Matrix metalloproteinases contribute to the blood-brain barrier disruption during bacterial meningitis

In this study, we investigated the involvement of matrix metalloproteinases (MMPs) in the pathophysiology of bacterial meningitis. By using an enzyme immunoassay, high concentrations of MMP-9 were detected in the cerebrospinal fluid (CSF) of adult patients with bacterial meningitis but not in controls, and in patients with Guillain-Barré syndrome. Moreover, we observed significantly elevated concentrations of the tissue inhibitor of metalloproteinase-1 (TIMP-1) in the CSF of patients with bacterial meningitis, compared with controls. In a rat model of meningococcal meningitis, intracisternal injection of heat-killed meningococci caused a disruption of the blood-brain barrier (BBB), an increase in intracranial pressure, and CSF pleocytosis paralleled by the occurrence of MMP-9 activity in the CSF 6 hours after meningococcal challenge. The MMP inhibitor batimastat (BB-94) significantly reduced the BBB disruption and the increase in intracranial pressure irrespective of the time of batimastat administration (15 minutes before and 3 hours after meningococcal challenge) but failed to significantly reduce CSF white blood cell counts. In conclusion, our results suggest that MMPs are involved in the alterations of BBB permeability during experimental meningococcal meningitis.     

Rat brain salsolinol and blood-brain barrier

Behavioral and anatomical consequences of particularly large intrastriatal injections of ibotenic acid are described. Only in the rat with the largest injection, which encompassed almost the entire frontal lobe, were enduring aphagia and adipsia observed; epileptic attacks were, however, not detectable in this or in any other of the rats. In spite of the massiveness of the lesion, neither remote lesions nor damage to passing fibers was observed. It is therefore suggested to substitute kainic acid by ibotenic acid for the production of local, discrete brain lesions.     

Interleukin-8 released into the cerebrospinal fluid after brain injury is associated with blood-brain barrier dysfunction and nerve growth factor production

Revision surgery of cemented implants is indicated when mechanical failure causes severe pain and/or loss of function for the patient. Successful revision arthroplasty of cemented implants requires complete removal of the existing cement. Removal of old cement is an arduous task often causing damage to the surrounding bone tissue. In this study, the authors investigate the use of an Argon laser and the addition of dyes to enhance the laser ablation of bone cement. Methylene blue and red dye #13 were each added separately to polymethylmethacrylate (PMMA) bone cement powder. A continuous wave Argon ion laser (lambda = 514 nm) was used for cement ablation. Cement samples were ablated at different power levels (1.5, 2.3, and 3.0 W) and exposure times (30, 60, 90, 120 sec). The results show that the Argon laser was unable to ablate undyed PMMA. However, the addition of either methylene blue or red dye #13 greatly improved cement ablation by altering the cements' absorption characteristics. Results of Student's t-tests show a statistical difference between red and blue dyed PMMA mean ablation areas at all energy levels tested (P < .0002). As expected, all red ablation areas were greater than blue ablation areas at each energy level tested since red dye absorbs more energy at 514 nm than methylene blue dye. The results of this study suggest that by selectively altering the absorption characteristics of PMMA, laser removal of bone cement can be achieved. In addition, this study also shows that bone tissue does not absorb visible light energy at 514 nm, suggesting that bone cement may be removed with minimal damage to the surrounding bone tissue.     

Transport of alpha-aminoisobutyric acid across the blood-brain barrier studied with in situ perfusion of rat brain

