In these investigations, evidence of neuroprotection mediated by agonism at the GLP-1 receptor was evaluated in animals with pyridoxine (PYR) induced peripheral sensory neuropathy. Data from functional (behavioral) evaluations and histological observations and analyses of nerve specimens, were referenced to similar data from saline-injected (SAL) animals without neuropathy. Compared to the SAL injected animals, PYR intoxicated animals exhibited a range of functional and morphological defects which were to varying degrees, ameliorated by treatment with GLP-1 or its longer-lasting analog Ex4.
We have previously reported (Perry et al., 2004
) electrophysiologic dysfunction resulting from pyridoxine intoxication; including sensorimotor neuropathy, characterized by nerve conduction deficits and absence of the H wave (H waves confirm intact sensorimotor circuitry). These PYR-induced electrophysiologic impairments were duplicated in this series of investigations (data not shown). We are now additionally able to report significant functional (behavioral) deficits following PYR-treatment and some evidence of amelioration of these effects (neuroprotection) with GLP-1 treatment. As we have previously shown (Perry et al., 2004
) pyridoxine intoxication is progressive, resulting in a pronounced hindlimb deficit leaving the animal unable to coordinate all four limbs simultaneously, resulting in severe ataxia. In spite of this, animals are able to move around the cage by adopting a shuffling gait, likely as a result of preserved muscle force and some residual proprioceptive function. The rotarod and inclined screen assessment paradigms represent measures of sensory and motor performance together with coordinating and integrative functions. Data indicate that GLP-1 treatment can restore incline screen performance in PYR-animals to levels observed in SAL treated controls. Furthermore, there was the suggestion of a dose-dependent protective effect of GLP-1 against PYR-induced functional deficits in rotarod performance.
Administration of exogenous of GLP-1 and Ex4 also appeared to support the maintenance of morphologic integrity of the individual axons within the sciatic nerve, and soma within the DRG of PYR-treated rats. Taken together with evidence that GLP-1 can enhance the survival and plasticity of neurons in the brain (Perry et al., 2002
; During et al., 2003
), these findings could indicate that GLP-1 has the ability to act at multiple targets to stimulate signaling pathways which enhance neuroprotection within the peripheral nervous system in addition to the central nervous system. As previously reported (Perry et al., 2004
) pyridoxine toxicity results in the degeneration of peripheral sensory ganglia, particularly large neurons with long, heavily myelinated processes. High dose pyridoxine produces ataxia with necrosis of DRG neurons, while lower doses produce cell body and axonal atrophy without discernable functional sequelae. Between these ranges exists a spectrum of injury, with the earliest manifestations occurring at the level of the cell body. Cell body atrophy is manifest as cytoplasmic alterations including vacuolization, increased dense bodies, neurofilament aggregates, and chromatolysis. Our observations at the light microscope level suggest that GLP-1 and Ex4 treatment offers some protection against PYR-induced axonopathy within the sciatic nerve. Pyridoxine treatment appears to result in an increase in the total number of myelinated nerve fibers, mediated in part by a shift of the size distribution from large to small fibers, and an increased endoneurial area. This latter change is likely to be related to the axonal degeneration together with the increased frequency of small diameters fibers. Treatment with GLP-1 or its longer-lasting analog Ex4 appeared support the integrity of neurofilaments, suggesting a direct neuroprotective role for these peptides. We observed that the GLP-1 receptor agonists seemed to mediate quite complete normalization of the mean size of all axons in PYR animals, with a lesser effect in the large myelinated fibers. This may reflect the relatively short duration of treatment with GLP-1 or Ex4 which may not have been sufficient to support the placticity required to combat the pyridoxine insult, particularly in the larger fibers.
