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A novel method for establishing daily in vivo concentration gradients of soluble nerve growth factor (NGF)

https://doi.org/10.1016/j.jneumeth.2007.05.032Get rights and content

Abstract

Despite the capacity for spontaneous axonal regeneration, recovery following injuries to the peripheral nervous system (PNS) following transection are often incomplete and limited to short distances. Nerve growth factor (NGF) has been previously shown to support neuron survival, and direct growth of both developing and regenerating nerve fibers along a concentration gradient, based largely on in vitro studies. Here, we present a novel in vivo model of administering daily concentration gradients of NGF by directly manipulating the placement of the catheter-nerve conduit junction. Our results show that a dose of 800 pg NGF can be reliably used to establish a chemotactic concentration gradient over both a transient time period, and chronically through repeated daily administrations of the drug. Results from these studies may lead to a better mechanistic understanding of how concentration gradients of soluble NGF influence in vivo peripheral nerve regeneration.

Introduction

Injuries to the peripheral nervous system (PNS) present a serious problem for society, affecting approximately 2.8% of all trauma cases, and often resulting in poor recovery of function and subsequent impaired quality of life for the patient (McAllister et al., 1996, Noble et al., 1998, Jaquet et al., 2001, Belkas et al., 2004, Lundborg, 2004). Spontaneous axonal regeneration in the PNS is often limited to short distances, with poor recovery of function being particularly prevalent in nerve injuries which sever peripheral nerves far from their targets, and require autologous nerve graft repair. Nevertheless, this method is inherently flawed due to donor site morbidity, a lack of donor tissue availability, incomplete, and non-specific regeneration, and subsequent poor recovery of function in both animal models and clinical cases (Dahlin and Lundborg, 1998, Bellamkonda, 2006, Nichols et al., 2006).

Following injury to the peripheral nervous system, regenerating axons are directed to reinnervate their appropriate target end organs through a combination of both stimulatory (neurotropic) and inhibitory molecular gradients (Goldberg and Barres, 2000). A number of experiments have supported the notion of directed growth of both developing and regenerating nerve fibers along a concentration gradient of soluble Nerve growth factor (NGF) released from a target-derived source (Charlwood et al., 1972, Letourneau, 1978, Dickson and Senti, 2002, Moore et al., 2006). For example, Letourneau (1978) showed that growth cones of sensory neurons orient themselves towards a source of NGF, and an enhanced extension of fibers occurs along an NGF gradient in vitro, which has recently been shown to be mediated through the high affinity trkA receptor (Gundersen and Barrett, 1979, Gundersen, 1985, Gallo et al., 1997). More recently, Rosoff et al. (2004) has shown that axons are capable of detecting a concentration difference of NGF as small as about one molecule across their spatial extent.

Despite the plethora of in vitro data supporting the idea of neurotropism in both developing and regenerating peripheral nerves, an in vivo model is largely deficient at the present time, especially in mammalian species. Although numerous models have been utilized to deliver NGF to the nerve injury site (e.g., osmotic pumps, subcutaneous injections), they do not allow for repeated localized injections to the regenerating microenvironment and tend to foster a time-dependent decline in the biological activity of NGF, perhaps owing to its short half-life (Tria et al., 1994, Poduslo and Curran, 1996). Recently, a new model of nerve regeneration has been developed by McDonald and Zochodne (2003), which allows for the strategic placement of NTF delivery at either the proximal, distal, or middle portion of the nerve conduit and is therefore theoretically capable of establishing a gradient of NGF within its lumen. In the present study, we analyzed: (1) the establishment of a reliable concentration gradient over two transient time periods, and (2) the ability to establish repeated, daily concentration gradients over a chronic time period. Our results indicate that a chemotactic NGF gradient can be established dependent on the placement of the catheter-nerve conduit junction over both a transient time period, and after repeated daily administrations of the drug.

Section snippets

Animals

Adult male Lewis rats (108 in total), weighing 250–300 g (Charles River, QC) were used in this study and all interventions were carried out under inhalation anaesthetic (Isofluroane, 99.9% Halocarbon Laboratories, River Edge, NJ). Animals were maintained in a temperature and humidity controlled environment, were allowed standard rat chow (Purina, Mississauga, ON) and water ad lib, with a 12:12 h light:dark cycle. All surgical procedures were carried out in an aseptic manner, and standard

A concentration gradient of NGF can be established over a transient time period in the T-tube model

To determine if a concentration gradient was established over a transient time period, proximal, distal, and middle located T-tube-catheter chambers underwent serial sampling from both the proximal and distal ends of each tube at 1, 2, 5, and 10 min, following administration of 0.1 ml NGF (800 pg). We found a significant multivariate effect for the interaction of time × location × group, using Wilks’ Lambda: F (6, 14) = 287, p < 0.05. Subsequent LSD pairwise comparison tests revealed that for proximal

Major findings

The major findings in this study were: (1) using an NGF dose of 800 pg, we were successfully able to establish a chemotactic concentration gradient of NGF depending on the placement of the catheter-nerve conduit junction over a transient (10 min) period; (2) a concentration gradient of NGF can be established over a 30 min period, and can then be re-established with an additional administration of NGF after 1 h; and (3) a concentration gradient of NGF was shown to be re-established with repeated

Acknowledgements

This research was supported by grants from CIHR and AHFMR to R.M. We would like to thank Dr. Qing-Gui Xu and Joanne Forden for technical expertise and assistance.

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