Cerebrospinal Fluid Of Rabbits Research Articles

Cerebrospinal Fluid Changes in Experimental Model of Extraparenchymal Neurocysticercosis: preliminary results

Introduction: Neurocysticercosis (NCC) is the most common parasitic disease affecting the CNS and is considered a public health problem in many countries around the world, including Brazil. The cerebrospinal fluid (CSF) changes provoked by the disorder in humans are well defined in literature and were first described in 1940 by Lange. These modifications include pleocytosis, hyperproteinorrachia and eosinophilia. The majority of the experimental models of NCC published are focused on the parenchymal form of the disease. Few models studied the extraparenchymal form or analyzed the CSF parameters. Objective: To analyze the temporal modifications in the CSF of rabbits after the experimental induction of extraparenchymal NCC. Method: We included 14 male rabbits in this study. Each animal underwent a suboccipital injection of 50 cysts of T. crassiceps into the cisterna magna. Just before the inoculation, CSF samples were collected (sham). The animals were observed for 6 months, with monthly suboccipital punctures for CSF sample collection. Cellularity and protein concentration changes were analyzed throughout the observation period. Results: The initial cell counting average was only 1 (±0.88) cell/μL, raising to 1138.9 (±624.1) cells/μL in the first month and then dropping to 17.2 (±14.4) cells/μL in the sixth month. The differential analysis proved that, after the inoculation, lymphocytes were the most common cells in the CSF (85.1%, ±14.6). A similar pattern was identified with the proteinorrachia: an initial average of 25.8 (±8.3) mg/dL, increasing to 66.5 (±22.5) mg/dL one month later and then gradually decreasing to 40.2 (±15.6) mg/dL in the last month. Conclusion: It was possible to identify a substantial increase in cellularity and protein concentration in the CSF after the induction of NCC in rabbits, followed by a gradual fall over the remaining months. These variations are comparable to the findings in humans. The authors are currently giving continuity to this study, and stronger data will be released in future publications.

Delamanid Central Nervous System Pharmacokinetics in Tuberculous Meningitis in Rabbits and Humans.

Central nervous system tuberculosis (TB) is devastating and affects vulnerable populations. Multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculous meningitis (TBM) specifically are nearly uniformly fatal, with little information being available to guide the treatment of these patients. Delamanid (DLM), a nitro-dihydro-imidazooxazole, is a new, well-tolerated anti-TB drug with a low MIC (1 to 12 ng/ml) against Mycobacterium tuberculosis It is used for the treatment of pulmonary MDR-TB, but pharmacokinetic (PK) data for DLM in the central nervous system (CNS) of patients with TBM are not available. In the present study, we measured DLM concentrations in the brain and cerebrospinal fluid (CSF) of six rabbits with and without experimentally induced TBM receiving single-dose DLM. We report the steady-state CSF concentrations from three patients receiving DLM as part of multidrug treatment who underwent therapeutic drug monitoring. Drug was quantified using liquid chromatography-tandem mass spectrometry. In rabbits and humans, mean concentrations in CSF (in rabbits, 1.26 ng/ml at 9 h and 0.47 ng/ml at 24 h; in humans, 48 ng/ml at 4 h) were significantly lower than those in plasma (in rabbits, 124 ng/ml at 9 h and 14.5 ng/ml at 24 h; in humans, 726 ng/ml at 4 h), but the estimated free CSF/plasma ratios were generally >1. In rabbits, DLM concentrations in the brain were 5-fold higher than those in plasma (means, 518 ng/ml at 9 h and 74.0 ng/ml at 24 h). All patients with XDR-TBM receiving DLM experienced clinical improvement and survival. Collectively, these results suggest that DLM achieves adequate concentrations in brain tissue. Despite relatively low total CSF drug levels, free drug may be sufficient and DLM may have a role in treating TBM. More studies are needed to develop a fuller understanding of its distribution over time with treatment and clinical effectiveness.

