Cerebrospinal fluid kappa and lambda free light chains in oligoclonal band-negative patients with suspected multiple sclerosis.

BACKGROUND AND PURPOSE: Cerebrospinal fluid (CSF) kappa free light chains (FLCs) may be a more sensitive marker of intrathecal immunoglobulin (Ig)G synthesis compared with oligoclonal bands (OCBs). Our aim was to retrospectively determine the additional value of the kappa and lambda index (CSF FLC/serum FLC)/(CSF albumin/serum albumin) in predicting a multiple sclerosis (MS) diagnosis in a group of OCB-negative patients with suspected MS. METHODS: The CSF and serum kappa and lambda FLCs were tested using the Freelite kit (serum) and Freelite Mx (CSF) assay (The Binding Site Group, Bimingham, UK) in 391 OCB-negative patients with suspected/possible MS and in 54 OCB-positive patients with MS. RESULTS: The CSF kappa FLC levels were below the detection limit (0.27 mg/L) in 61% of patients. Using quantitative data, we found the best kappa index cut-off value for the prediction of MS to be 5.8. A kappa index ≥5.8 was present in 25% of OCB-negative MS (23/92) and in 98% of OCB-positive patients with MS. Using a qualitative approach and a kappa index cut-off of 5.9, based on literature data, we likewise found that 24% of OCB-negative patients with MS had a kappa index ≥5.9, compared with 5.4% of OCB-negative patients without MS (P < 0.001). No reliable data could be obtained for the lambda index; lambda FLCs were below the detection limit (0.68 mg/L) in 90% of CSF samples. CONCLUSIONS: The kappa index could contribute to the identification of OCB-negative patients with a high probability of an MS diagnosis. Using more sensitive techniques might even improve the diagnostic performance of the kappa index and better define the role of the lambda index.

Ageing and inflammation in patients with HIV infection.

Nowadays, HIV+ patients have an expected lifespan that is only slightly shorter than healthy individuals. For this reason, along with the fact that infection can be acquired at a relatively advanced age, the effects of ageing on HIV+ people have begun to be evident. Successful anti-viral treatment is, on one hand, responsible for the development of side effects related to drug toxicity; on the other hand, it is not able to inhibit the onset of several complications caused by persistent immune activation and chronic inflammation. Therefore, patients with a relatively advanced age, i.e. aged more than 50 years, can experience pathologies that affect much older citizens. HIV+ individuals with non-AIDS-related complications can thus come to the attention of clinicians because of the presence of neurocognitive disorders, cardiovascular diseases, metabolic syndrome, bone abnormalities and non-HIV-associated cancers. Chronic inflammation and immune activation, observed typically in elderly people and defined as 'inflammaging', can be present in HIV+ patients who experience a type of premature ageing, which affects the quality of life significantly. This relatively new condition is extremely complex, and important factors have been identified as well as the traditional behavioural risk factors, e.g. the toxicity of anti-retroviral treatments and the above-mentioned chronic inflammation leading to a functional decline and a vulnerability to injury or pathologies. Here, we discuss the role of inflammation and immune activation on the most important non-AIDS-related complications of chronic HIV infection, and the contribution of aging per se to this scenario.

p-Cresol and Cardiovascular Risk in Kidney Transplant Recipients.

