Aging of the myenteric plexus: neuronal loss is specific to cholinergic neurons

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Abstract

Neuron loss occurs in the myenteric plexus of the aged rat. The myenteric plexus is composed of two mutually exclusive neuronal subpopulations expressing, respectively, nitrergic and cholinergic phenotypes. The goal of the present study, therefore, was to determine if neuron loss is specific to one phenotype, or occurs in both. Ad libitum fed virgin male Fischer 344 rats of 3 and 24 months of age were used in each of two neuronal staining protocols (n=10/age/neuron stain). The stomach, duodenum, jejunum, ileum, colon, and rectum were prepared as whole mounts and processed with either NADPHd or Cuprolinic Blue to stain, respectively, the nitrergic subpopulation or the entire population of myenteric neurons. Neuron numbers and sizes were determined for each preparation. Neuron counts from 24-month-old rats were corrected for changes in tissue area resulting from growth. There was no age-related loss of NADPHd-positive neurons for any of the regions sampled, whereas significant losses of Cuprolinic Blue-labeled neurons occurred in the small and large intestines of 24-month-old rats. At the two ages, the average neuron sizes were similar in the stomach and small intestine for both stains, but neurons in the large intestine were significantly larger at 24 months. In addition, numerous swollen NADPHd-positive axons were found in the large intestine at 24 months. These findings support the hypothesis that age-related cell loss in the small and large intestines occurs exclusively in the cholinergic subpopulation. It appears, however, from the somatic hypertrophy and the presence of swollen axons that the nitrergic neurons are not completely spared from the effects of age.

Introduction

Neuronal loss occurs with age in the myenteric plexus of the gastrointestinal (GI) tract. This age-related attrition has been observed in a variety of species including humans (e.g., De Souza et al., 1993, Gomes et al., 1997), guinea pigs Gabella, 1989, Wade et al., 2002b, rats Santer and Baker, 1988, Cowen et al., 2000, Phillips and Powley, 2001, and mice (El-Salhy et al., 1999). Though the time course and the regional patterns of the cell losses have been described (Phillips and Powley, 2001), it is unclear whether cell death affects all classes of myenteric neurons nonselectively or is confined to specific phenotypes (Wade, 2002).

For an initial analysis of the issue of cell death selectivity, estimates of the two broadest chemical phenotypes, the nitrergic and cholinergic neurons, are the obvious choice. Nitrergic neurons [i.e., those using nitric oxide (NO) synthesized by nitric oxide synthase (NOS)] and cholinergic neurons [i.e., those using acetylcholine synthesized by choline acetyltransferase (ChAT)] represent two mutually exclusive subpopulations that together constitute essentially the entire myenteric population. This pattern holds for a variety of species (guinea pig: Vanden Berghe et al., 1999, Chiocchetti et al., 2003; and mouse: Sang and Young, 1996, Sang and Young, 1998) and is especially true for the rat Mann et al., 1999, Nakajima et al., 2000.

To date, attempts to evaluate age-related cell losses by chemical phenotype have focused primarily on the nitrergic subpopulation, and have resulted in conflicting and limited observations. Three studies report no age-related loss of nitrergic neurons Santer, 1994, Johnson et al., 1998, Cowen et al., 2000. A fourth (Belai et al., 1995) found either no change or an increase in the percentage of nitrergic neurons, depending on the region of the GI tract sampled. In contrast, two studies described age-related losses of nitrergic neurons Takahashi et al., 2000, Wade et al., 2002b. Further convoluting the issue of phenotypical selectivity is the difficulty of extrapolating these initial results to the entire GI tract, since observations were limited to only one or two regions of the GI tract (jejunum: Santer, 1994; ileum: Belai et al., 1995, Johnson et al., 1998, Cowen et al., 2000; and colon: Belai et al., 1995, Takahashi et al., 2000, Wade et al., 2002b).

