Devic's neuromyelitis optica: a prospective study of seven patients treated with prednisone and azathioprine.

Seven newly diagnosed patients with Devic's neuromyelitis optica were treated with long-term prednisone and azathioprine, and were followed every 2 months for at least 18 months. Their Expanded Disability Status Scale score improved significantly (mean at baseline, 9; mean at 18 months, 3; p < 0.005), and no relapses occurred for more than 18 months. Multicenter controlled studies are needed to prove the efficacy of this therapeutic regimen.

Matrix metalloproteinases and TIMPs are associated with blood-brain barrier opening after reperfusion in rat brain.

BACKGROUND AND PURPOSE: Reperfusion disrupts cerebral capillaries, causing cerebral edema and hemorrhage. Middle cerebral artery occlusion (MCAO) induces the matrix-degrading metalloproteinases, but their role in capillary injury after reperfusion is unknown. Matrix metalloproteinases (MMPs) and tissue inhibitors to metalloproteinases (TIMPs) modulate capillary permeability. Therefore, we measured blood-brain barrier (BBB) permeability, brain water and electrolytes, MMPs, and TIMPs at multiple times after reperfusion. METHODS: Adult rats underwent MCAO for 2 hours by the suture method. Brain uptake of 14C-sucrose was measured from 3 hours to 14 days after reperfusion. Levels of MMPs and TIMPs were measured by zymography and reverse zymography, respectively, in contiguous tissues. Other rats had water and electrolytes measured at 3, 24, or 48 hours after reperfusion. Treatment with a synthetic MMP inhibitor, BB-1101, on BBB permeability and cerebral edema was studied. RESULTS: Brain sucrose uptake increased after 3 and 48 hours of reperfusion, with maximal opening at 48 hours and return to normal by 14 days. There was a correlation between the levels of gelatinase A at 3 hours and the sucrose uptake (P<0.05). Gelatinase A (MMP-2) was maximally increased at 5 days, and TIMP-2 was highest at 5 days. Gelatinase B and TIMP-1 were maximally elevated at 48 hours. The inhibitor of gelatinase B, TIMP-1, was also increased at 48 hours. Treatment with BB-1101 reduced BBB opening at 3 hours and brain edema at 24 hours, but neither was affected at 48 hours. CONCLUSIONS: The initial opening at 3 hours correlated with gelatinase A levels and was blocked by a synthetic MMP inhibitor. The delayed opening, which was associated with elevated levels of gelatinase B, failed to respond to the MMP inhibitor, suggesting different mechanisms of injury for the biphasic BBB injury.

Differential expression of extracellular matrix remodeling genes in a murine model of bleomycin-induced pulmonary fibrosis.

Exposure to the chemotherapeutic drug bleomycin leads to pulmonary fibrosis in humans and has been widely used in animal models of the disease. Using C57BL/6 bleomycin-sensitive mice, pulmonary fibrosis was induced by multiple intraperitoneal injections of the drug. An increase in the relative amounts of steady-state alpha1(I) procollagen, alpha1(III) procollagen, and fibronectin mRNA as well as histopathological evidence of fibrosis was observed. The effect of bleomycin on the expression of the enzymes responsible for extracellular matrix degradation, the matrix metalloproteinases (MMPs), and their inhibitors (TIMPs), was selective and showed temporal differences during the development of fibrosis. Of the MMPs tested, bleomycin treatment resulted in the up-regulation of gelatinase A and macrophage metalloelastase gene expression in whole-lung homogenates, whereas gelatinase B, stromelysin-1, and interstitial collagenase gene expression was not significantly changed. Timp2 and Timp3, the murine homologues of the respective TIMP genes, were constitutively expressed, whereas Timp1 was markedly up-regulated during fibrosis. The strong correlation between enhanced extracellular matrix gene expression, differential MMP and TIMP gene expression, and histopathological evidence of fibrosis suggest that dysregulated matrix remodeling is likely to contribute to the pathology of bleomycin-induced pulmonary fibrosis.

Trimethyltin induces gelatinase B and urokinase in rat brain.

The neurotoxicant trimethyltin (TMT) increases mRNA levels for cytokines, tumor necrosis factor-alpha, interleukin-1alpha, and interleukin-6. Cytokines induce matrix metalloproteinases (MMPs) and urokinase-type plasminogen activator (uPA). MMPs and uPA disrupt extracellular matrix. Since matrix damage may play a role in the neuropathological changes seen with TMT toxicity, we determined the effect of TMT on proteolytic enzyme production. Adult rats were injected with 8.0 mg TMT/kg. At different times after TMT injection, tissue samples from frontal lobe and hippocampus were assayed for MMPs and uPA, using gelatin-substrate and casein/plasminogen-substrate zymography. Gelatinase B (92 kDa type IV collagenase) production increased significantly in frontal lobe tissue at 24, 48 and 96 h, and in hippocampus at 48 h compared to saline-injected controls. Gelatinase A (72 kDa type IV collagenase) was significantly decreased at 12 and 24 h in frontal lobe compared to controls. Urokinase-type PA was significantly increased in hippocampus at 12 and 96 h, and in frontal lobe at 96 h compared to controls. Gelatinase B and uPA are up-regulated by TMT in frontal lobe and hippocampus, suggesting that they may contribute to the neuropathology of TMT.

Effect of steroids on CSF matrix metalloproteinases in multiple sclerosis: relation to blood-brain barrier injury.