p-Cresol Sulphate (pCS) is a uremic toxin that originates exclusively from dietary sources and has a high plasma level related to chronic kidney disease (CKD) and cardiovascular disease (CVD). The aim of our study was to evaluate the plasma levels of pCS in kidney transplant recipients (KTRs) related to estimated glomerular filtration rate (eGFR), traditional risk factors, cardiovascular clinical events and endothelial progenitor cells (EPCs), bone marrow-derived cells for the vascular repair system. We considered 51 KTRs and 25 healthy blood donors (HBDs). pCs levels were analyzed using high-performance liquid chromatography (HPLC) coupled with mass spectrometry with an electrospray ionization (ESI) (LC/ESI-MS/MS) on a triple-quadrupole; EPCs were analyzed using flow cytometric analysis. eGFR was 52.61 ± 19.9 mL/min/1.73 m(2) in KTRs versus 94 ± 21 mL/min/1.73 m(2) in HBDs. We did not find differences in pCS levels between KTRs and HBDs. Levels of pCS were inversely related with eGFR in KTRs and pCS levels were significantly lower in KTRs with eGFR <30 mL/min/1.73 m(2) versus eGFR >30 mL/min/1.73 m(2). Furthermore, there was a difference in pCS levels between eGFR <30 mL/min/1.73 m(2) of KTRs compared with HBDs. Levels of pCS were almost significantly influenced by the presence of a previous vascular event and were inversely related with mature EPCs. These findings suggest that KTRs should not have higher CVD risk than HBDs and their physiological vascular repair system appears to be intact. In KTRs the reduction of eGFR also increased pCS levels and reduced EPCs numbers and angiogenesis capacity. In summary, pCS acts as an emerging marker of a uremic state, helping assess the global vascular competence in KTRs.

Dynamics of adaptive and innate immunity in patients treated during primary human immunodeficiency virus infection: results from Maraviroc in HIV Acute Infection (MAIN) randomized clinical trial.

We evaluated the dynamics of innate and adaptive immunity in patients treated with combined antiretroviral therapy (cART) during primary human immunodeficiency virus infection (PHI), enrolled in a prospective randomized trial (MAIN, EUDRACT 2008-007004-29). After 48 weeks of cART, we documented a reduction in activated B cells and CD8(+) T cells. Natural killer cell and dendritic cell frequencies were measured and a decrease in CD16(+) CD56(dim) with a reciprocal rise in CD56(high) natural killer cells and an increase in myeloid and plasmacytoid dendritic cells were recorded. In conclusion, 48 weeks of cART during PHI showed significant benefits for both innate and adaptive immunity.

Inhibition of autophagy, lysosome and VCP function impairs stress granule assembly.

Stress granules (SGs) are mRNA-protein aggregates induced during stress, which accumulate in many neurodegenerative diseases. Previously, the autophagy-lysosome pathway and valosin-containing protein (VCP), key players of the protein quality control (PQC), were shown to regulate SG degradation. This is consistent with the idea that PQC may survey and/or assist SG dynamics. However, despite these observations, it is currently unknown whether the PQC actively participates in SG assembly. Here, we describe that inhibition of autophagy, lysosomes and VCP causes defective SG formation after induction. Silencing the VCP co-factors UFD1L and PLAA, which degrade defective ribosomal products (DRIPs) and 60S ribosomes, also impaired SG assembly. Intriguingly, DRIPs and 60S, which are released from disassembling polysomes and are normally excluded from SGs, were significantly retained within SGs in cells with impaired autophagy, lysosome or VCP function. Our results suggest that deregulated autophagy, lysosomal or VCP activities, which occur in several neurodegenerative (VCP-associated) diseases, may alter SG morphology and composition.

Castleman's disease mimicking a parotid gland tumor: report of a case and review of the literature.

Castleman's disease (giant lymph node hyperplasia, angiofollicular hyperplasia, lymphoid hamartoma, benign giant lymphoma), is a quite rare and complex disease of lymphoid tissue that causes progressive lymph node enlargement, typically presenting as a solitary mediastinal mass. It was first described by Castleman et al in 1956. The head and neck regions are the second, less common site for this lesion. The preoperative diagnosis is very often extremely difficult and the routine investigations are often inconclusive. A multicentric extension of this disease shows poorer prognosis compared to the unifocal Histologically, three types do exist: the hyaline-vascular type, the plasma cell type and the mixed type. The etiology and pathogenesis is still unclear. In the literature, to our knowledge, only 112 cases have been reported involving head and neck, and only 22, including our own, interesting the parotid gland. In this report we describe a rare singular of Castleman's disease presenting as a tumor of the inferior pole of the parotid gland extending in the submandibular region, in a 35-year-old woman. The patient undergone a surgical treatment and, therefore, the mass was successfully totally removed.

Randomized trial to evaluate cardiometabolic and endothelial function in patients with plasma HIV-1 RNA suppression switching to darunavir/ritonavir with or without nucleoside analogues.