ENDOTOXINEMIA, DYSREGULATION AND THE PRE-ILLNESS FORMATION

The study of the conditions of pre-illness and early stages of the disease, as well as their mechanisms, are of particular relevance for modern preventive medicine. It is known that dysregulation is the basis of many pathological processes, the leading link in the pathogenesis of which is endotoxinemia, the severity of which is largely determined by the state of detoxification liver function. The aim of the study was to elucidate the significance of the liver detoxification function and endotoxinemia in the occurrence of dysregulation and the pre-illness formation. In experiments on rats and rabbits it was is established that the direction and nature of changes in the processes of heat exchange, energy and plastic supply of the organism, their hormonal and humoral regulation under the action of E. сoli endotoxin (LPS) depend on the severity of endotoxinemia, the state of detoxification liver function. It is shown that in conditions of functional liver failure, the action of LPS in the body in trace concentrations is accompanied by an increase, and with severe endotoxinemia, a decrease in the activity of the processes of energy and plastic supply of the organism, detoxification. It was found that the ambiguous direction and nature of the changes in the processes of heat exchange, energy and plastic supply of the organism, their neurotransmitter, hormonal and humoral regulation under conditions of endotoxinemia depend on its severity and are largely due to a decrease in the level of arginine in blood plasma and cerebrospinal fluid. In experiments on rats it was revealed that the central action of 1-noradrenaline (10 μg) under the conditions of preliminary introduction of L-arginine hydrochloric acid (100 μg) into the brain ventricles (for 15 min) exerts a more pronounced and prolonged decrease in body temperature in comparison with control, while the duration and severity of the hypothermic effect from the introduction of acetylcholine into the brain ventricles at a dose of 2.5 μg with eserine (5 μg) decreased. It was found that in conditions of bacterial endotoxinemia, accompanied by an increase in body temperature and a decrease in the level of arginine in blood plasma and cerebrospinal fluid in rabbits, intensive utilization of 14C-arginine hydrochloric acid from tissues of the hypothalamus takes place. Endotoxinemia is a key link in the pathogenesis of disregulatory pathology and a universal factor in the development of such a condition as pre-illness.

Proteomic profiling of heat acclimation in cerebrospinal fluid of rabbit

Heat acclimation (AC) is a phenotypic adaptation to the high ambient temperatures. So far, the physiological effects of AC have been well studied, but the molecular mechanisms underlying it, especially the proteomic studies have been rarely reported. Conducting a protein profile of cerebrospinal fluid (CSF) can facilitate the understanding of molecular pathways involved in AC and identifying stress-specific proteins as a laboratory biomarker. In this study we carried out proteomic profiling of the AC in CSF of rabbit, which would allow a deep insight into molecular signals underlying the AC. For this purpose, rabbits were subjected to AC (dry bulb temperature of (36±1)°C, wet bulb temperature of (29±0.5)°C, black-bulb temperature of (40±1.0)°C, 100min per day for 21days, untreated rabbits were used as controls. We adopted a gel-free proteomic approach (iTRAQ) method to identify protein composition in CSF of rabbits with AC. In total, 1310 proteins were identified. Among which 127 were significant up-regulated and 77 were down-regulated. According to the functions, all AC-induced proteins were classified into 8 categories, including plasma protein factors, metabolism-related proteins, energy metabolism-related proteins, cell surface/intercellular matrix proteins, stress related proteins, tumor-related proteins, as well as housekeeping proteins and putative proteins. Meanwhile, a total of 21 pathways were found involved in the developing of AC. Further analysis indicated that proteins mostly close to AC were grouped into two signal pathways, the immune-related signal pathways and the carbohydrate/lipoprotein metabolism-related signal pathways. Our study was first to carry out the whole proteomic picture of AC, and screen out the critical signaling pathways involved in this physical procedure. Biological significanceThis study reported the comparative proteomic analysis of cerebrospinal fluid of rabbits between heat acclimation and normal conditions using the gel-free proteomic mass-spectrometry approach with isotope-labeled samples (iTRAQ) techniques. Mass spectrometry analysis of the proteins from heat acclimated rabbits resulted in the identification of a total of 1310 proteins, among these, 204 proteins were related to the formation of heat acclimation. These proteins were assigned to 8 categories according to their functions. Additionally, 21 pathways involved in infectious diseases, metabolism, immunology, blood circulation, transcriptional regulation and renin-angiotensin were identified by pathway analysis in heat acclimation. This study was the first to use rabbits as a model for unraveling the molecular pathways underlying the establishment of integrative heat acclimation.

