The effect of age and injury on the expression of inducible nitric oxide synthase in facial motor neurons in F344 rats
Introduction
In young and adult animals, the cell body response to axonal injury is characterized by a series of morphological, physiological and biochemical phenomena that vary in magnitude because of differences in species, type of neuron as well as distance between the lesion and the cell body (Watson, 1968; Lieberman, 1974; Grafstein and Forman, 1980). For example, there is an increase in enzymatic activity supporting RNA synthesis and growth-associated proteins (Nandy, 1968; Jacob and McQuarrie, 1991, Jacob and McQuarrie, 1993, Jacob and McQuarrie, 1996; Bisby and Tetzlaff, 1992; Jacob, 1995). The synthesis of messenger RNAs that code for the cytoskeletal elements actin and tubulin increase, while the activity of choline acetyltransferase decreases (Grafstein and Forman, 1980; Hoffman and Cleveland, 1988; Tetzlaff et al., 1991; Lund and McQuarrie, 1996). Thus, there is a generalized up-regulation of the cellular growth program concomitant with a down-regulation in neurotransmission-related activities in the cell.
While little is known about the regulation of axonal repair and nerve regeneration in the aging animal, both the expression of cellular proteins as well as levels of cytoskeletal mRNAs appear to be altered with age. In old mice, partial denervation results in collateral sprouting of peripheral motor axons and normal muscle function, however morphological analyses of the expanded axonal arbor show axonal integrity is compromised (Jacob and Robbins, 1990a, Jacob and Robbins, 1990b). In addition, the rate of nerve regeneration is slower in old animals compared with young animals (Drahota and Gutmann, 1961; Black and Lasek, 1969; Komiya, 1980; Pestronk et al., 1980; Vaughan, 1990, Vaughan, 1992).
The mediators of the cell body response to injury, in animals of any age, have not been well defined. Nitric oxide is a bifunctional mediator in the mammalian nervous system, serving as both an intra- and intercellular messenger. Nitric oxide may be an important mediator in both normal neuronal aging as well as nerve repair events because of its known roles in synaptic plasticity, synaptogenesis and neuropathologic processes (Minc-Golomb et al., 1994; Schmidt and Walter, 1994; Galea et al., 1995; Merrill et al., 1995). Despite the important physiologic functions of nitric oxide, there is evidence that nitric oxide is a neurotoxin in the central nervous system (Dawson et al., 1992). Nitric oxide-mediated neurotoxicity appears to play a role in the pathogenesis of both central nervous system damage following acute injury (Whittle et al., 1995), as well as chronic neurodegenerative diseases (Koprowski et al., 1993).
Nitric oxide is produced by the conversion of l-arginine to nitric oxide and citrulline by a family of electron-transferring enzymes, collectively referred to as nitric oxide synthase (NOS) (Marletta, 1993). Because nitric oxide is a labile, short-lived mediator, its activity is terminated by molecule instability and not catabolic processes. Therefore, regulation of nitric oxide production usually occurs by modulation of the synthetic enzyme. Nitric oxide synthases are a family of enzymes, of which three are well-characterized: neuronal NOS, endothelial NOS and inducible NOS. Neuronal NOS and eNOS are constitutively expressed and are calcium regulated while iNOS is transcriptionally regulated.
The majority of literature available on the expression of NOS in neurons describes the expression of nNOS in sensory neurons located in the thoracic and lumbar spinal ganglia, as well as neuronal structures in the central nervous system. Neuronal NOS expression is increased in sensory neurons after peripheral nerve transection (Verge et al., 1992) or in response to noxious stimuli (Schmidt and Walter, 1994). Similarly, nNOS was induced in sensory ganglia after nerve injury, although nNOS protein immunoreactivity could also be detected in cranial motor neurons after injury if regeneration was prevented and massive cell death ensued (Yu, 1994). The effect of age or injury on the expression of iNOS has not been examined to date, thus the objectives of this study were to examine the effect of age as well as injury on iNOS protein immunoreactivity in facial motor neurons and in the cerebral vasculature of the cortex from adult and old Fischer 344 rats.
Section snippets
Animals
Male Fischer 344 (F344) rats at 6–10 months (adult) and 22–26 months (old) of age were obtained from the NIA breeding colony at Harlan Sprague–Dawley Labs (Indianapolis, IN) and used for all studies. The adult rats weighed 356±13 g (S.D.) at the time of retrograde tracer application; the old rats weighed 420±7 g (S.D.) at this time (P=0.03, unpaired Student’s t-test). Groups of 24 F344 rats at each age (adult and old) were used for the microvessel isolation (see below). Rats were anesthetized
The effect of age on iNOS expression in FMNs and cerebral microvessels
A comparison of FMNs from adult and old rats showed divergent patterns of iNOS protein expression. In the facial nucleus of the adult rat, motor neurons were clearly immunopositive (Fig. 1A, arrows) while the blood vessels coursing through the substance of the tissue as well as the surrounding neuropil showed weak to no iNOS protein immunoreactivity (Fig. 1A). In contrast, in the facial nucleus of the old rat, iNOS protein immunoreactivity was not found in the FMNs, but rather was only apparent
Discussion
In this paper, we demonstrate that iNOS protein immunoreactivity is present in facial motor neurons during normal aging and that injury can induce the expression of iNOS in either motor neurons or blood vessels in the central nervous system. The presence of iNOS has been described previously only in adult sensory neurons and in cranial motor neurons after massive cell death has occurred (Yu, 1994). Further, we show that there is an upregulation of iNOS expression in blood vessels with aging.
Our
Acknowledgements
The authors wish to thank Ms. Toya Botchlet and Mr. Pete Moore for their expert technical assistance. Supported by grants to J.M.J. (American Federation for Aging Research) and P.G. (NIH 30457 and the Alzheimer’s Association).
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