BACKGROUND: We performed a study to evaluate change in cardiometabolic and endothelial function in HIV-infected patients switching to darunavir/ritonavir (DRV/r) monotherapy versus triple therapy. METHODS: The MONARCH trial recruited 30 patients who were taking triple combination therapy and with HIV RNA<40 copies/ mL. Patients were randomized to either DRV/r 800/100 mg once daily (OD) monotherapy or DRV/r 800/100 mg OD plus 2 nucleoside reverse transcriptase inhibitors (NRTIs). The primary objective was to assess endothelial function change from baseline to 24 and 48 weeks in brachial artery flow-mediated dilation (FMD) test; changes in endothelial precursor cells (EPCs) and circulating endothelial cells (CECs) were secondary objectives. RESULTS: At baseline, the median age of participants was 43 years, 77% were men, and median CD4 cell count was 585 cells/µL. The median FMD (%) decreased in both arms in the study period (P ≯ .05), with no statistically significant difference between arms (10.7% at baseline and 6.7% at week 48 in the DRV/r + 2 NRTIs arm; 11.1% at baseline and 8.8% at week 48 in the DRV/r arm). The changes at week 48 were similar in the 2 arms for EPCs and CECs. Total cholesterol and low-density lipoprotein (LDL) cholesterol showed larger rises to week 48 in the DRV/r arm monotherapy group than in the triple-therapy group (+26 vs +9 mg/dL for total cholesterol and +14 vs +5 mg/dL for LDL cholesterol). CONCLUSIONS: In the MONARCH trial, switching from triple combination treatment to DRV/r, with or without nucleoside analogues, did not translate into clinically meaningful reductions in endothelial function as measured by FMD.

Enrichment in c-Kit+ enhances mesodermal and neural differentiation of human chorionic placental cells.

OBJECTIVE: Human term placenta (HTP) has attracted increasing attention as an alternative source of stem cells for regenerative medicine since the amniochorionic membrane harbors stem cells populations that are easily accessible, abundantly available without ethical objections. In the chorionic side of HTP we found a progenitor perivascular "niche" in which rare cells co-express Oct-4 and c-Kit. We investigated the stem cell characteristics and differentiation potential of a chorionic derived population enriched in c-Kit(+) cells and compared this to the unenriched population. STUDY DESIGN: Cells, isolated from the chorion of HTP, were expanded and enriched in c-Kit(+) cells (Chorionic Stem Cells-CSC). Histological staining, immunofluorescence, Western blot and flow cytometry were used to verify the stem cells characteristics of the populations and to compare the differentiation capability towards mesodermal and neural lineages in vitro. RESULTS: The expression of the pluripotent marker Oct-4 was greater in the CSCs compared to the unselected cells (Chorionic Cell-CC) but both Oct-4 and c-Kit expression decreased during passages. After differentiation, CSC displayed stronger chondrogenic and osteogenic potential and a greater adipogenic forming capacity compared to unselected ones. CSC differentiated better into immature oligodendrocytes while CC showed a neuronal progenitor differentiation potential. Moreover, both populations were able to differentiate in hepatogenic lineage. CONCLUSION: CSC display improved Oct-4 expression and a high differentiation potential into mesodermal lineages and oligodendrocytes.

T cell homeostasis in centenarians: from the thymus to the periphery.

The immune system undergoes a process of profound remodelling during aging, referred to as immunosenescence, and characterized by complex modifications of several components. In this review, we discuss recent developments and observations regarding the generation of T cells in the thymus during aging and longevity, and the regulation and maintenance of peripheral blood lymphocytes. The generation of new T cells is indeed crucial to maintain a functional immune system, and is a fundamental step to avoid unsuccessful aging, thus reaching longevity in good health. Mechanisms will be described that are related to the production and maintenance of those lymphocytes defined "recent thymic emigrants", and to the detection of the so called "T cell receptor rearrangement excision circles (TREC)", along with the presence in the periphery of naïve and memory T cells, that can be influenced and regulated by several different mechanisms. Several strategies aimed at improving thymic functionality are currently receiving a growing interest, and some of them are based on molecules that are produced by, and/or act on immune cells. Data on the possible use of these molecules, including cytokines like interleukin (IL)-7, IL-15 and keratinocyte growth factor, to restore thymic function are reviewed and discussed.