Identification of absorbed constituents in the rabbit plasma and cerebrospinal fluid after intranasal administration of Asari Radix et Rhizoma by HS-SPME-GC-MS and HPLC-APCI-IT-TOF-MSn.

Traditional Chinese Medicine (TCM) nasal therapy has been utilized to treat numerous diseases for over two millennia. It has many advantages compared with other routes. In this article, headspace-solid phase microextraction-gas chromatography-mass spectrometry and high performance liquid chromatography-atmospheric pressure chemical ionization-ion trap-time of flight-multistage mass spectrometry were applied for the first time to analyze the absorbed constituents in rabbit plasma and cerebrospinal fluid (CSF) after intranasal administration of Asari Radix et Rhizoma (AR). In total, 47 absorbed AR constituents including 14 monoterpenes, 10 phenylpropanoids, four benzene derivatives, two alkanes, nine N-alkylamides and eight lignans were tentatively identified in the rabbit plasma and CSF. Thirty-three absorbed constituents are found to have different bioactivities related to the pharmacological actions of AR through bibliography data retrieval. These indicated that many types of constituents of TCM can be absorbed at the nasal cavity into both rabbit blood and CSF. This is the first study to explore the absorption of AR, and comprehensively analyze the absorbed constituents after intranasal administration of TCM. These findings extend our understanding of the effective substances of AR, and inspire us to make a hypothesis on the mechanism of additive effect of multiple constituents of TCMs, which is very worthy of further investigation.

107 DETECTION OF BIOTINYLATED-OVULATION-INDUCING FACTOR (OIF) IN CEREBROSPINAL FLUID AND ITS ABILITY TO INDUCE OVULATION

Ovulation-inducing factor (OIF) is a protein in the seminal plasma of llamas that induces a preovulatory LH surge by acting directly or indirectly on the hypothalamic GnRH neurons (Silva et al. 2011 Reprod. Biol. Endocr. 9, 74). We hypothesize that OIF crosses the blood–brain barrier and reaches the hypothalamus via secretion into the cerebro-spinal fluid (CSF) through the choroid plexus. Two experiments were designed to determine whether biotinylation of OIF (as a tracer) alters its bioactivity in a llama model (Experiment 1) and whether it crosses the blood–brain barrier in a rabbit model (Experiment 2). In Experiment 1, llamas with a follicle ≥8 mm in diameter that had grown for 3 consecutive days were assigned randomly to 5 groups (n = 2/group) and given an IV dose of 1) 800 µg of OIF, 2) 800 µg of OIF biotinylated at the amino end; 3) 1600 µg of OIF biotinylated at the amino end, 4) 800 µg of OIF biotinylated at the carboxyl end, or 5) phosphate buffered saline (control). The ovaries were examined daily by transrectal ultrasonography on Day 3 and 8 after treatment (Day 0 = treatment) to detect ovulation and corpus luteum formation. In Experiment 2, adult female rabbits were assigned randomly to 3 groups and given an IV dose of (1) 250 µg of OIF, (2) 250 µg of OIF biotinylated at the amino end, or (3) 250 µg of OIF biotinylated at the carboxyl end. A 50-µL sample of CSF was collected from the cisterna magna under general anesthesia before (0 min) and 10, 20, 30, and 45 min after treatment. The presence of biotinylated OIF in CSF samples was determined by dot blot, using streptavidin-peroxidase and diaminobenzidine. In Experiment 1, the diameter of the follicle at the time of the treatment did not differ among groups (9.7 ± 0.2, 9.4 ± 0.0, 10.5 ± 1.0, 10.1 ± 0.2, 10.3 ± 0.4). Ovulation was detected in all llamas except one llama treated with 800 µg OIF biotinylated at the carboxyl end and both llamas given PBS. The diameter of the corpus luteum did not differ among OIF-treated groups. In Experiment 2, OIF biotinylated at both amino and carboxyl ends was detected in CSF samples at 10, 20, 30, and 45 min after IV administration. No signal was recorded before IV administration (0 min) or in samples from rabbits that were given nonbiotinylated OIF. We conclude that the biotinylation process did not affect OIF bioactivity, and OIF crosses the blood–brain barrier and reaches the CSF in rabbits. Research supported by FONDECYT 1120518, the Natural Sciences and Engineering Research Council of Canada, and the Alpaca Research Foundation.

