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A gelatin in situ-overlay technique localizes brain matrix metalloproteinase activity in experimental focal cerebral ischemia

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Title: A gelatin in situ-overlay technique localizes brain matrix metalloproteinase activity in experimental focal cerebral ischemia
Authors: Loy, M.1, Burggraf, D.1, Martens, K.H.1, Liebetrau, M.1, Wunderlich, N.1, Bültemeier, G.1, Nemori, R.2, Hamann, G.F.1 hamann@brain.nefo.med.uni-muenchen.de
Source: Journal of Neuroscience Methods. May2002, Vol. 116 Issue 2, p125. 9p.
Subjects: Photographic gelatin, Cerebral ischemia, Cerebrovascular disease
Abstract: To determine the activity of matrix metalloproteinases (MMP), especially MMP-2 and MMP-9, which play an important role in ischemic stroke and intracerebral hemorrhage, we adapted a simple and rapid method for localizing gelatinase activity to a gelatin film in situ-overlay technique previously used in cancer research. Ten micrometer cryosections of rat brain from controls and animals subjected to 3 h of ischemia and 48 h of reperfusion (suture model for transient cerebral ischemia) were used. After thawing, a gelatin film with a polyester base was put on the slide, incubated for 24 h at 37 °C, stained with Ponceau S, and then discolored in bi-distilled water. Non-staining areas on the film corresponded to lysis zones, caused by activated MMPs. This was proven by MMP incubation at various concentrations on the plain gelatin film and pretreatment with EDTA (an MMP inhibitor), which prevents lysis zones in normal and ischemic brains. As confirmatory tests, SDS-PAGE zymography was used to define MMP activity, and also MMP-2 immunohistochemistry to detect the possibly cellular origin of MMPs. Normal rat brain exhibited a low background activity, which was visible as a light halo-like lysis zone over and around the brain. Areas in normal brain with medium MMP activity were within the white matter (corpus callosum, anterior commissure, and cerebellum). Ischemic brain exhibited high activity lysis zones within the infarcted area (detected by microtubuli associated protein-2 staining). These zones consisted of microscopically small lysis holes with a diameter of about 10–20 μm. Immunohistochemistry showed that especially microvessels expressed MMP antigen. SDS-PAGE zymography differentiated between a high level of activated MMPs in the ischemic area and a low level in the non-ischemic basal ganglia. The gelatin film in situ-overlay technique is able to localize MMP activity in ischemic rat brain tissue on a microscopic level. [Copyright &y& Elsevier]
Copyright of Journal of Neuroscience Methods is the property of Elsevier B.V. and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)
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Header DbId: egs
DbLabel: Engineering Source
An: 7814045
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PubTypeId: academicJournal
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Items – Name: Title
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  Data: A gelatin in situ-overlay technique localizes brain matrix metalloproteinase activity in experimental focal cerebral ischemia
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  Data: <searchLink fieldCode="AR" term="%22Loy%2C+M%2E%22">Loy, M.</searchLink><relatesTo>1</relatesTo><br /><searchLink fieldCode="AR" term="%22Burggraf%2C+D%2E%22">Burggraf, D.</searchLink><relatesTo>1</relatesTo><br /><searchLink fieldCode="AR" term="%22Martens%2C+K%2EH%2E%22">Martens, K.H.</searchLink><relatesTo>1</relatesTo><br /><searchLink fieldCode="AR" term="%22Liebetrau%2C+M%2E%22">Liebetrau, M.</searchLink><relatesTo>1</relatesTo><br /><searchLink fieldCode="AR" term="%22Wunderlich%2C+N%2E%22">Wunderlich, N.</searchLink><relatesTo>1</relatesTo><br /><searchLink fieldCode="AR" term="%22Bültemeier%2C+G%2E%22">Bültemeier, G.</searchLink><relatesTo>1</relatesTo><br /><searchLink fieldCode="AR" term="%22Nemori%2C+R%2E%22">Nemori, R.</searchLink><relatesTo>2</relatesTo><br /><searchLink fieldCode="AR" term="%22Hamann%2C+G%2EF%2E%22">Hamann, G.F.</searchLink><relatesTo>1</relatesTo><i> hamann@brain.nefo.med.uni-muenchen.de</i>
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  Data: <searchLink fieldCode="JN" term="%22Journal+of+Neuroscience+Methods%22">Journal of Neuroscience Methods</searchLink>. May2002, Vol. 116 Issue 2, p125. 9p.
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  Data: <searchLink fieldCode="DE" term="%22Photographic+gelatin%22">Photographic gelatin</searchLink><br /><searchLink fieldCode="DE" term="%22Cerebral+ischemia%22">Cerebral ischemia</searchLink><br /><searchLink fieldCode="DE" term="%22Cerebrovascular+disease%22">Cerebrovascular disease</searchLink>
– Name: Abstract
  Label: Abstract
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  Data: To determine the activity of matrix metalloproteinases (MMP), especially MMP-2 and MMP-9, which play an important role in ischemic stroke and intracerebral hemorrhage, we adapted a simple and rapid method for localizing gelatinase activity to a gelatin film in situ-overlay technique previously used in cancer research. Ten micrometer cryosections of rat brain from controls and animals subjected to 3 h of ischemia and 48 h of reperfusion (suture model for transient cerebral ischemia) were used. After thawing, a gelatin film with a polyester base was put on the slide, incubated for 24 h at 37 °C, stained with Ponceau S, and then discolored in bi-distilled water. Non-staining areas on the film corresponded to lysis zones, caused by activated MMPs. This was proven by MMP incubation at various concentrations on the plain gelatin film and pretreatment with EDTA (an MMP inhibitor), which prevents lysis zones in normal and ischemic brains. As confirmatory tests, SDS-PAGE zymography was used to define MMP activity, and also MMP-2 immunohistochemistry to detect the possibly cellular origin of MMPs. Normal rat brain exhibited a low background activity, which was visible as a light halo-like lysis zone over and around the brain. Areas in normal brain with medium MMP activity were within the white matter (corpus callosum, anterior commissure, and cerebellum). Ischemic brain exhibited high activity lysis zones within the infarcted area (detected by microtubuli associated protein-2 staining). These zones consisted of microscopically small lysis holes with a diameter of about 10–20 μm. Immunohistochemistry showed that especially microvessels expressed MMP antigen. SDS-PAGE zymography differentiated between a high level of activated MMPs in the ischemic area and a low level in the non-ischemic basal ganglia. The gelatin film in situ-overlay technique is able to localize MMP activity in ischemic rat brain tissue on a microscopic level. [Copyright &y& Elsevier]
– Name: AbstractSuppliedCopyright
  Label:
  Group: Ab
  Data: <i>Copyright of Journal of Neuroscience Methods is the property of Elsevier B.V. and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract.</i> (Copyright applies to all Abstracts.)
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      – Type: doi
        Value: 10.1016/S0165-0270(02)00037-7
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      – Code: eng
        Text: English
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        StartPage: 125
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      – SubjectFull: Photographic gelatin
        Type: general
      – SubjectFull: Cerebral ischemia
        Type: general
      – SubjectFull: Cerebrovascular disease
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      – TitleFull: A gelatin in situ-overlay technique localizes brain matrix metalloproteinase activity in experimental focal cerebral ischemia
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              Text: May2002
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              Y: 2002
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