Extraskeletal chondroma of the masseter muscle: a case report with review of the literature.

The authors report a case of soft tissue chondroma of the masseter muscle in a 49-year-old man. The tumour was entirely composed of lobules of hyaline cartilage. The literature on head and neck soft tissue chondroma is also reviewed. To the authors' knowledge, this is the first case of muscular soft tissue chondroma in the head and neck region.

Chronic erythropoietin-mediated effects on the expression of astrocyte markers in a rat model of contusive spinal cord injury.

Using a standardized rat model of contusive spinal cord injury (SCI; [Gorio A, Gokmen N, Erbayraktar S, Yilmaz O, Madaschi L, Cichetti C, Di Giulio AM, Vardar E, Cerami A, Brines M (2002) Recombinant human erythropoietin counteracts secondary injury and markedly enhances neurological recovery from experimental spinal cord trauma. Proc Natl Acad Sci U S A 99:9450-9455]), we previously showed that the administration of recombinant human erythropoietin (rhEPO) improves both tissue sparing and locomotory outcome. In the present study, to better understand rhEPO-mediated effects on chronic astrocyte response to SCI in rat, we have used immunocytochemical methods combined with confocal and electron microscopy to investigate, 1 month after injury, the effects of a single rhEPO administration on the expression of a) aquaporin 4 (AQP4), the main astrocytic water channel implicated in edema development and resolution, and two molecules (dystrophin and syntrophin) involved in its membrane anchoring; b) glial fibrillary acidic protein (GFAP) and vimentin as markers of astrogliosis; c) chondroitin sulfate proteoglycans of the extracellular matrix which are upregulated after SCI and can inhibit axonal regeneration and influence neuronal and glial properties. Our results show that rhEPO administration after SCI modifies astrocytic response to injury by increasing AQP4 immunoreactivity in the spinal cord, but not in the brain, without apparent modifications of dystrophin and syntrophin distribution. Attenuation of astrogliosis, demonstrated by the semiquantitative analysis of GFAP labeling, was associated with a reduction of phosphacan/RPTP zeta/beta, whereas the levels of lecticans remained unchanged. Finally, the relative volume of a microvessel fraction was significantly increased, indicating a pro-angiogenetic or a vasodilatory effect of rhEPO. These changes were consistently associated with remarkable reduction of lesion size and with improvement in tissue preservation and locomotor recovery, confirming previous observations and underscoring the potentiality of rhEPO for the therapeutic management of SCI.

Erythropoietin-mediated preservation of the white matter in rat spinal cord injury.

We investigated the effect of a single administration of recombinant human erythropoietin (rhEPO) on the preservation of the ventral white matter of rats at 4 weeks after contusive spinal cord injury (SCI), a time at which functional recovery is significantly improved in comparison to the controls [Gorio A, Necati Gokmen N, Erbayraktar S, Yilmaz O, Madaschi L, Cichetti C, Di Giulio AM, Enver Vardar E, Cerami A, Brines M (2002) Recombinant human erythropoietin counteracts secondary injury and markedly enhances neurological recovery from experimental spinal cord trauma. Proc Natl Acad Sci U S A 99:9450-9455; Gorio A, Madaschi L, Di Stefano B, Carelli S, Di Giulio AM, De Biasi S, Coleman T, Cerami A, Brines M (2005) Methylprednisolone neutralizes the beneficial effects of erythropoietin in experimental spinal cord injury. Proc Natl Acad Sci U S A 102:16379-16384]. Specifically, we examined, by morphological and cytochemical methods combined with light, confocal and electron microscopy, i) myelin preservation, ii) activation of adult oligodendrocyte progenitors (OPCs) identified for the expression of NG2 transmembrane proteoglycan, iii) changes in the amount of the chondroitin sulfate proteoglycans neurocan, versican and phosphacan and of their glycosaminoglycan component labeled with Wisteria floribunda lectin, and iv) ventral horn density of the serotonergic plexus as a marker of descending motor control axons. Injured rats received either saline or a single dose of rhEPO within 30 min after SCI. The results showed that the significant improvement of functional outcome observed in rhEPO-treated rats was associated with a better preservation of myelin in the ventral white matter. Moreover, the significant increase of both the number of NG2-positive OPCs and the labeling for Nogo-A, a marker of differentiated oligodendrocytes, suggested that rhEPO treatment could result in the generation of new myelinating oligodendrocytes. Sparing of fiber tracts in the ventral white matter was confirmed by the increased density of the serotonergic plexus around motor neurons. As for chondroitin sulfate proteoglycans, only phosphacan, increased in saline-treated rats, returned to normal levels in rhEPO group, probably reflecting a better maintenance of glial-axolemmal relationships along nerve fibers. In conclusion, this investigation expands previous studies supporting the pleiotropic neuroprotective effect of rhEPO on secondary degenerative response and its therapeutic potential for the treatment of SCI and confirms that the preservation of the ventral white matter, which contains descending motor pathways, may be critical for limiting functional deficit.