The comparison of three methods of drawing cerebrospinal fluid in rabbit

Component analysis of cerebrospinal fluid (CSF) is frequently required to probe the cause of disease, pathologic mechanisms and effective drugs in the experimental study of central nervous system. An ideal way of drawing CFS is very important for the successful analysis. However, pros and cons of types of CSF drawing have never been analyzed and compared. The purpose of this study was to choose an ideal method of obtaining CSF in rabbits by comparing the three usual ones: skull drilling, lumbar puncture and atlanto-occipital membrane puncture. In the study, the rabbits were randomly divided into three groups: skull drilling group, lumbar puncture group and atlanto-occipital membrane puncture group, and our modified puncture needles were used. The rates of success, operating time and repeatability, etc. of the three methods were compared. The results suggest that the atlanto-occipital membrane puncture method is the best. The method was the simplest, fastest, and most repeatable of the three, furthermore, with this method, the most CSF was drawn and achieved the highest success rate.

Studies of pathology and VEGF expression in rabbit cerebrospinal fluid metastasis: application of dynamic contrast-enhanced MRI

Objective The objective was to analyze the correlation of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) with vascular endothelial growth factor (VEGF) protein expression and to assess the potential application of DCE-MRI to the rabbit cerebrospinal fluid (CSF) metastasis model. Methods Thirty New Zealand rabbits were divided into experimental and control groups. In the experimental group, VX2 tumor cells were injected into the subarachnoid space at the plane of cisterna magna in 24 rabbits. In the control group, physiological saline was injected into the subarachnoid space at the plane of cisterna magna in six rabbits. DCE-MRI was performed at multiple time points, and several pharmacokinetic parameters, including K trans, K ep and V e, were calculated. Also, VEGF levels in plasma and CSF were evaluated by enzyme-linked immunosorbent assay prior to DCE-MRI examination. After DCE-MRI examination, the rabbits were sacrificed, and the corresponding tumor specimens were harvested. Hematoxylin–eosin staining and VEGF immunohistochemical staining were carried out, and VEGF expression in the specimens was evaluated by the immunohistochemical scoring system. Results Vascular endothelial growth factor positive staining was localized in the cytoplasm and cell membranes of tumor cells, as well as in a subset of epithelial cells. Both VEGF immunohistochemical scores and VEGF expression in CSF and plasma exhibited positive correlations with K trans and K ep values as demonstrated by rank correlation statistical analysis. Conclusions Vascular endothelial growth factor expression in plasma and CSF in the CSF metastasis model was higher than in normal tissues. Therefore, DCE-MRI reliably indicated VEGF expression in the rabbit CSF metastasis model.

Cryptococcus neoformans Requires a Functional Glycolytic Pathway for Disease but Not Persistence in the Host

ABSTRACTCryptococcus neoformans is an important fungal pathogen of immunocompromised individuals, with a close relative, Cryptococcus gattii, emerging as a serious threat for the immunocompetent. During initial infection, C. neoformans colonizes the airspaces of the lungs, resulting in pneumonia, and subsequently migrates to the central nervous system (CNS). We sought to understand fungal carbon utilization during colonization of these fundamentally different niches within the host, in particular the roles of gluconeogenesis and glycolysis. We created mutants at key points in the gluconeogenesis/glycolysis metabolic pathways that are restricted for growth on lactate and glucose, respectively. A phosphoenolpyruvate carboxykinase mutant (the pck1∆ mutant), blocked for entry of 2- and 3-carbon substrates into gluconeogenesis and attenuated for virulence in a murine inhalation model, showed wild-type (WT) persistence in a rabbit cerebrospinal fluid (CSF) model of cryptococcosis. Conversely, both the pyruvate kinase (pyk1∆) and the hexose kinase I and II (hxk1∆/hxk2∆) mutants, which show impaired glucose utilization, exhibited severely attenuated virulence in the murine inhalation model of cryptococcosis and decreased persistence in the CNS in both the rabbit CSF and the murine inhalation models while displaying adequate persistence in the lungs of mice. These data suggest that glucose utilization is critical for virulence of C. neoformans and persistence of the yeast in the CNS.

