Tissue inhibitor of matrix metalloproteinases-3 (TIMP-3) lacks involvement in bacterial collagenase-induced intracerebral hemorrhage in mouse.

Intracerebral hemorrhage (ICH) leads to delayed cell death in the regions around the hemorrhagic mass. Apoptosis has been identified in the dying cells, but the mechanism involved is unclear. Others and us have shown that matrix metalloproteinases (MMPs) are increased in ICH and could directly contribute to cell death. Tissue inhibitor to metalloproteinases-3 (TIMP-3) facilitates apoptosis in cancer cells and neurons by inhibiting the shedding of tumor necrosis factor-alpha (TNF-alpha) death receptors, Fas and p55TNF receptor 1, by MMP-3 and TNF-alpha converting enzyme (TACE), respectively. Therefore, TIMP-3 may contribute to cell death in ICH. We adapted the bacterial collagenase-induced hemorrhage (CIH) model to the mouse. Adult C57Bl/6 and Timp-3 knockout mice had CIH. Expression of mRNA for TIMP-3 was determined by real-time PCR. Hemorrhage volume and numbers of apoptotic cells were measured by unbiased stereology. Timp-3 mRNA was similar in the knockout and wild-type mice prior to injury and induction of CIH failed to cause an increase in Timp-3 mRNA in the wild-type. Furthermore, there were no differences found in the hemorrhage size or in the numbers of apoptotic cells between the Timp-3 knockout or wild-type. We were unable to prove the hypothesis that TIMP-3 is involved cell death in CIH in the mouse.

Immunohistochemistry of matrix metalloproteinases in reperfusion injury to rat brain: activation of MMP-9 linked to stromelysin-1 and microglia in cell cultures.

Reperfusion damages the blood-brain barrier (BBB). Matrix metalloproteinases (MMPs) are associated with the opening of the BBB, but their cellular localization and activation mechanisms are uncertain. We used immunohistochemistry to determine the cellular localization of the MMPs in reperfused rat brain, and cell cultures to study their activation. Spontaneously hypertensive rats (SHR) had a 90 min middle cerebral artery occlusion (MCAO) followed by reperfusion for times from 3 h to 21 days. Frozen sections were immunostained with antibodies to gelatinase A (MMP-2), stromelysin-1 (MMP-3), and gelatinase B (MMP-9). Sham-operated control rats showed MMP-2 immunostaining in astrocytic processes next to blood vessels. After 3 h of the onset of reperfusion MMP-2 immunostaining increased in astrocytes. At 24 h immunoreactivity for MMP-3 and MMP-9 appeared. MMP-3 co-localized with activated microglia (Ox-42+) and ischemic neurons (NeuN+). MMP-9 immunostaining was seen at 48 h in endothelial cells, neutrophils, and neurons. At 5 and 21 days intense MMP-2 staining was seen in reactive astrocytes around the ischemic core. Studies of activation of the MMP were done in lipopolysaccharide (LPS)-stimulated astrocyte and microglia cultures. Stimulated astrocytes produced an activated form of MMP-2. When microglia were stimulated, they activated MMP-9. Immunostaining showed MMP-3 in cultures of enriched microglial cells. The hydroxymate-type, MMP inhibitor, BB-1101, blocked the activation of MMP-2 and MMP-9 by LPS in mixed glial cultures. We propose that MMP-2 is normally present in astrocytic end feet, and that during ischemia MMP-9 and MMP-3 are produced. MMP-3 in microglia/macrophages may be activating proMMP-9. Our results show that a differential expression of MMPs by astrocytes, microglia, and endothelial cells at the blood vessels is involved in the proteolytic disruption of the BBB.

Neutral proteases and disruption of the blood-brain barrier in rat.

Blood-brain barrier disruption is common in many neurological diseases. Matrix metalloproteinases are induced in brain injury and increase capillary permeability by attacking the extracellular matrix around cerebral capillaries. Other neutral proteases are also increased in sites of secondary injury, and may contribute to the proteolysis of the blood-brain barrier. Therefore, we studied capillary permeability and histological tissue damage after intracerebral injection of neutrophil elastase, cathepsin G, heparatinase and plasmin. Adult rats were injected intracerebrally with an enzyme. After 1, 4 or 24 h, measurements were made of brain uptake of a radiolabeled tracer, [14C]sucrose. Enzymes that significantly increased capillary permeability were injected into other rats for histological assessment of tissue damage. Elastase increased capillary permeability significantly when compared with controls; maximal damage was seen at 4 h. Plasmin produced smaller increases in permeability at 4 h, exerting its maximal effect on sucrose uptake at 24 h. Cathepsin G had a small effect at 4 h. Heparitinase had no effect. Histologic examination of elastase-injected brains at 24 h revealed multifocal perivascular and intraparenchymal acute hemorrhages accompanied by a polymorphonuclear cell infiltrate. Elastase-injected brains were microscopically similar to saline-injected brains at 1 and 4 h. Plasmin produced fibrinoid changes in the blood vessels at 24 h, coinciding with the maximal increase in capillary permeability. We conclude that neutrophil elastase attacks the capillary extracellular matrix, causing extensive hemorrhage, while plasmin leads to increased vascular permeability and fibrinoid necrosis of blood vessel walls. Differential effects of neutral proteases released secondary to injury could be important in both the acute changes in blood vessel permeability and long-term alterations in vessel structure.

Isolation of an immunosuppressive fraction in ultrafiltrate from uremic sera.

An improved purification method for isolating an immunosuppressive fraction from uremic ultrafiltrates was described. Lyophilized ultrafiltrates were fractionated on Sephadex G-15 and the active fraction was further purified using high-performance liquid chromatography. The inhibitory effect of eluates on in vitro lymphocyte proliferation was assessed at the different steps of purification. Results obtained after enzyme treatment suggested that the substance(s) responsible for the immunosuppressive activity is (are) of peptidic nature. This peptide appears to be different from the other peptides previously reported, which have a much lesser activity in the lymphocyte proliferation assay.