Transport of alpha-aminoisobutyric acid (AIB) from blood to brain in pentobarbital-anesthetized rats was examined using in situ perfusion. In situ perfusion with washed sheep red blood cells allowed the precise control of the composition of the perfusate that was necessary for a detailed examination of the transport of AIB. Retrograde perfusion at 4 ml/min through the left external carotid artery with oxygenated, artificial blood (hematocrit = 0.3) maintained a normal electroencephelogram during a 10 min experiment. The perfusate cerebral blood flow, at a value of 1.2 +/- 0.1 ml/g/min, and the perfusate cerebral plasma volume, at a value of 5.4 +/- 1.9 microliter/g, in the left frontal cortex were within the range of reported in vivo values. The in situ PS product for AIB (3.8 +/- 0.4 microliter/g/min) was higher than the value observed in vivo. AIB uptake was reduced to the in vivo value by 2 mM phenylalanine (1.3 +/- 0.3 microliter/g/min) and equally well by a mixture of neutral amino acids at their normal plasma concentrations but was unaffected by 2 mM methyl-AIB or removal of sodium from the perfusate. A kinetic analysis showed that the apparent Ki for phenylalanine inhibition of AIB transport was 19.8 +/- 4.9 microM. Thus, although AIB has affinity for the large neutral amino acid carrier in the blood-brain barrier, brain uptake by this mechanism in vivo is negligible due to competition by other amino acids in the plasma.     

Lack of autoregulatory response of the cerebral circulation after osmotic opening of the blood-brain barrier in dogs

The reversibility of osmotic opening of the blood-brain barrier was studied in dogs one hour after intracarotid 3 M urea injection. At that time the permeability of cerebral blood vessels to albumin is restored as evidenced by lack of Evans blue extravasation. Despite that, the response of the urea-perfused hemisphere to changes of perfusion pressure was abnormal. Blood flow in that hemisphere followed passively blood pressure changes in contrast to the contralateral hemisphere in which the blood flow remained independent of the perfusion pressure.     

Outwitting the blood-brain barrier for therapeutic purposes: osmotic opening and other means

OBJECTIVE: This article reviews historical aspects of the blood-brain barrier (BBB) and recent advances in mechanisms to deliver therapeutic agents across the BBB for the treatment of intracerebral tumors and other neurological diseases. METHODS: The development of the osmotic BBB disruption procedure as a clinically useful technique is described. Osmotic BBB disruption is contrasted with alternative methods for opening or bypassing the BBB, including pharmacological modification of the BBB with bradykinin and direct intracerebral infusion. RESULTS: Laboratory studies have played a fundamental role in advancing our understanding of the BBB and delivery of agents to brain. Preclinical animal studies will continue to serve an integral function in our efforts to improve the diagnosis and treatment of a number of neurological disorders. Techniques involving the modification of the BBB and/or blood-tumor barrier to increase delivery of therapeutic agents have been advanced to clinical trials in patients with brain tumors with very favorable results. CONCLUSION: Improving delivery of agents to the brain will play a major role in the therapeutic outcome of brain neoplasms. As techniques for gene therapy are advanced, manipulation of the BBB also may be important in the treatment of central nervous system genetic disorders.     

Reversible blood-brain barrier dysfunction to peroxidase after a small stab wound in the rat cerebral cortex

Small stab wounds were made in the rat frontal lobe. The animals were injected with horseradish peroxidase intravenously at different times after the injury in order to study the extravasation of this tracer. There was a leakage of peroxidase into the brain during the first 3 days after the injury. The route of passage from the vessel lumen into the brain was through disrupted blood vessels in the injured region. Endothelial pinocytosis and formation of thin, trans-endothelial channel-like structures with or without a content of peroxidase were two other possible routes of passage across the blood vessels. Occasionally, badly damaged endothelial cells displayed a diffuse cytoplasmic distribution of peroxidase, indication a diffusion into and possibly across these injured cells. No widened tight junctions were seen. Thus, this study indicated four possible routes of passage of horseradish peroxidase across the endothelial cells: cellular gross damage with disrupture of the cells, diffusion across badly injured endothelial cells, possibly pinocytosis and formation of trans-endothelial channel-like structures. The cellular uptake of the tracer was vesicular in most neurons, astrocytes, oligodendrocytes and hematogeneous phagocytes. However, a diffuse distribution of the tracer was seen in some "dark" neurons near leaking vessels in the vicinity of the stab wound.     