Effect of dexamethasone on vascular endothelial growth factor in cerebrospinal fluid of rabbits with bacterial meningitis

Objective To investigate the effects of dexamethasone (DEX) on vascular endothelial growth factor( VEGF) in cerebrospinal fluid of rabbits with bacterial meningitis. Methods A total of 36 rabbits were assigned to study,which were randomly divided into meningitis model group (MOD) .dexametha-sone-treated group (DEXT) and control group( CON). CSF was sampled for determining at 6 h, 12 h and 24 h after injection of E. coli suspension. The concentration of VEGF in every CSF sample was determined quantitatively by ELISA. Results There were higher concentrations of CSF VEGF at6h,12h and 24hin M0D(( 1219 ±176) ng/L,( 1343 ±160) ng/L,(981 ±134) ng/L) than that in CON( (374 ±172) ng/L, (370 ± 169) ng/L,(367 ± 171) ng/L) (P<0.01). There was higher brain water content in M0D( (80.8 ± 0.5) % ) than that in CON( (80.0 ± 0.5) % ) (P < 0.01). There was positive correlation between the brain water content and the concentration of CSF VEGF at 24 h( r - 0.919,P < 0.01). Compared with MOD, the concentrations of CSF VEGF in DEXT at 6 h, 12 h,24 h ((941 ±147) ng/L, (1083 ± 123) ng/L, (825 ± 66) ng/L) were decreased significantly(P <0.05), the brain water content was less ((80.4 ±0.5) %) (P < 0.05). Conclusion The secretion of VEGF markedly increases in the pathological process of bacterial meningitis. VEGF contributes to the damage of blood brain barrier and the formation of brain edema. DEX can decrease the degree of brain edema by suppressing the generation of VEGF and lightening the damage of blood brain barrier. Key words: Vascular endothelial growth factor; Meningitis; Cerebrospinal fluid; Dexamethasone; Rabbit

UPLC–MS–MS Analysis of Baicalin in the Cerebrospinal Fluid of Rabbits: Application to a Pharmacokinetic Study

A sensitive and selective ultra-performance liquid chromatographic–tandem mass spectrometric method has been developed for analysis of baicalin in the cerebrospinal fluid of rabbits. Samples were separated on a C18 column with a gradient prepared from acetonitrile and 0.3% aqueous formic acid solution as mobile phase. The target compounds were quantified by multiple reaction monitoring (MRM) using electrospray ionization (ESI). Intra-and inter-day accuracy, precision, and linear range were investigated in detail. The lower limit quantification (LOQ) was 0.108 ng mL−1. The method has been successfully used for evaluation of the pharmacokinetics of baicalin in 12 rabbits.

Corticosteroids, 11β‐Hydroxysteroid Dehydrogenase Isozymes and the Rabbit Choroid Plexus

The epithelial cells of the choroid plexus (CP) are responsible for cerebrospinal fluid (CSF) secretion into the ventricles of the brain. The balance between CSF production and drainage, in part, facilitates a normal intracranial pressure. The secretion of Na(+) and anions by the CP creates an osmotic gradient driving water into the ventricles. This is opposite to classical Na(+) transporting tissues, such as the kidney, where Na(+) and water reabsorption is mediated by 11beta-hydroxysteroid dehydrogenase type 2 that protects the mineralocorticoid receptor by abrogating active cortisol to inactive cortisone. In the human ocular ciliary epithelium, Na(+) and water secretion is dependent on a novel mediator of ciliary epithelial Na(+) transport, 11beta-HSD type 1 (11beta-HSD1), that generates intraocular cortisol. In a mechanism analogous to that of the embryologically related ocular ciliary epithelium, we propose that autocrine regulation of intracranial cortisol is dependent on 11beta-HSD1 expression in the CP epithelial cells. By conducting immunolocalisation studies on brains from New Zealand White Albino rabbits, we defined the expression of 11beta-HSD1 in the secretory CP epithelial cells. Enzyme assays performed on intact rabbit CP whole tissue explants confirmed predominant 11beta-HSD1 activity, generating cortisol that was inhibited by glycyrrhetinic acid (an 11beta-HSD inhibitor). Using the real time-polymerase chain reaction, rabbit CP tissue was found to express levels of 11beta-HSD1, glucocorticoid receptor alpha and serum and glucocorticoid-regulated kinase 1 mRNA comparable to that expressed in rabbit ocular ciliary body, thereby highlighting the similarity between these two tissues. Furthermore, an enzyme-linked immunosorbent assay of rabbit CSF revealed a median cortisol concentration of 1.7 nmol/l (range 1.4-4.3 nmol/l, n = 9). Our data have identified a functional 11beta-HSD1 within the CP, mediating intracranial cortisol bioavailability. Expression of 11beta-HSD1 may be fundamental in the regulation of CSF secretion and the local generation of cortisol may represent a pathophysiological mechanism underlying cortisol-dependent neuroendocrine diseases.