Even less is known about the patterns of aging of cholinergic neurons in the myenteric plexus. The only study to address the question (Cowen et al., 2000) reported that cell death in the aging ileum was restricted to the cholinergic phenotype with nitrergic neurons showing no age-related losses. This finding, however, remains to be replicated and extended to the rest of the GI tract. The lack of observations may be, at least in part, a consequence of the fact that available labeling protocols for cholinergic enteric neurons are better adapted for qualitative, rather than quantitative, analyses Mann et al., 1999, Chiocchetti et al., 2003. The difficulties hampering counts of cholinergic neurons include issues of specificity of the available antibodies, problems of reliability and reproducibility, and apparent differences between central and peripheral variants of ChAT Nakajima et al., 2000, Chiocchetti et al., 2003. When these complications are compounded by the fact that any immunohistochemical protocol used across different ages must be able to detect down-regulated levels of ChAT or other cholinergic markers and to distinguish such reduced levels from complete loss of cells, it is particularly difficult to establish that labeling is reliable and that decreases are sensitive indices of cell loss.

Given the difficulties of quantifying markers for the cholinergic phenotype, an estimate of this subpopulation based on calculation seems more reliable and practical: quinolinic phthalocyanine, or Cuprolinic Blue, is a pan-neuronal stain for enteric neurons that has proved reliable, sensitive, and complete in quantitative analyses Heinicke et al., 1987, Holst and Powley, 1995, Karaosmanoglu et al., 1996, Phillips and Powley, 2001, whereas other stains such as reduced nicotinamide adenine dinucleuotide (NADH) diaphorase and protein gene product 9.5 have been found to be nonselective, difficult to reproduce, and likely to underestimate the entire neuronal population Heinicke et al., 1987, Eaker and Sallustio, 1994, Karaosmanoglu et al., 1996, Johnson et al., 1998. Comparable quantitative comparisons between Cuprolinic Blue and other putative panneuronal markers (e.g., antihuman neuronal protein HuC/HuD or neuron-specific nuclear protein) have not been done. Similarly, nicotinamide adenine dinucleuotide phosphate diaphorase (NADPHd) has proved a reliable marker for nitrergic enteric neurons Dawson et al., 1991, Hope et al., 1991, Belai et al., 1992, Ward et al., 1992. Since, as discussed above, nearly all myenteric neurons are either nitrergic or cholinergic, subtraction of the nitrergic cell counts from Cuprolinic Blue cell counts will yield an estimate of the size of the cholinergic subpopulation in the myenteric plexus. For cell losses of the magnitude observed with aging (i.e., 13–41%), these calculations should be particularly reliable. Furthermore, errors from ignoring any minor population of nonnitrergic–noncholinergic neurons and from failing to distinguish any small population with a nitrergic-plus-cholinergic phenotype would be minor in proportion to the overall size of the age-related cell losses.

Based on these considerations, the present work was designed to (a) determine if age-related cell loss occurs in nitrergic (i.e., NADPHd-positive) myenteric neurons; (b) determine if age-related cell loss occurs in cholinergic neurons (calculated from the counts of cells with a pan-neuronal stain and counts of nitrergic neurons), either in addition to or instead of any reduction in nitrergic neurons; (c) evaluate whether loss or sparing of nitrergic and cholinergic neurons varied across the different organs of the aged gut; and (d) assess whether surviving neurons exhibit age-related changes in soma size.

Section snippets

Subjects

Virgin male Fischer 344 rats (n=40) were obtained from Harlan Laboratory (Indianapolis, IN): 24 months olds from the National Institute on Aging colony at Harlan and 3 months olds from the general colony. Upon arrival, rats were housed in polypropylene cages (three per cage) at 22–24 °C and 40–60% humidity on a 12:12-h light/dark cycle. Solid chow (laboratory diet no. 5001; PMI Feeds, Brentwood, MO) and tap water were available ad libitum. All procedures were conducted in accordance with the

Stomach

There were no significant differences in surface area of the stomach between the NADPHd and Cuprolinic Blue groups at 3 months of age or 24 months of age (p values >0.05). The NADPHd-stained stomachs of 24-month-old rats were slightly larger, however, than the stomachs of NADPHd-stained 3-month-old rats (p<0.05). In contrast, there was no difference in stomach areas between the Cuprolinic Blue groups at the two ages (p>0.05). Because (a) an increase was not seen in both 24-month groups and (b)

Discussion

The present results confirm and extend the complementary hypotheses (a) that age-related cell loss in the myenteric plexus does not occur in nitrergic neurons and (b) that the losses occur exclusively in the cholinergic subpopulation of enteric neurons. At the same time, the results illustrate that nitrergic neurons, though they survive, are not completely spared the damaging effects of aging. Specifically, throughout the intestines, some nitrergic neurons develop swollen or dilated axons, and,