Distinct expression pattern of microtubule-associated protein/microtubule affinity-regulating kinase 4 in differentiated neurons.

Protein kinases of the microtubule affinity-regulating kinase (MARK) family were originally discovered because of their ability to phosphorylate tau protein and related microtubule-associated proteins (MAPs), and their role in the establishment of cell polarity in different contexts. Recent papers have indicated that microtubule affinity-regulating kinase 4 (MARK4) is a gene that is finely regulated at transcriptional level and expressed in two spliced isoforms called MARK4L and MARK4S. We here describe the characterization of the mouse orthologue of the human MARK4 gene. Interestingly, MARK4S is predominantly expressed in the brain, whereas MARK4L shows lower transcript levels in this organ. Using MARK4 antibodies specific for each isoform, we found that both isoforms have an identical expression pattern in the mouse CNS, and are present in a number of neuronal populations. We also found that human microtubule affinity-regulating kinase 4S (hMARK4S), whose expression is not detectable in human neural progenitor cells (HNPCs) and NTera2 (NT2) cells, is up-regulated in both cell systems from the very early stages of neuronal differentiation. This indicates that neuronal commitment is marked by MARK4S up-regulation. In conclusion, this study provides the first direct evidence suggesting that MARK4 is a neuron-specific marker in the CNS, and the up-regulation of MARK4S during neuronal differentiation suggests that it plays a specialized role in neurons.

Jejunal creatine absorption: what is the role of the basolateral membrane?

The mechanism of the intestinal creatine absorption is not well understood. Previous studies have established the involvement of a CT1 carrier system in jejunal apical membrane. The current research was aimed at completing the picture of creatine absorption. To investigate the process supporting creatine exit from enterocyte, basolateral membrane vesicles isolated from rat jejunum were used. The presence of various symport and antiport mechanisms was searched and a NaCl-dependent electrogenic transport system for creatine was evidenced, which shares some functional and kinetic features with the apical CT1. However, Western blot and immunohistochemical experiments ruled out the presence of a CT1 transporter in the basolateral membrane. Further studies are required to identify the basolateral transport mechanism. However, in the in vivo conditions, the NaCl gradient is inwardly directed, therefore such a mechanism cannot energetically mediate the exit of creatine from the cell into the blood during the absorptive process, but rather it may drive creatine into the enterocyte. To shed more light on the creatine absorption process, a possible creatine movement through the paracellular pathway has been examined using the jejunal tract everted and incubated in vitro. A linear relationship between creatine transport and concentration was apparent both in the mucosa-to-serosa and serosa-to-mucosa directions and the difference between the two slopes suggests that paracellular creatine movement by solvent drag may account for transintestinal creatine absorption. As a matter of fact, when transepithelial water flux is reduced by means of a mucosal hypertonic solution, the opposite creatine fluxes tend to overlap. The findings of the present study suggest that paracellular creatine movement by solvent drag may account for transintestinal creatine absorption.

Expression of GABA transporters, GAT-1 and GAT-3, in the cerebral cortex and thalamus of the rat during postnatal development.