CXC chemokines generate age-related increases in neutrophil-mediated brain inflammation and blood-brain barrier breakdown

Children are at greater risk than adults of permanent brain damage and mortality following head injury or infection [1-5]. Rodent models have demonstrated a 'window of susceptibility' in young animals during which the brain parenchyma is at greater risk of acute neutrophil-mediated breakdown of the blood-brain barrier [6-7]. The exact mechanism of this age-related susceptibility to brain inflammation has yet to be defined, but animal models have revealed that the potent pro-inflammatory cytokine interleukin-1beta (IL-1beta) initiates an intense acute neutrophil-mediated inflammatory response in the brains of young rats and mice that is not seen in adults [6]. Here, we demonstrate the rapid induction of CXC chemokines (which contain a Cys-X-Cys motif), in particular the cytokine-induced neutrophil chemoattractant CINC-1, following the intracerebral administration of IL-1beta. The CXC chemokines produced a more intense neutrophil response in young rats than in adults. The IL-1beta-induced blood-brain barrier breakdown in young rats could be attenuated by an anti-CINC-1 neutralising antibody. These results show that the immature central nervous system (CNS) is dramatically more susceptible to the chemotactic effects of CXC chemokines. Blocking the CXC chemokine activity associated with brain inflammation inhibits neutrophil-mediated blood-brain barrier damage and represents a significant therapeutic possibility.     

MAP2, synaptophysin immunostaining in rat brain and behavioral modifications after cerebral postischemic reperfusion

Infection rates are important markers for clinical quality assurance. For internal control, they may only be used under the condition of homogeneous data collection and evaluation according to identical standard operating procedures during the entire investigation period. For inter-hospital comparison, they may only be used if additionally the observed patient groups are well defined and comparable. A survey of the infection rates published during the last 6 years in the German traumatological literature (n = 71) indeed shows (concerning series later than 1985) similar infection rates for procedures in less and in more problematic anatomical regions and in clean and contaminated situations of about 2-3%, after open injuries sporadically max. 10%. Finally, it is demonstrated that conclusions concerning a general "risk of infection" based on infection rates for specific surgical procedures are not possible and vice versa. We strongly recommend the future application of a standardized definition of wound infection. The differentiation between deep and superficial infection should be abandoned. For all mentioned "infection rates" it should be indicated whether it is with reference to the risk of infection of a specific procedure or only a general statement.     

Glucose transporter isoforms in brain: absence of GLUT3 from the blood-brain barrier

Two glucose transporter (GLUT) isoforms have been identified in brain. The GLUT1 isoform is abundant in cerebral microvessels and may be present in glia and neurons, whereas GLUT3 is probably the major neuronal glucose transporter. This study investigates whether GLUT3 is also present in microvessels from rat, human, and canine brain, by means of antisera directed against the divergent C-terminal sequences of mouse and human GLUT3. GLUT1 was detected in whole brain as two molecular mass forms: 55 kDa in microvessels and 45 kDa in cortical neuronal/glial membranes. With the aid of the appropriate antisera to the species-specific sequences, GLUT3 was detected in rat and human cortical membranes but not in isolated rat or human microvessels. These antisera failed to detect GLUT3 in either canine cortical membranes or canine microvessels, implying additional species specificity in the C-terminal sequence.     

Matrix metalloproteinases and tissue inhibitors of metalloproteinases in loose artificial hip joints

The role of extracellular matrix metalloproteinase enzymes and the tissue inhibitors of metalloproteinase in the periprostetic connective tissue matrix of loose artificial hip joints is reviewed. In the periprosthetic granulomatous interface connective tissues between bone and implants and inner cellular regenerating pseudocapsular tissues, matrix metalloproteinase 1, matrix metalloproteinase 2, matrix metalloproteinase 3, matrix metalloproteinase-9, and membrane type 1 matrix metalloproteinase enzymes can be shown in the light of immunohistochemistry, enzyme activity analysis, and messenger ribonucleic acid levels. Tissue inhibitors of metalloproteinase 1 and tissue inhibitors of metalloproteinase 2 also are found in the corresponding tissues. Analysis of matrix metalloproteinase and tissue inhibitors of metalloproteinase interaction shows imbalance between the enzymes and the endogenous inhibitors in favor of matrix metalloproteinase. This induces pathologic connective tissue remodeling in the interface and pseudocapsule. The data suggest that matrix metalloproteinase and tissue inhibitors of metalloproteinase system participate in the extracellular matrix degradation and tissue remodeling in artificial hip joints, and may contribute to the periprosthetic weakening, implant loosening, and osteolysis around implants. More evidence for their active involvement is sought by intervention studies with type specific matrix metalloproteinase inhibitors.     

