The effect of age and injury on the expression of inducible nitric oxide synthase in facial motor neurons in F344 rats

https://doi.org/10.1016/S0047-6374(98)00150-XGet rights and content

Abstract

Nitric oxide has been implicated in both normal neuronal aging as well as nerve repair events because of its known roles in synaptic plasticity, synaptogenesis and neuropathologic processes. In this study, we have determined the effect of aging, by comparing brainstem facial motor neurons (FMNs) as well as blood vessels, from adult F344 rats to those in old animals. Inducible nitric oxide synthase (iNOS) expression was determined both by immunohistochemistry using an antibody to iNOS on tissue sections and slot blots. In adult rats, iNOS expression was detectable only in FMNs and not in blood vessels. In old rats, there were robust levels of iNOS protein in blood vessels, while iNOS protein was not detectable in FMNs from old rats. There was also a 12-fold increase in iNOS expression in isolated blood vessels from old rats compared to vessels from adult animals. To determine the effect of injury on iNOS expression, the facial nerve was transected and immunocytochemistry performed as above. After nerve transection in adult rats, iNOS was demonstrable only in blood vessels after 1 day, but by 7 days iNOS protein immunoreactivity was robust in FMNs. In old animals, iNOS protein expression was observed only in FMNs at 1 day, but by 7 days after injury, protein immunoreactivity was localized to the blood vessels. These data suggest that aging and injury differentially affect the expression of iNOS and that the up-regulation of iNOS may be important for the availability of nitric oxide in the aged or injured nervous system.

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).

References (45)

  • M.A. Bisby et al.

    Changes in cytoskeletal protein synthesis following axon injury and during axon regeneration

    Mol. Neurobiol.

    (1992)
  • M.M. Black et al.

    Slowing of rate of axon regeneration during growth and maturation

    Exp. Neurol.

    (1969)
  • T.M. Dawson et al.

    A novel neuronal messenger molecule in brain: the free radical, nitric oxide

    Ann. Neurol.

    (1992)
  • C.A. Diglio et al.

    Rat cerebral microvascular smooth muscle cells in culture

    J. Cell. Physiol.

    (1982)
  • Z. Drahota et al.

    The influence of age on the course of reinnervation of muscle

    Gerontologia

    (1961)
  • O. Durieu-Trautmann et al.

    Nitric oxide and endothelin secretion by brain microvessel endothelial cells: regulation by cyclic nucleotides

    J. Cell. Physiol.

    (1993)
  • M. Farooque et al.

    Astrocytic reaction after graded facial cord compression in rats: immunohistochemical studies on glial fibrillary acidic protein and vimentin

    J. Neurotrauma

    (1995)
  • E. Galea et al.

    Transient expression of calcium-independent nitric oxide synthase in blood vessel during brain development

    FASEB J.

    (1995)
  • B. Grafstein et al.

    Intracellular transport in neurons

    Physiol. Rev.

    (1980)
  • S.S. Gross et al.

    Nitric oxide: pathophysiological mechanisms

    Ann. Rev. Physiol.

    (1995)
  • P.N. Hoffman et al.

    Neurofilament and tubulin expression recapitulates the developmental program during axonal regeneration: induction of a specific beta-tubulin isotype

    Proc. Natl. Acad. Sci. U.S.A.

    (1988)
  • T. Imai et al.

    Induction of nitric oxide synthase by cyclic AMP in rat vascular smooth muscle cells

    J. Clin. Invest.

    (1994)
  • Cited by (6)

    • Ageing-related role of nitric oxide in the brain

      2004, NeuroImmune Biology
      Citation Excerpt :

      In human, age-dependent increase in the production of NOS-II-derived NO was supposed by measuring 3-nitrotyrsine/tyrosine in the cerebrospinal fluid [33]. In other animal models, age-dependent increase in NO production, activity and expression of the enzyme was largely observed in different regions of the aged-brain [24,26,34–41]. Briefly, an increase of NOS-II expression has been demonstrated in diencephalum, in cerebral cortex, and in the cerebellum hemispheres.

    • NADPH-Diaphorase Activity and NO Synthase Expression in the Olfactory Epithelium of the Bovine

      2010, Journal of Veterinary Medicine Series C: Anatomia Histologia Embryologia
    View full text