The cellular and subcellular localization of two GABA transporters, GAT-1 and GAT-3, was investigated using immunocytochemical methods in the rat cerebral cortex and thalamus during postnatal development. The distribution of the transporters is compared with that of the neuronal marker GABA, and with that of vimentin and of glial fibrillary acidic protein, which identify immature and mature astrocytes, respectively. Our observations show that the two transporters are already expressed at birth in both brain areas with the same cellular localization as in adult rats, as GAT-1 is present in growth cones and terminals only in the cortex, whereas both transporters are expressed in astrocytes in the cortex and thalamus. The distribution of GAT-1 and GAT-3 undergoes postnatal changes reflecting in general the neurogenetic events of the neocortex and thalamus and, more specifically, the maturation of GABAergic innervation. The adult-like pattern of expression is achieved in the third postnatal week in the cortex and in the second postnatal week in the thalamus. The early expression of GAT-1 in GABAergic terminals confirms previous studies showing the existence of neuronal mechanisms of GABA uptake from the embryonic stages. As for the glial localization, the precocious existence of two astrocytic GABA transporters suggests that they operate through different functional mechanisms from birth, whereas their exclusively glial expression in the thalamus indicates that the astroglia plays a major role in the transport, recycling and metabolism of thalamic GABA.

In vitro effect of leptin on LH release by anterior pituitary glands from female rats at the time of spontaneous and steroid-induced LH surge.

OBJECTIVE: The purpose of this work was to study the direct effect of leptin on LH release by anterior pituitary glands from female rats at the time of spontaneous and steroid-induced LH surge. METHODS: LH responsiveness to leptin by pituitaries from rats killed in the afternoon (1500 h) at different stages of the 4-day estrous cycle (diestrus-1: D1; diestrus-2: D2; proestrus; estrus), ovariectomized (OVX; 15 days post-castration) and ovariectomized steroid-primed (OVX-E(2)/Pg; pretreated with 5 microg estradiol and 1 mg progesterone), was evaluated in vitro. Hemi-adenohypophyses were incubated in the presence of synthetic murine leptin for 3 h. RESULTS: Addition of increasing concentrations of leptin (0.1-100 nmol/l) to the incubation medium of proestrus pituitaries produced a dose-related stimulation of LH release; the maximal increase to 315% of control was obtained with 10 nmol/l leptin. Leptin (10 nmol/l) enhanced LH release at all days of the estrous cycle, the greatest response occurring in proestrus (318%) and the lowest at D1 (123%). In order to evaluate the role of nitric oxide (NO) in the action of leptin on LH release, glands from proestrus rats were incubated in the presence of 10 nmol/l leptin with or without 0.3 mmol/l N(G)-monomethyl-l-arginine (NMMA), a competitive inhibitor of NO synthase (NOS). NMMA completely suppressed the stimulation of LH release induced by leptin. Leptin also stimulated LH release by pituitaries from OVX rats, and treatment with steroid hormones led to a marked increase in the response (OVX: 162% compared with OVX-E(2)/Pg: 263%; P<0.05). For comparative analysis, a similar experimental procedure was carried out using GnRH (10 nmol/l). Leptin acts at the pituitary level in a similar manner as GnRH, although with significantly lower potency. CONCLUSIONS: These results confirm and extend previous reports regarding a direct action of leptin at the pituitary level, stimulating LH release by anterior pituitaries from female rats at the time of spontaneous and steroid-induced LH surge. In the female rat pituitary this leptin action is controlled by gonadal steroids and mediated by NO.

Early vacuolization and mitochondrial damage in motor neurons of FALS mice are not associated with apoptosis or with changes in cytochrome oxidase histochemical reactivity.