Contrast-enhanced MRI in patients with MS shows that increased permeability of the blood-brain barrier (BBB) commonly occurs. The changes in capillary permeability often precede T2-weighted MRI evidence of tissue damage. In animal studies, intracerebral injection of the matrix metalloproteinase (MMP) 72-kDa type IV collagenase (gelatinase A) opens the BBB by disrupting the basal lamina around capillaries. Steroids affect production of endogenous MMPs and tissue inhibitors to metalloproteinases (TIMPs). To determine the role of MMP activity in BBB damage during acute exacerbations of MS, we measured MMPs in the CSF of patients with MS. Patients (n = 7) given steroids to treat an acute episode of MS had CSF sampled before and after 3 days of methylprednisolone (1 g/day). Patients had a graded neurologic examination and gadolinium-enhanced MRI before treatment. CSF studies included total protein, cell count, and a demyelinating profile. We measured levels of MMPs, urokinase-type plasminogen activator (uPA), and TIMPs by zymography, reverse zymography, and Western blots. The MMP, 92-kDa type IV collagenase (gelatinase B), fell from 216 +/- 70 before steroids to 54 +/- 26 relative lysis zone units (p < 0.046) after treatment. Similarly, uPA dropped from 3880 +/- 800 to 2655 +/- 353 (p < 0.03). Four patients with gadolinium enhancement on MRI had the most pronounced drop in gelatinase B and uPA. Western immunoblots showed an increase in a complex of gelatinase B and TIMPs after treatment, suggesting an increase in a TIMP (p < 0.05). Reverse zymography of CSF samples showed that steroids increased a TIMP with a molecular weight similar to that of mouse TIMP-3 (p = 0.053). Our results suggest that increased gelatinase B is associated with an open BBB on MRI. Steroids may improve capillary function by reducing activity of gelatinase B and uPA and increasing levels of TIMPs.

Tumor necrosis factor-alpha-induced gelatinase B causes delayed opening of the blood-brain barrier: an expanded therapeutic window.

Proteolytic damage is a late event in the molecular cascade initiated by brain injury. Earlier, we proposed that matrix metalloproteinases (MMPs) and urokinase-type plasminogen activator (uPA) are important in secondary brain injury. We have shown that intracerebral injection of activated 72-kDa type IV collagenase (gelatinase A) opens the blood-brain barrier, and that during hemorrhagic brain injury there is endogenous production of 92-kDa type IV collagenase (gelatinase B) and uPA. Therefore, to study the functional link between proteolytic enzymes and blood-brain barrier damage, we induced MMP expression by infusing tumor necrosis factor-alpha (TNF) intracerebrally in rats. Initially, the effect on capillary permeability of increasing doses of TNF, using [14C]sucrose uptake, was measured. Then, the time-course of the capillary permeability change was studied at 4, 16, 24 and 72 h. Expression of MMP and uPA was measured by zymography at 24 h after TNF injection and compared to saline-injected controls. A dose-dependent increase in capillary permeability was seen 24 h after TNF injection. Maximal uptake of [14C]sucrose occurred at 24 h compared to saline-injected controls (P < 0.05). Zymography showed production of gelatinase B, which was significantly greater than in saline-injected controls at 24 h (P < 0.05). Batimastat, a synthetic inhibitor to metalloproteinases, reduced sucrose uptake at 24 h (P < 0.0001), and was effective even when given 6 h after TNF (P < 0.01). Thus, gelatinase B is the intermediate substance linking TNF to modulation of capillary permeability. Agents that interfere with transcription of proteolytic enzymes or block their action may reduce delayed capillary injury, extending the therapeutic window.

Injury-induced 92-kilodalton gelatinase and urokinase expression in rat brain.

BACKGROUND: Proteolytic disruption of the extracellular matrix is important in pathologic processes. We have shown that activated 72-kilodalton (kd) type IV collagenase injected intracerebrally attacks brain extracellular matrix and opens the blood-brain barrier. Therefore, we tested the hypothesis that endogenous production of matrix-degrading proteases may be a factor in secondary brain injury. EXPERIMENTAL DESIGN: Adult rats had a hemorrhagic injury produced by injection of 0.4 units of bacterial collagenase into the caudate/putamen. Endogenous production of matrix metalloproteinases and plasminogen activators (PA) was measured by substrate-gel sodium dodecyl sulfate-polyacrylamide gel electrophoresis (zymography) at 1, 4, 8, 16, 24, and 48 hours, and 7 and 14 days after the injury. RESULTS: Gelatin-containing zymograms had bands of the expected molecular weights from the injected bacterial collagenase at 1 hour. By 8 hours a new 92-kd gelatinase was seen in zymograms. EDTA eliminated the 92-kd band, indicating that it was a metalloproteinase. The 92-kd type IV collagenase/gelatinase was maximally increased by 24 hours (p < 0.0001). Plasminogen-casein zymography showed 40- and 60-kd bands from PA. The 40-kd PA reached a maximum at 24 hours (p < 0.05) and remained elevated for 7 days. Amiloride completely eliminated the 40-kd band and reduced the 60-kd band, suggesting that they were a urokinase-type PA. CONCLUSIONS: Hemorrhagic injury induces both 92-kd type IV collagenase/gelatinase and 40-kd urokinase-plasminogen activator expression in brain. We propose that metalloproteinases and serine proteases take part in a proteolytic cascade that breaks down extracellular matrix, opening the blood-brain barrier with secondary brain edema and cell death.