Age-related changes in myenteric neuron density

Cell loss did not occur in neurons stained for NADPHd (i.e., nitrergic neurons), so all age-related cell loss in the myenteric plexus must take place in the subpopulation of neurons that are NADPHd-negative (i.e., cholinergic neurons). We have calculated the percent cell loss for the cholinergic neurons to illustrate the impact of cell loss on this single subpopulation (see Table 4). When neuron losses, which have previously been attributed to the entire population of myenteric neurons, are

Additional observations on myenteric neuron density

The densities of nitrergic neurons in the regions sampled were comparable to previous reports for the adult rat Nichols et al., 1993, Cracco and Filogamo, 1994, Belai et al., 1995, Berthoud, 1995, Wilhelm et al., 1998, Jarvinen et al., 1999, Mann et al., 1999, Timmermans et al., 1999. Notable, however, were the high densities of nitrergic neurons observed in the forestomach, duodenal bulb, and jejunum, which may indicate some functional specialization(s). The role of the forestomach in gastric

Age-related change in neuron size

In the present study, the mean myenteric neuron sizes for 3-month-old rats stained with either Cuprolinic Blue or NADPHd are very similar to previous reports. Gabella (1971), for example, using a histochemical technique to detect DPNH-tetrazolium reductase activity, reported that, in 6-month-old rats, the peak neuron size for the stomach and rectum was between 175 and 275 μm2 (we found a mean neuron size of 267 and 215 μm2, respectively), and in the small intestine, the peak neuron size was

Age-related swelling of nitrergic axons

A pathological alteration in aged human sympathetic ganglia (e.g., Schmidt et al., 1990) is neuroaxonal dystrophy. This distinct pathology is characterized by axonal swellings and is consistent with a dying back of the axon (Schmidt, 2002). In the present experiment, 24-month-old rats exhibited numerous swollen NADPHd-positive axons in the circular muscle of the small and large intestines and some swollen axons within individual ganglia. These swollen axons were several times their normal

Age-related neuron loss reflects deterioration of the myenteric plexus

We have posited (Phillips and Powley, 2001) that neuron loss in the myenteric plexus is characteristic of a degenerative process that would eventually results in a decline in function Varga, 1976, McDougal et al., 1984, Smits and Lefebvre, 1996b, Wade et al., 2002a, and not simply a normal part of a developmental process (i.e., changes in the nervous system that result in functionally appropriate cell numbers) as suggested by Gabella (1989). The findings of the current study of: (a) selective

Potential mechanism for NADPHd-neuron survival

Survival of aged nitrergic neurons is consistent with a protective role for NO in myenteric neurons (Belai et al., 1995) similar to the protective role for NO that has been described in the autonomic (Blottner, 1999) and central nervous system (Wolf, 1997). Cowen et al. (2000) suggested that neurons that use NO constitutively may have enhanced defense mechanisms against free radical damage. Any hypothesis of selective cell death in the enteric nervous system will have to be able to explain;

Predicted deterioration of function

The cell bodies of nitrergic inhibitory motor neurons are located oral to the circular muscle that they innervate (e.g., Brookes et al., 1991, Brookes et al., 1997, Pfannkuche et al., 1998, Schemann et al., 2001), and electrical stimulation of this descending pathway results in relaxation of the circular muscle Neunlist et al., 1999, Furness, 2000, Schemann et al., 2001. In contrast, the cell bodies of cholinergic excitatory motor neurons are located aboral to the circular muscle that they

Conclusion

Based on our findings and the findings of others (Cowen et al., 2000), it appears that age-related neuron loss occurs exclusively in the cholinergic subpopulation of myenteric neurons, whereas nitrergic neurons survive Santer, 1994, Belai et al., 1995, Cowen et al., 2000. It remains to be determined, however, if all neurons of the cholinergic phenotype are affected or if cell loss is restricted to specific chemical or functional subclasses. In addition, we have yet to understand what feature(s)

Acknowledgements

This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases (NIH DK27627 and DK61317), and a National Institute on Aging pilot project award.

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