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. 2011;34(3):257-64.
doi: 10.1179/107902611X12972448729648.

Immunohistochemical analysis of TIMP-2 and collagen types I and IV in experimental spinal cord ischemia-reperfusion injury in rats

Ihsan Anik  1 , Sibel Kokturk, Hamza Genc, Burak Cabuk, Kenan Koc, Sadan Yavuz, Sureyya Ceylan, Savas Ceylan, Levent Kamaci, Yonca Anik
Affiliations

Immunohistochemical analysis of TIMP-2 and collagen types I and IV in experimental spinal cord ischemia-reperfusion injury in rats

Ihsan Anik et al. J Spinal Cord Med. 2011.

Abstract

Background: Thoracic and thoracoabdominal aortic intervention carries a significant risk of spinal cord ischemia. The pathophysiologic mechanisms that cause hypoxic/ischemic injury to the spinal cord have not been totally explained. In normal spinal cord, neurons and glial cells do not express type IV collagen. Type IV collagen produced by reactive astrocytes is reported to participate in glial scar formation. Tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors that regulate the activity of the matrix metalloproteinases (MMPs). TIMP-2 binds strongly with MMP-2, facilitating activation by membrane-type MMP. Imbalance between TIMPs and MMPs can lead to excessive degradation of matrix components. Type IV collagen involved in the blood-brain barrier disruption and glial scar formation, TIMP-2 influences MMP-2 that controls degradation of collagen I and IV.

Objective: To examine the immunohistochemical analysis of TIMP-2 and collagen types I-IV in experimental spinal cord ischemia-reperfusion in rats.

Methods: Thirty-two male Wistar rats weighing 250-300 g were divided into four groups: group S: sham group (n = 8); group 0P: 30-minute occlusion without perfusion (n = 8); group 3P: 30-minute occlusion and 3-hour perfusion (n = 8); and group 24P: 30-minute occlusion and 24-hour perfusion (n = 8). Infrarenal aorta was cross-clamped at two sites by using two aneurysm clips for 30 minutes. Reperfusion was provided after removal of the clips. Lumbar spinal cord segments were removed for immunohistochemical analysis.

Results: TIMP-2 and collagen staining in 3-hour perfused (3P) group were nearly the same with sham group (S). TIMP-2 and collagen staining increased in the 24-hour perfused group.

Conclusion: Alterations in collagen levels may relate to the biphasic breakdown of the blood-brain barrier and collagen staining in new cell types with relation to glial scar formation. Our results demonstrate that 3-hour perfusion after occlusion in hypoxic/ischemic spinal cord injury seems to be the critical reversible period.

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Figures

Figure 1
Figure 1
The anterior horn of spinal cord was positive for type IV collagen (A–D). Immunohistochemical staining for type IV collagen in 0P group (A). Strong vascular basement membrane staining of type IV collagen. Immunohistochemical staining for type IV collagen in 24P group (B). Weak vascular staining of type IV collagen in spinal cord in 3P (C) and S group (D). Asterisk (*) indicates neurons and arrows indicate type IV collagen positive stained blood vessels. Magnification ×20 in all panels.
Figure 2
Figure 2
Immunohistochemical staining for type I collagen. Insets show a close-up of the spinal cord. Strong staining of type I collagen in 0P and 24P groups (A, B). Weak staining of type I collagen in spinal cord in 3P and S groups (C, D). Type I collagen staining is indicated by arrowheads. Asterisk indicates neurons. Magnification ×20 in all panels and ×40 in insets.
Figure 3
Figure 3
The anterior horn of spinal cord was positive for TIMP-2. Immunohistochemical staining for type TIMP-2 in 0P group (A). Strong positive staining of TIMP-2 (A). Immunohistochemical staining for TIMP-2 in 24P group (B). Weak vascular staining of TIMP-2 in spinal cord in 3P (C) and S groups (D). Asterisk (*) indicates neurons and arrowheads indicate TIMP-2 positive stained glia cells. Magnification ×40 in all panels.
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