Research ReportDevelopmental and injury-induced expression of α1β1 and α6β1 integrins in the rat spinal cord
Introduction
After a contusive spinal cord injury, blood vessels are lost over the first 3 days in adult rats, followed by an angiogenic response between day 3 and 7, and the subsequent regression of the new blood vessels by 14 days post-injury, the time when secondary tissue loss begins (Imperato-Kalmar et al., 1997, Casella et al., 2002, Casella et al., 2006, Loy et al., 2002, Whetstone et al., 2003). The loss of the original and new blood vessels is thought to contribute to secondary spinal cord damage through a loss of essential nutrients and molecules for metabolism and removal of metabolic by-products. One potential factor contributing to the loss of blood vessels is the disruption of the endothelial cells from their laminin-containing basement membrane, which can induce apoptosis through intracellular signaling cascades by unbound integrin receptors (Frisch and Screaton, 2001, Zhan et al., 2004). Furthermore, integrin activation can promote endothelial cell survival in vitro (Scatena and Giachelli, 2002, Stupack and Cheresh, 2004). Therefore, it will be important to identify specific integrins that are associated with the vasculature during development and after spinal cord injury, when endothelial cells need to grow, mature and survive to promote tissue integrity.
Integrins are a family of 24 known heterodimers consisting of one of 18 α and one of 8 β subunits which bind to extracellular matrix molecules and regulate proliferation, growth, migration, differentiation and survival of a variety of cell types (Milner and Campbell, 2002b, Clegg et al., 2003, Stupack and Cheresh, 2004). Two predominant laminin-binding integrins are α1β1 (Ignatius et al., 1990, Tawil et al., 1990) and α6β1 (Sonnenberg et al., 1990). The α1β1 integrin can also bind collagen (preferentially collagen-IV; Ignatius et al., 1990, Tawil et al., 1990). The α1, α6 and β1 integrin subunits are expressed by the vasculature in rodent brain (Kloss et al., 1999, Milner and Campbell, 2002a). Integrins containing the α1, α6 and β1 subunits have been shown to promote angiogenesis in vitro and in vivo (Davis and Camarillo, 1995, Senger et al., 1997, Senger et al., 2002, Babic et al., 1998, Pozzi et al., 2000, Gonzalez et al., 2002, Grzeszkiewicz et al., 2002, Leu et al., 2002, Leu et al., 2003). A switch from predominant expression of α4 and α5 by blood vessels in postnatal day 1 (P1) mouse brain to α1, α6 and β1 integrin subunits from P7 into adulthood has previously been reported (Milner and Campbell, 2002a). However, their presence in the postnatally developing spinal cord has yet to be fully described. Additionally, no studies have investigated changes in the expression of integrins by the vasculature following spinal cord injury. Moreover, little is known about the distribution of the α1β1 and α6β1 integrin heterodimers, i.e., the functional forms of integrins. Previous studies have primarily used antibodies directed against individual integrin subunits and relied on co-localization to suggest the possible existence of functional heterodimers.
To confirm the importance of the α1β1 and α6β1 integrins for the spinal cord vasculature, we investigated their expression during postnatal development in rats using heterodimer-specific antibodies. Moreover, to determine whether these integrins might be modulated after spinal cord injury, we documented their changes after contusive injury in adult rats.
Section snippets
α1β1 integrin is present only in endothelial cells of normal developing and contused adult rat spinal cord
Within the spinal cord, immunostaining for the α1β1 integrin heterodimer appeared to be associated exclusively with blood vessels as early as the first day after birth (P1) and throughout adulthood (Fig. 1). Immunostaining was also evident in the meninges from P15 to adulthood, likely representing meningeal fibroblasts as previously reported (Milner and ffrench-Constant, 1994). The number and complexity of blood vessels increased throughout development, most notably between P10 and P15 to reach
Discussion
In this study, we show in the rat spinal cord that: (1) during development, α1β1 and α6β1 integrin expression increases in blood vessels between P10 and P15, suggesting that they have a role in the formation and/or maintenance of the blood–spinal barrier, although the earlier and more extensive expression of α1β1 integrin suggests different functions between the two; (2) after spinal cord contusion in the adult, α1β1 integrin is upregulated in blood vessels over a greater distance from the
Experimental procedures
All experiments were conducted in accordance with the National Institutes of Health's guidelines on the care and use of laboratory animals and were approved by the Animal Care Committee of the University of Louisville. Rat pups were randomly selected at P1 (first day of birth), P5, P10, P15 and P20 from Sprague-Dawley dams housed singly (n = 4 for each time-point; Harlan, Indianapolis, IN). Twelve young adult female Sprague-Dawley rats (Harlan; 8–9 weeks and weighing 180–200 g) were used to study
Acknowledgments
The authors would like to thank Kimberley Jenkins-Milton for technical assistance, Christine Nunn for surgical assistance, Leigh-Ann Wilson for animal care and Drs. Richard Benton and Jason Talbott for their helpful suggestions. We would also like to thank Carol Birmingham of Chemicon International for the generous gift of α6β1, α1β1, GFAP and NG2 antibodies. Dr. Scott Whittemore is thanked for his helpful comments, the SMI-71 antibodies and use of his cryostat. This work was supported by a
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Current address: Department of Neurology and Neurosurgery, Montréal Neurological Institute, McGill University, Montréal, Québec, Canada.