Overexpression of mutated superoxide dismutase (SOD1) in transgenic mice causes a progressive motor neuron degeneration in the spinal cord similar to that in human amyotrophic lateral sclerosis (ALS). Ultrastructural analysis of motor neurons at different stages of the disease in transgenic C57BL/6 mice carrying the G93A mutation of SOD1 showed, at about 2 weeks of age, much earlier than the initial symptoms of the disease, microvacuoles in the cytoplasm, with marked swelling of the mitochondria. Nuclei with an apoptotic morphology were never observed in these motor neurons. Swollen mitochondria were also seen in the distal part of motor axons of phrenic nerves and in the large axons of sciatic nerves before the onset of the disease, but no mitochondrial alterations were seen in skeletal muscles or in the small sciatic nerve axons. Moreover, we found no apparent changes in the histochemical reactivity of cytochrome oxidase in motor neurons of transgenic mice even at the advanced stage of the disease, suggesting that partial neuronal activity in these cells may be maintained despite the altered mitochondria. Immunoreactivity for human SOD1 was high around vacuoles in the motor neurons of transgenic mice but no cytoplasmic intracellular SOD1 aggregates were observed. Our data indicate that mitochondrial swelling may be an important factor triggering the cascade leading to progressive motor neuron death. Activation of the mitochondrial permeability transition pore may be involved in this process, through excitotoxicity or other neurotoxic stimuli.

Blood-brain barrier preservation in the in vitro isolated guinea pig brain preparation.

The morphofunctional preservation of the blood-brain barrier (BBB) was evaluated in the isolated guinea pig brain maintained in vitro by arterial perfusion. Electron microscopy evaluation after 5 hr in vitro demonstrated that cerebral capillaries and BBB specializations in this preparation retain features compatible with structural integrity. BBB-impermeable and -permeable atropine derivatives arterially perfused to antagonize carbachol-induced fast oscillatory activity confirmed the functional preservation of the BBB in vitro. To study BBB function further, changes in extracellular K+ concentration during arterial perfusion of a high-K+ solution were measured with K+-sensitive electrodes positioned in the cortex and, as control, at the brain venous outlet, where the solution perfused through the brain arterial system was collected. After 5 hr in vitro, the [K+](o) values measured during high-K+ perfusion in the piriform and entorhinal cortices were 5.02 +/- 0.17 mM (mean +/- SE) and 5.2 +/- 0.21 mM, respectively (n = 6). Coperfusion of the high-K+ solution with the Na+/K+ pump blocker ouabain (10 microM; n = 4) induced consistently spreading depression preceded by a rise in [K+](o). Finally, sporadic, isolated spots of extravasation of the fluorescent marker fluorescein isothiocyanate (FITC)-dextran preferentially circumscribed to deep cortical layers was observed in brains perfused with FITC-dextran after 5 hr in vitro. The study demonstrates that the in vitro isolated guinea pig brain is viable for studying cerebrovascular interactions and BBB permeability of compounds active in the central nervous system.

Chondroitin sulfate proteoglycans in the rat thalamus: expression during postnatal development and correlation with calcium-binding proteins in adults.

Postnatal expression of chondroitin sulfate proteoglycans was studied in the rat thalamus by immunocytochemistry and Western immunoblotting techniques with monoclonal antibodies that recognize carbohydrate epitopes (clones CS-56, 1-B-5, 2-B-6). The complex of the results shows that these antibodies recognize mostly nonoverlapping molecules whose expression is regulated during postnatal development. Chondroitin sulfate proteoglycans, recognized by antibody CS-56, and hyaluronan, identified by antibody 1-B-5 after hyaluronidase digestion, are abundant in the neuropil of most thalamic nuclei at the perinatal stage and progressively decrease during the second week of life, attaining levels barely detectable by immunocytochemistry at the end of the third week. In adult thalamus, chondroitin sulfate proteoglycans of high molecular mass, bearing glycosaminoglycans unsulfated in the linking region, and recognized by antibody 1-B-5 are confined to perineuronal nets around neurons chiefly localized in thalamic reticular nucleus. The immunoreactvity for antibody 2-B-6, specific for chondroitin-4-sulfate, is low at the perinatal stage and is not detectable in adult thalamus. Double-immunolabeling has shown that, along the rostrocaudal extension of reticular nucleus, the most developed perineuronal nets are associated with a subset of neurons expressing calretinin, and not with parvalbumin-positive neurons, which represent the largest neuronal population of the nucleus. The distribution of perineuronal nets supports the presence, in thalamic reticular nucleus, of neuronal subpopulations with different morphological and physiological features.