An in vitro blood-brain barrier model: cocultures between endothelial cells and organotypic brain slice cultures

This communication describes a novel in vitro blood-brain barrier (BBB) model: organotypic slice cultures from the central nervous system were overlaid on endothelial cell monolayers grown on permeable membranes. Morphological, electrophysiological, and microdialysis approaches were carried out to characterize and validate this model. After 10 days in coculture, morphological studies reveal the presence of tight junctions. Electrophysiological recordings of neuronal activity performed on organotypic cultures with or without an endothelial cell monolayer show that amplitude of evoked responses were comparable, indicating good viability of cocultures after 2 weeks. Perfusion of known BBB permeable or nonpermeable molecules was used to test the coculture tightness in conjunction with electrophysiological or microdialysis approaches: application of glutamate (Glu), which doesn't easily cross the BBB, triggers off rhythmic activity only in control cultures, whereas epileptogenic activity was observed in both control cultures and cocultures during perfusions with picrotoxin, a molecule that can diffuse through the BBB. Finally, the microdialysis technique was used to determine the permeability of molecules coming from the perfusion chamber: L-dopa, dopamine, and Glu were employed to assess the selective permeability of the coculture model. Thus, these results indicate that the in vitro model described possesses characteristics similar to those of the BBB in situ and that cocultures of organotypic slices and endothelial cell monolayers have potential as a powerful tool for studying biochemical mechanisms regulating BBB function and drug delivery to the central nervous system.     

Matrix metalloproteinases and their inhibitors in human vitreous

PURPOSE: To conduct zymographic analysis to study the matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) in vitreous samples of patients undergoing pars plana vitrectomy as part of the treatment of vitreoretinal disease. METHODS: Forty-two vitreous samples were collected at the time of pars plana vitrectomy. Diagnoses included severe (exudative) age-related macular degeneration (AMD) (12), macular hole (10), presumed ocular histoplasmosis syndrome (6), proliferative diabetic retinopathy (PDR) (5), epiretinal membrane (4), vitreomacular traction syndrome (2), macroaneurysm with subretinal hemorrhage (1), central retinal vein occlusion with vitreous hemorrhage (1), and proliferative vitreoretinopathy (1). Gelatin zymography, reverse gelatin-zymography, carboxymethylated transferrin zymography, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis were performed on the liquid vitreous samples to assess for MMP and TIMP activity. RESULTS: Progelatinase A occurred in all vitrectomy samples. In addition, a band consistent with TIMP-2 occurred in all samples on reverse zymography. An inhibitor of MMP of a lower molecular weight than TIMP-1 was found in all the samples. A serine proteinase with a broad band around 180 kDa was found in 2 of the 11 AMD vitreous samples. A 75-kDa metalloproteinase was found in several AMD samples, but it was much more abundant in the PDR samples. CONCLUSIONS: Metalloproteinases and their endogenous inhibitors are present in human vitreous and may be involved in the pathogenesis of PDR and other vitreoretinal diseases.     

Myocardial ischemia and reperfusion are associated with an increased stiffness of remote nonischemic myocardium

During and after an ischemic injury, maintenance and recovery of cardiac function may critically depend on remote nonischemic myocardium. Graded myocardial ischemia is associated with an approximately 50% increase in stiffness of nonischemic myocardium. We determined whether this increase in stiffness is unique to the ischemic period or persists during reperfusion. Ten anesthetized (isoflurane 1.0% vol/vol) open-chest dogs were instrumented to measure left ventricular pressure and dimensions (sonomicrometry) in ischemic and nonischemic myocardium. Regional chamber stiffness and myocardial stiffness were assessed using the end-diastolic pressure-length relationship which was modified by stepwise infusion and withdrawal of 200 mL of the animals' own blood during baseline, 45 min low flow ischemia (systolic bulge), and 60 min after the onset of reperfusion. In remote nonischemic myocardium, regional myocardial ischemia was associated with a significant (P < 0.05) increase in chamber stiffness (+44%) and myocardial stiffness (+48%). Sixty minutes after the onset of reperfusion, chamber stiffness (+54%, P < 0.05 versus baseline) and myocardial stiffness (+55%, P < 0.05 versus baseline) remained increased. Thus, the ischemia-induced increase in stiffness of remote nonischemic myocardium persists for at least 60 min after reperfusion.     