Activin A concentrations in human cerebrospinal fluid are age-dependent and elevated in meningitis

Objective Activin A, and its binding protein, follistatin (FS), are expressed in the central nervous system (CNS). We have previously shown elevated concentrations of FS in the cerebrospinal fluid (CSF) of patients with meningitis and increased concentrations of activin A in the CSF of rabbits with bacterial meningitis. Methods We measured CSF and serum concentrations of activin A and FS in normal subjects and in patients with various neurological diseases using previously validated immunoassays specific for activin A or FS. Results In healthy persons, serum concentrations of both activin A and FS were age-dependent. In CSF, concentrations of activin A ranged from 0.03 to 0.33 ng/ml and were strongly correlated with age in both sexes, whereas FS CSF concentrations were below the assay detection limit in most of the patients. Activin A concentrations in CSF of patients with various neurological diseases, including meningitis, chronic inflammatory CNS diseases, neurodegenerative diseases, tumors in the CNS, cerebral ischemia, intracerebral/subarachnoid hemorrhages, subdural hemorrhages and epileptic seizures, were compared with age- and sex-matched control patients. The comparisons revealed significantly elevated concentrations of activin A in patients with meningitis ( P = 0.017). Serum concentrations of activin A or FS were not affected by any of the neurological diseases examined. Conclusions Our results show for the first time that in normal subjects concentrations of activin A in CSF are correlated with age, and furthermore, that activin A CSF concentrations are elevated in patients with meningitis. The latter underlines a role for activin A in acute inflammatory processes within the CNS.

Temporal expression of inflammatory mediators in brain basilar artery vasculitis and cerebrospinal fluid of rabbits with coccidioidal meningitis.

Strokes due to transmural vasculitis associated with coccidioidal meningitis result in significant morbidity and mortality. The immunological and inflammatory processes responsible are poorly understood. To determine the inflammatory mediators, i.e. cytokines, chemokines, iNOS, matrix metalloproteinase-9 (MMP-9), that possibly contribute to vasculitis, temporal mRNA expression in brain basilar artery samples and MMP-9 protein in the CSF of male NZW rabbits infected intracisternally with 6.5 x 10(4) arthroconidia of Coccidioides immitis were assessed. Five infected and 3 sham-injected rabbits at each time point were euthanized 4, 9, 14 and 20 days post infection. All infected rabbits had neurological abnormalities and severe vasculitis in the basilar arteries on days 9-20. In basilar arteries of infected animals versus controls, mRNAs encoding for IL-6, iNOS, IFN-gamma, IL-2, MCP-1, IL-1beta, IL-10, TNF-alpha, CCR-1, MMP-9, TGF-beta, as well as MMP-9 protein in CSF, were found to be significantly up-regulated. Thus, this study identified inflammatory mediators associated with CNS vasculitis and meningitis due to C. immitis infection. Assessment of the individual contribution of each mediator to vasculitis may offer novel approaches to the treatment of coccidioidal CNS infection. This study also provides unique methodology for immunology studies in a rabbit model.

Increased activin levels in cerebrospinal fluid of rabbits with bacterial meningitis are associated with activation of microglia.

