsequently, the level of NS-7 in brain was compared between sham-operated and middle cerebral artery (MCA)-occluded rats. In MCA-occluded rats, the concentrations of NS-7 in the ischemic cerebral cortex and striatum were 64–71 % of those in sham-operated group at 1 h after injection, although the initial concentrations (at 2–5 min) were much lower (about 20 %) in MCA-occluded rats. The t max was observed at 1 h after injection, which was later than that (5 min) determined in sham-operated rats. Moreover, its elimination half-life was longer in MCA-occluded rats than in sham-operated animals. From these results it is suggested that peripherally administered NS-7 readily penetrates into brain, in which it exists for the most part in parenchymal fraction. In addition, substantial amount of NS-7 may distribute to the ischemic brain regions when it was injected after MCA occlusion.

The distribution of non-specific carboxylesterases and glutathione S-transferases in different rat liver cells : Effects of vitamin a deficiency

Non-specific carboxylesterases (carboxylesterases) and glutathione s-transferases (GSTs) are two groups of drug metabolizing enzymes responsible for hydrolysis and glutathione conjugation of xenobiotics. This study was conducted to determine the following: (1) the distribution of carboxylesterase and GST activities in different rat liver cells, (2) the effects of vitamin A deficiency (A —) on the absolute activities and on the distribution of carboxylesterases and GSTs in rat liver. Rat livers were fractionated into parenchymal and non-parenchymal cells by means of collagenase perfusion and differential centrifugation. Non-parenchymal cells were further fractionated by means of Percoll density gradient centrifugation into a layer of Kupffer cells and another layer containing stellate and endothelial cells. Carboxylesterase and GST activities were determined in these fractions. The results of the study show that: (1) both carboxylesterases and GSTs were mainly localized in the parenchymal fraction, (2) there was no significant difference between male and female rats with regard total activity or distribution of carboxylesterases and GSTs in rat liver cells, (3) A — caused a highly significant reduction in carboxylesterase and GST activities in total liver homogenates and parenchymal cells. This reduction was not ameliorated by administration of retinoic acid 18 hr before sacrifice of animals. These results open up a new era of investigations about the potential role of vitamin A in the regulation of detoxification enzymes.