The pathophysiology of the blood-brain barrier dysfunction induced by severe hypercapnia and by epileptic brain activity

Antiserum to fentanyl was obtained in rabbits repeatedly injected with carboxyfentanyl conjugated to bovine serum albumin. Using the antiserum, a highly sensitive radioimmunoassay has been developed, based on the dextran-coated charcoal method. It proved possible to assay the drug directly in plasma, in amounts as small as 30 picogram in 0.5 ml. The antibody was highly specific for fentanyl and no cross-reaction was observed with its major metabolites. This sensitive and specific radioimmunoassay method was employed to determine fentanyl in plasma from six volunteers after an intravenous bolus of 0.2 mg, and in plasma from dogs treated both intravenously and subcutaneously with 0.02 mg/kg. The plasma level of fentanyl could be followed for up to 6 h after a therapeutic dose in dogs and man.     

Matrix metalloproteinases and metalloproteinase inhibitors in choroidal neovascular membranes

PURPOSE: Matrix metalloproteinases (MMP) are a family of extracellular matrix degrading enzymes associated with the development of neovascularization. To investigate the possible role of these enzymes in choroidal neovascularization, the mRNA expression of MMPs and tissue inhibitors of metalloproteinases (TIMPs) were analyzed in subfoveal fibrovascular membranes from patients with age-related macular degeneration (AMD). METHODS: Surgically removed subfoveal fibrovascular membranes from five eyes were analyzed for the expression of MMP and TIMP mRNA. In situ hybridization anti-sense and sense riboprobes were generated using DNA complementary to human collagenase (MMP-1), 72 kDa gelatinase (MMP-2), stromelysin (MMP-3), 92-kDa gelatinase (MMP-9), TIMP-1, TIMP-2, and TIMP-3. Vascular endothelial cells were detected using immunostaining for von Willebrand factor. RESULTS: MMP-2 and MMP-9 mRNA were detected in all specimens. Most of the membranes also expressed TIMP-1 and TIMP-3 mRNA, and two of the membranes expressed TIMP-2 mRNA. MMP-2, TIMP-1, and TIMP-2 mRNA had a similar overall distribution that was relatively uniform within the vascularized membrane stroma. MMP-2 expression appeared to be localized mainly to the vascular endothelial cells, whereas TIMP-1 and TIMP-3 were detected in other cell types such as fibroblastlike cells. MMP-9 expression was distinctly expressed by cells at the margins of the membranes and often in proximity to a thickened Bruch's membrane-like layer under the retinal pigment epithelial cells. TIMP-3 mRNA was strongly expressed within the retinal pigment epithelial cell layer and also in the stroma of one membrane. None of the membranes showed detectable MMP-1 or MMP-3 expression. CONCLUSIONS: The results support a role for MMPs in the development of choroidal neovascularization in AMD. The localization of MMP-2 and MMP-9 to the areas of new vessel formation and to the enveloping Bruch's-like membrane, respectively, suggests that MMP-2 and MMP-9 may be cooperatively involved in the progressive growth of choroidal neovascular membranes in AMD.     

The role of the blood-brain barrier transporter PTS-1 in regulating concentrations of methionine enkephalin in blood and brain