Within the DRG, A and B cell integrity and cytoplasmic appearance were improved in PYR animals that received concurrent GLP-1 or Ex4, with appearances under light microscopy similar to SAL animals. Pyridoxine is a toxin known to target large fiber sensory neurons (Albin et al., 1987
); Krinke and Fitzgerald, 1988
; Schaeppi and Krinke, 1982
; Windebank et al., 1985
; Xu et al., 1989
). As such, we have previously demonstrated that pyridoxine intoxication causes a reduction in the mean area of A- and B-cells in the DRG (Perry et al., 2004
). This was confirmed here, together with evidence that treatment with GLP-1 or Ex4 may offer some protection against the PYR-induced A and B cell area reductions and internal morphologic disruption. There appears to be a morphological correlation between the integrity of the DRG and the axonal dysruption in the sciatic nerve. Light microscopic observation of the neurofilament-positive immunoreactivity (NFI) was entirely supportive of the staining for myelin (LFB) in demonstrating a morphologic appearance in the PYR animals receiving GLP-1 or EX4 which more closely resembled the SAL treated animals than PYR - IAP .
The rationale for selecting pyridoxine to produce an animal model of large-fiber neuropathy is based on several factors, including the selective and severe neurotoxic actions of this compound on large DRG neurons in rodents (Xu et al., 1989
), dogs (Schaeppi and Krinke, 1982
), and humans (Albin et al 1987
). Our interest in this particular animal model of sensory neuropathy is that the rapidly developing, large fiber neurodegeneration may be considered to model one aspect of clinical diabetic peripheral neuropathy. This model could be of use as a screen for evaluating neurotrophic / neuroprotective properties of novel compounds currently in development for type 2 diabetes mellitus.
GLP-1 receptor agonism has been characterized as a therapeutic option in diabetes. Ex4 (Exenatide; Amylin Pharmaceuticals Inc), is the first of a new class of pharmaceutics known as incretin mimetics, now in use for the treatment of type 2 diabetes (Estall & Drucker, 2006
; Holst 2006
). Ex4 binds at the putative GLP-1 receptor and is structurally similar to GLP-1, while providing a more long-lasting effect than GLP-1. Clinical data suggest that Ex4 treatment decreases blood glucose toward target levels, improves markers of beta cell function and is associated with weight loss. It has also been demonstrated to exhibit neurotrophic properties both in vitro and in vivo (Perry et al
). Binding sites for Ex4 have been identified throughout the rat central nervous system (Goke et al 1995
) which leads to speculation that sustainable central GLP-1 receptor agonism may have a therapeutic role in the treatment of a number of central and peripheral neurodegenerative disorders, such as Alzheimer’s disease, vascular and post-stroke dementia and Parkinson’s diseases and peripheral neuropathies, such as that associated with type 2 diabetes.
About 60-70% of type 2 diabetics have mild to severe forms of nervous system damage. The results of such damage include impaired sensation or pain in the hands and feet, slowed digestion of food in the stomach, carpel tunnel syndrome, and other nerve problems. Severe forms of diabetic neuropathy are a major contributing cause of lower-extremity amputations. Improved glycemic control and/or trophic support can help to reduce neural toxicity and minimize or eliminate subsequent diabetic complications, including distal symmetric polyneuropathy (UKPDS 33). However, no current therapy is capable of reversing the nerve degeneration induced by uncontrolled hyperglycemia. Aldose reductase inhibitors (ARI) have demonstrated beneficial effects on nerve function in rodent studies, by blocking neural accumulation of sorbitol and downstream toxic effects. Unfortunately, these findings have not been reproduced clinically for diabetic patients. The lack of success of ARI’s in the clinic may, in part, be related to the failure of a number of these compounds to penetrate the blood-nerve barrier. Similarly trials of the antioxidant, alpha-lipoic acid, have not demonstrated consistent beneficial effects in humans. To date, symptomatic relief is the only option available to diabetic neuropathy patients. It is possible that GLP-1 agonists such as Ex4, which hold the promise of neurotrophic or neuroprotective effects in addition to a favorable profile for glycemic and energy balance regulation may hold promise for the management of diabetic peripheral neuropathies.
In summary, we have presented preliminary evidence that GLP-1 receptor agonism can be neuroprotective in an experimental model of sensory neuropathy. Since it is has been demonstrated that GLP-1 receptor agonists can promote neural plasticity and protection in animal models of other neurological indications, we propose that GLP-1 agonists may hold promise as therapeutic agents for the treatment of many different neurodegenerative conditions throughout the central and peripheral nervous systems.