Abstract
Assays for proteolytic activity are often used to confirm a functional role for enzymes involved with CNS pathobiology. In studies focused on matrix metalloproteinases (MMPs), specifically those investigating the role of gelatinases (MMP-2, MMP-9), gelatin zymography is used to assess relative changes in enzyme activity. The advantages of this method lie in its gel separation of pro and active enzyme and the option for generation of semiquantitative data. Gelatin zymography is applied with success in studies of traumatic brain injury (TBI), but may require modifications for best results. In this chapter, we first present an overview of different zymographic approaches taken to assess the role of gelatinases in various forms of TBI and related CNS pathologies. Next, we focus on the specific method developed in our laboratory to optimize zymographic signal from injured CNS tissue. Finally, we include points of technique modification which have worked well for us, emphasizing tissue dissection, extraction method, and gel incubation period. This information is presented to offer support for those seeking to apply gelatin zymography with samples from injured brain tissue, where pathology can vary considerably. Individual models and experimental design may require further zymographic modifications for optimization of MMP detection.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Ding JY, Kreipke CW, Schafer P, Schafer S, Speirs SL, Rafols JA (2009) Synapse loss regulated by matrix metalloproteinases in traumatic brain injury is associated with hypoxia inducible factor-1alpha expression. Brain Res 1268:125–134
Grossetete M, Phelps J, Arko L, Yonas H, Rosenberg GA (2009) Elevation of matrix metalloproteinases 3 and 9 in cerebrospinal fluid and blood in patients with severe traumatic brain injury. Neurosurgery 65:702–708
Grossetete M, Rosenberg GA (2009) Bacterial collagenase injection intracerebral hemorrhage rat model. In: Chen J, Xu X-M, Xu ZC, Zhang JH (eds) Animal models of acute neurological injuries. Humana Press, New York, NY, pp 337–348
Homsi S, Federico F, Croci N, Palmier B, Plotkine M, Marchand-Leroux C, Jafarian-Tehrani M (2009) Minocycline effects cerebral edema: relations with inflammatory and oxidative stress markers following traumatic brain injury in mice. Brain Res 1291:122–132
Ranasinghe HS, Williams CE, Christophidis LJ, Mitchell MD, Fraser M, Scheepens A (2009) Proteolytic activity during cortical development is distinct from that involved in hypoxic ischemic injury. Neuroscience 158:732–744
Reyes R, Guo M, Swann K, Shetgeri SU, Sprague SM, Jimenez DF, Barone CM, Ding Y (2009) Role of tumor necrosis factor-alpha and matrix metalloproteinase-9 in blood brain barrier disruption after peripheral thermal injury in rats. J Neurosurg 110:1218–1226
Vilalta A, Sahuguillo J, Rosell A, Poca MA, Riveiro M, Montaner J (2008) Moderate and severe traumatic brain injury induce early over expression of systemic and brain gelatinases. Intensive Care Med 34:1384–1392
Yu F, Kamada H, Niizuma K, Endo H, Chan PH (2008) Induction of MMP-9 expression and endothelial injury by oxidative stress after spinal cord injury. J Neurotrauma 25:184–195
Siffinger M, Stefovska V, Zentner I, Hansen B, Stepulak A, Knaute C, Marzahan J, Ikonomidou C (2007) The role of matrix metalloproteinases in infant traumatic brain injury. Neurobiol Dis 25:526–535
Yamaguchi M, Jaddhav V, Obenaus A, Colohan A, Zhang JH (2007) Matrix metalloproteinase inhibition attenuates brain edema in an in vivo model of surgically-induced brain injury. Neurosurgery 61:1067–1075
Kim HJ, Fillmore HL, Reeves TM, Phillips LL (2005) Elevation of MMP-3 expression and activity during trauma-induced synaptogenesis. Exp Neurol 192:60–72
Truettner JS, Alonso OF, Dietrich DW (2005) Influence of therapeutic hypothermia on matrix metalloproteinase activity after traumatic brain injury in rats. J Cereb Blood Flow Metab 25:1505–1516
Sheba FA, Mostafa G, Knopman J, Friedrichh V Jr, Bederson JB (2004) Acute alterations in microvascular basal lamina after subarachnoid hemorrhage. J Neurosurg 101:633–640
Mori T, Wang X, Aoki T, Lo EH (2002) Down regulation of matrix metalloproteinase-9 and attenuation of edema via inhibition of ERK mitogen activated protein kinase in traumatic brain injury. J Neurotrauma 19:1411–1419
Wang X, Mori T, Jung JC, Fini ME, Lo EH (2002) Secretion of matrix metalloproteinase-2 and −9 after mechanical trauma injury in rat cortical cultures and involvement of MAP kinase. J Neurotrauma 19:615–625
Phillips LL, Reeves TM (2001) Interactive pathology following traumatic brain injury modifies hippocampal plasticity. Res Neurol Neurosci 19:213–235
Vecil GG, Larsen PH, Corley SM, Herx LM, Besson A, Goodyer CG, Yong VW (2000) Interleukin-1 is a key regulator of matrix metalloproteinase-9 expression in human neurons in culture and following mouse brain trauma. J Neurosci Res 61:212–224
Zao X, Postmantur R, Kampfl A, Liu SJ, Wang KK, Newcomb JK, Pike BR, Clifton GL, Hayes RL (1998) Subcellular localization and duration of mu-calpain and m-calpain activity after traumatic brain injury in the rat: a casein zymography study. J Cereb Blood Flow Metab 18:161–167
Walker EJ, Rosenberg GA (2010) Divergent role for MMP-2 in myelin breakdown and oligodendrocyte death following transient global ischemia. J Neurosci Res 88:764–773
Hawkes SP, Hongxia LI, Taniguchi GT (2010) Zymography and reverse zymography for detecting MMPs and TIMPs. Meth Mol Biol 622:257–269
Phillips LL, Lee NN, Black RT, Harris LK, Colley BS, Reeves TM (2009) Acute postinjury minocycline treatment attenuates both gray and white matter gelatinase activity and promotes recovery following traumatic brain injury. J Neurotrauma 26:A30
Woessner JF Jr (1995) Quantification of matrix metalloproteinases in tissue samples. Meth Enzymol 248:510–529
Zhang JW, Gottschall PE (1997) Zymographic measurement of gelatinase activity in brain tissue after detergent extraction and affinity-support purification. J Neurosci Meth 76:15–20
Acknowledgments
Studies are supported by funding from NIH NS044372 and NS056247.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Harris, L.K., Black, R.T., Reeves, T.M., Phillips, L.L. (2012). Application of Zymographic Methods to Study Matrix Enzymes Following Traumatic Brain Injury. In: Chen, J., Xu, XM., Xu, Z., Zhang, J. (eds) Animal Models of Acute Neurological Injuries II. Springer Protocols Handbooks. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-576-3_12
Download citation
DOI: https://doi.org/10.1007/978-1-61779-576-3_12
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-575-6
Online ISBN: 978-1-61779-576-3
eBook Packages: Springer Protocols