Activin, a member of the transforming growth factor superfamily, is upregulated in a number of inflammatory episodes such as septicemia and rheumatoid arthritis. In the CNS, activin has been predominantly assessed in terms of a neuroprotective role. In this report we characterized the activin response in the CNS in a rabbit model of meningitis. In normal animals, cerebrospinal fluid (CSF) activin levels were higher than those in serum, indicating an intracranial secretion of this cytokine. Following intracisternal inoculation with Streptococcus pneumoniae, activin in CSF was unchanged for the first 12 h and then rose progressively; levels were increased approximately 15-fold within 24 h. Activin levels were correlated positively with CSF protein content and with the number of apoptotic neurons in the dentate gyrus. No apparent correlation was observed between CSF activin concentrations and bacterial titer, lactate concentrations or leukocyte density. Using immunohistochemistry, activin staining was localized to epithelial cells of the choroid plexus, cortical neurons and the CA3 region of the hippocampus, with similar staining intensities in both normal and meningitic brains. However, in meningitic brains there was also strong staining in activated microglia and infiltrating macrophages. Taken together, these results demonstrate that activin forms part of the CNS response to immune challenge and may be an important mediator to modulate inflammatory processes in the brain.

Calpain Inhibitor-like Fraction in Rabbit Cerebrospinal Fluid is probably an Oligopeptide

We have proved before that μ-calpain can be a promising autogenous agent to replace chymopapain in chemonucleolysis treatment. Even in the case of accidental intrathecal injection, the undesirable neural damage of calpaincan be neutralized by normal cerebrospinal fluid (CSF). The aim of this study was to characterize this unknown calpain inhibitor-like component in the CSF of normal rabbits. A heat-stable low molecular weight fraction in the CSF was identified. It was not extracted by chloroform solution, but labile to protease digestion. It inhibited the calpain proteolysis of microtubule-associating protein-2 (MAP-2) and membrane structure proteins of platelets and PC12 cells. It can block the autolytic activation of native human erythrocyte μ-calpain, where the conversion of the large subunit of μ-calpain from the 80-kDa form to the 76-kDa form was obstructed, with accumulation of the intermediate 78-kDa form. A possible calpain inhibition mechanism was discussed. After purification by paper chromatography, gel filtration and reverse phase chromatography, the composition of the calpain inhibitor was investigated. An oligopeptide with molecular weight about 800 Da was identified as a novel calpain inhibitor by partial amino acid sequence.

Evaluation of T-3811ME (BMS-284756), a new des-F(6)-quinolone, for treatment of meningitis caused by penicillin-resistant Streptococcus pneumoniae in rabbits.

T-3811ME (BMS-284756) is a new des-F(6)-quinolone with high levels of activity against gram-positive bacteria, including penicillin-resistant Streptococcus pneumoniae (PRSP) strains. T-3811, the free base of T-3811ME, exhibited potent activity against 28 clinical strains of PRSP isolated clinically (MIC at which 90% of the isolates tested are inhibited, 0.0625 microg/ml). After the intravenous dosing of T-3811ME (20 mg/kg of body weight as T-3811) in rabbits with meningitis caused by PRSP, the area under the concentration-time curve (AUC) of T-3811 in cerebrospinal fluid (CSF) was 5.79 microg. h/ml and was 4.5-fold higher than that of T-3811in the CSF of rabbits without meningitis. In addition, the AUC/MIC for T-3811ME (20 mg/kg as T-3811) in CSF was 185, which was 4.3-fold higher than that for ceftriaxone (administered intravenously at 100 mg/kg). After the administration of any dose of T-3811ME (5, 10, and 20 mg/kg as T-3811), the viable cell counts in CSF decreased in a dose-dependent manner. In particular, after dosing of 20 mg/kg (as T-3811), the viable cell counts in CSF were significantly less than those in the nontreated group (P < 0.01). By histopathological evaluation, 6 h after the administration of T-3811ME (20 mg/kg as T-3811), the thickening of the cerebral meninx and the infiltration of neutrophils into the cerebral meninx were less severe in the treated group than in the nontreated group. T-3811ME (BMS-284756) may be expected to be evaluated for the management of meningitis caused by highly penicillin-resistant pneumococci.