The entrainment of the circadian rhythm of locomotor activity was studied in the field mouse Mus booduga in order to examine the relationship between the free-running period (tau) and minimum tolerable light pulse interval of the skeleton photoperiods. The animals were entrained under three different light/dark (LD) schedules, each out of phase with the other. They were then subjected to various skeleton photoperiods created by two repeated light pulses (LPs) interrupting darkness. Animals that selected the shorter interval between the LPs as their "subjective night" had significantly shorter tau (23.13 +/- 0.38 h) as compared to those that selected the longer dark interval as subjective night (tau = 23.87 +/- 0.18 h). When the longer dark interval was 12 h, animals selecting that interval as their subjective night included both long-tau and short-tau individuals. When both intervals of darkness were of equal duration, no difference in the selection of subjective night was seen between short and long-tau animals. When the "dusk" LP for the animals that selected the longer dark interval as subjective night was advanced by 2 h to create a new skeleton photoperiod, the number of transient cycles appearing before steady-state entrainment was found to depend on the duration of the photoperiods. When the night defined by the two LPs was reduced below 6h, a dramatic "phase jump" in the activity rhythm was observed, and the initial phase relationship was restored after a relaxation in the night duration. We observed considerable interindividual variation in the "minimum tolerable light pulse interval of skeleton photoperiods," which we suggest may be due to the observed variation in tau among individuals.     

Specific changes in human brain following reperfusion after cardiac arrest

BACKGROUND AND PURPOSE: Very few reports are available on serial changes in human brain after cardiac arrest. The primary objective of this study is to investigate sequential neuroradiological changes in patients remaining in a persistent vegetative state following resuscitation after cardiac arrest. METHODS: We repeatedly studied eight vegetative patients resuscitated from unexpected out-of-hospital cardiac arrest using computed tomographic (CT) scanning and high-field magnetic resonance (MR) imaging at 1.5 T. RESULTS: In seven of the eight patients, CT scans obtained between days 2 and 6 features symmetrical low-density lesions in the bilateral caudate, lenticular, and/or thalamic nuclei. These ischemic lesions were persistently of low density on serial CT scans. In these seven patients, MR images demonstrated what were thought to be hemoglobin degradation products derived from minor hemorrhages localized in the bilateral basal ganglia, thalami, and/or substantia nigra. Diffuse brain edema in the acute stage and diffuse brain atrophy in the chronic stage were consistent neuroradiological findings. No abnormal enhanced lesions were demonstrated by CT scans. CONCLUSIONS: The most characteristic findings on high-field MR images were symmetrical lesions in the bilateral basal ganglia, thalami, and/or substantia nigra with specific changes suggestive of minor hemorrhages that were not evident on CT scans. We speculate that these minor hemorrhages result from diapedesis of red blood cells in these regions during the reperfusion period through the endothelium disrupted by ischemia-reperfusion insult.     

Beneficial effects of S-adenosyl-L-methionine on blood-brain barrier breakdown and neuronal survival after transient cerebral ischemia in gerbils

In the present study, the effect of bradykinin on basal and precontracted mouse-isolated trachea was investigated. In basal conditions mouse-isolated tracheal rings do not respond to bradykinin. However, when the tracheal rings were precontracted with carbachol (10(-7) M) a relaxation with bradykinin (3 x 10(-9)-3 x 10(-7)) was found. The maximal response amounted 69.7+/-4.1% (n=15) with a pD2 value of 7.2+/-0.21. The selective bradykinin B2 receptor antagonist HOE 140 (10(-10)-10(-8) M) antagonized the bradykinin-induced relaxation, while the bradykinin B1 receptor antagonist des-Arg9-Leu8-bradykinin (10(-6) M) had no influence. The selective bradykinin B1 receptor agonist des-Arg9-bradykinin (10(-6) M) caused a small relaxation (8.4+/-2.5%, n=6), which could be antagonized completely by the selective bradykinin B1 receptor antagonist des-Arg9-Leu8-bradykinin (10(-6) M) while addition of the selective bradykinin B2 receptor antagonist HOE 140 (10(-8) M) was without effect. In the presence of indomethacin (10(-6) M) the relaxation of bradykinin was completely abolished. Pretreatment of the tracheal rings with capsaicin, or the presence of the selective NK1 receptor antagonist RP 67851 (10(-6) M) or the presence of the nitric oxide synthase inhibitor L-NAME (3 x 10(-4) M) had no effect on the bradykinin-induced relaxation. In conclusion, these results demonstrate that the mouse-isolated tracheal is a preparation in which bradykinin exerts a relaxant response via stimulation of bradykinin B2 receptors. This response is probably mediated by prostaglandins.