Gene transfer of extracellular superoxide dismutase increases superoxide dismutase activity in cerebrospinal fluid.

Copper-zinc superoxide dismutase (CuZnSOD) is expressed intracellularly, while extracellular SOD (EC-SOD) is released from cells. The purpose of this study was to determine whether gene transfer of CuZnSOD increases SOD activity predominantly in tissues, and gene transfer of EC-SOD increases SOD activity in cerebrospinal fluid (CSF). We also determined whether heparin or dextran sulfate releases EC-SOD into CSF. We injected recombinant adenoviruses expressing EC-SOD (AdEC-SOD), CuZnSOD (AdCuZnSOD), or beta-galactosidase (Adbeta-gal) into the cisterna magna of rabbits. Total SOD activity in CSF was 39+/-11 U/mL (mean+/-SE) before virus injection. Three days later, total SOD activity in CSF increased to 148+/-22 U/mL after AdEC-SOD and 92+/-10 U/mL after AdCuZnSOD (P:<0.05 versus AdEC-SOD), with no change after Adbeta-gal (49+/-5 U/mL). EC-SOD protein was detected in CSF after AdEC-SOD but not AdCuZnSOD or Adbeta-gal. Injection of heparin or dextran sulfate into the cisterna magna increased total SOD activity 27-fold and 32-fold over basal values, respectively, in CSF of rabbits that received AdEC-SOD. In contrast to effects in CSF, total SOD activity in basilar artery and meninges was significantly higher after AdCuZnSOD and tended to be higher after AdEC-SOD than after Adbeta-gal. -We have developed a method for intracranial gene transfer of CuZnSOD and EC-SOD. After gene transfer, CuZnSOD was expressed mainly in tissues, and EC-SOD was released into the CSF, especially after injection of heparin or dextran sulfate. Gene transfer of different isoforms of SOD may be useful in studies of cerebral vascular physiology and pathophysiology.

Systemic neutralization of interleukin-8 markedly reduces neutrophilic pleocytosis during experimental lipopolysaccharide-induced meningitis in rabbits.

Interleukin-8 (IL-8) is elevated in the cerebrospinal fluid (CSF) of patients with meningitis and is proposed to participate in subarachnoid-space pleocytosis. However, intracisternal injection of IL-8 into rabbits failed to induce indices typical of meningitis (leukocyte, tumor necrosis factor, or protein accumulation in the CSF or histopathological changes), indicating that merely increasing the CSF level of this chemokine is insufficient to induce inflammation in this anatomical site. IL-8 treatment did not affect inflammatory responses to subsequently intracisternally administered lipopolysaccharide (LPS). IL-8 was chemotactic for rabbit neutrophils in vitro, and subcutaneous injection of IL-8 (diluted in buffer or CSF) proved the in vivo activity of this peptide and suggested the absence of an IL-8 inhibitor in normal rabbit CSF. LPS-dependent pleocytosis was only slightly diminished by intracisternally administered murine anti-rabbit IL-8 monoclonal antibody (MAb) WS-4 but was dramatically reduced by intravenously administered MAb. Therefore, elevated CSF IL-8 levels may contribute to, but cannot solely account for, neutrophil influx into the subarachnoid space during meningitis. However, inhibition of IL-8 activity of the bloodstream side of the blood-brain barrier effectively reduces pleocytosis, indicating a central role of IL-8 in neutrophil influx into CSF during bacterial meningitis. Thus, inhibition of IL-8 is a possible therapeutic target for adjunct treatment of meningitis.

Disposition of propofol between red blood cells, plasma, brain and cerebrospinal fluid in rabbits.

The disposition of propofol in the blood and brain of New Zealand rabbits was studied in three groups of six rabbits. One group received a single anaesthetic dose; a second group received a 1-h infusion; and a third group was studied after the rabbits were judged to have recovered from a 1-h infusion. There was a high concentration of propofol in the red blood cell fraction and in the brain, however, the red blood cell concentration largely exceeded the one found in the brain in all groups of animals. This is consistent with the high fat solubility of diisopropylphenol. The possible effects of propofol sequestered in red blood cells is discussed.