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

Autonomic Neuroscience: Basic and Clinical 106 (2003) 69 – 83 www.elsevier.com/locate/autneu

Aging of the myenteric plexus: neuronal loss is specific to cholinergic neurons Robert J. Phillips *, Elizabeth J. Kieffer, Terry L. Powley Department of Psychological Sciences, Purdue University, 703 Third Street, West Lafayette, IN 47907-2004, USA Received 10 January 2003; received in revised form 16 March 2003; accepted 24 March 2003

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. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Enteric nervous system; Choline acetyltransferase; Gastrointestinal tract; Nitric oxide synthase

1. 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. * Corresponding author. Tel.: +1-765-494-6268; fax: +1-765-4961264. E-mail address: [email protected] (R.J. Phillips).

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, 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 con-

1566-0702/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S1566-0702(03)00072-9

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voluting 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 panneuronal 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 panneuronal 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.

2. Materials and methods 2.1. 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 jC 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 NIH Guide for the Care and Use of Laboratory Animals and were approved by the Purdue University Animal Care and Use Committee. 2.2. Tissue preparation Rats were killed with a lethal dose of methohexital sodium (Brevital sodium, Eli Lilly, Indianapolis, IN; 10 mg/kg, i.p.) and then perfused through the left ventricle of the heart with 200 ml of 0.9% NaCl followed by 500 ml of 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4 (4 jC). The entire GI tract was removed, and the separate lengths of the small and large intestines were determined, respectively, by measuring from the pyloric sphincter to the ileocaecal junction and from the ceacal – colonic junction to the anal sphincter. Based on the criteria of Hebel and Stromberg (1976), the intestines were further divided into five whole mounts consisting of the duodenum, jejunum, ileum, colon, and rectum. The duodenum (first 5 cm anal to the pyloric sphincter), the jejunum (5 cm segments taken from the midpoint of the small intestine), and the ileum (first 5 cm oral to the ileocaecal junction) were sampled from the small intestines, and the colon (5 cm segment taken from 5 to 10 cm distal to the cecum) and the rectum (5 cm segment taken from 3 to 8 cm oral to the anal sphincter) were

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collected from the large intestines.1 For all specimens, whole mounts of the circular and longitudinal smooth muscle, containing the myenteric plexus, were prepared as described previously (Phillips and Powley, 2001) and stored in 4% paraformaldehyde. 2.3. NADPHd staining Neuronal NO is synthesized by the constitutive enzyme NOS, which requires nicotinamide adenine dinucleuotide phosphate (NADPH) for its activity (cf. Wolf, 1997). In the myenteric plexus, neuronal NOS is identical to NADPH diaphorase (NADPHd) in aldehyde-fixed tissue and can thus be demonstrated histochemically (Scherer-Singler et al., 1983). Therefore, neuronal NOS-positive neurons (i.e., nitrergic neurons) were selectively stained using a histochemical reaction for NADPHd (Dawson et al., 1991; Hope et al., 1991; Belai et al., 1992; Ward et al., 1992). After 2 – 24 h post-fixation, whole mounts (n = 10/age) were rinsed with 0.1 M Tris – HCl (pH 7.9) and then incubated for 1 h in 0.1 M Tris – HCl (pH 7.6) containing 1.0 mg/ml h-NADPH (Sigma, St Louis, MO), 0.33 mg/ml nitroblue tetrazolium (Sigma), and 0.5% Triton X-100 at 37 jC, followed by rinses in Tris – HCl (pH 7.9). The tissue was mounted on gelatin-coated slides, air-dried overnight, and coverslipped with cytoseal (Stephens Scientific, Kalamazoo, MI). 2.4. Cuprolinic Blue staining After 24 h post-fixation (n = 10/age), whole mounts were processed according to the Cuprolinic Blue protocol of Holst and Powley (1995) adapted from Heinicke et al. (1987). The tissue was mounted on gelatin-coated slides, air-dried overnight, dehydrated in a series of graded alcohol rinses, cleared in two separate rinses of xylene, and coverslipped with cytoseal. 2.5. Quantification and sampling of myenteric neurons All counts of myenteric neurons were made at  80 (objective  10, eyepiece  8) with a Leitz widefield Orthoplan II microscope (Leitz, Wetzlar, Germany) by an experimenter blind to the age of the rat from which the tissue originated. The quantification and sampling protocols 1

For the large intestine, which averages 19 cm in length at 3 months of age, Hebel and Stromberg (1976) defined the colon as the first 9 – 11 cm distal to the cecum and the remaining 8 cm as the rectum. Two issues, however, arise when partitioning the large intestine. First, the colon can be further divided into proximal and distal portions (e.g., Butt et al., 1993). Second, the boundary between the distal colon and the rectum might better be delineated in relation to known structures [e.g., the symphysis pubis (McDougal et al., 1984) or the pelvic brim (Butt et al., 1993)] than by gross length. Unfortunately, no clear morphological landmark exists to clearly dictate the transition zone from one region to the other. If, however, studies clearly define the region(s) sampled in relation to known landmarks (e.g., the cecum, the pelvic brim, the anus, etc.), researchers should be able to accurately compare across studies.

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have been described previously (Jarvinen et al., 1999; Phillips and Powley, 2001). Briefly, a grid defining sampling sites was adjusted to fit each whole mount, and then a counting grid (area = 1.0 mm2) was positioned at the sites. The neurons within the counting grid and those overlapping the upper and left edges were counted; those neurons overlapping the lower and right edges were excluded. For the stomach, the counting grid was positioned at 124 equidistant sampling sites. Only the dorsal side of the stomach was quantified, because previous studies have reported no difference in myenteric neuron density between the dorsal and ventral stomach for neurons stained with either NADPHd/NOS or Cuprolinic Blue (Jarvinen et al., 1999; Timmermans et al., 1999; Phillips and Powley, 2001). Several loci from three different regions of the stomach were sampled: the forestomach (55 sites), the corpus (59 sites), and the antrum (10 sites). For the intestines, the tissue was sampled in transverse strips (i.e., columns) every 1.0 cm along the length of the whole mounts. Each sampling column was divided into seven sampling sites equidistant around the circumference of organ. In this way, the mesenteric attachment and antimesenteric aspect of the tissue were sampled. 2.6. Correction for differences in intestinal area The length and width of the small and large intestines increase with age, resulting in a reduction in the overall packing density of neurons (i.e., a ‘‘dilution’’ effect; Gabella, 1989), so we and others use a correction factor (e.g., Johnson et al., 1998; Cowen et al., 2000; Phillips and Powley, 2001). Typically, the correction factor is derived from the ratio of mean intestinal areas between the ‘‘young’’ control group and the ‘‘aged’’ experimental group(s). In addition, to account for the large range of differences in the circumference of the tissue within a region (e.g., when maximally stretched the width of the duodenum can vary by as much as 30%), we generated a correction factor for each column surveyed based on the length of the entire organ and the width of that column (Phillips and Powley, 2001). Neuron counts were expressed per cm2 after making corrections for the differences in the area of the tissue. 2.7. Computer-assisted mapping of myenteric neuron size Measurements of the cross-sectional area of neuronal somata were made on randomly chosen cells using Stereo Investigator 2000 (V. 4.30; MicroBrightFiled, Colchester, VT) by an individual who was blind to the age of the rat from which the tissue originated. The microscopic image was projected to a monitor and the somata were outlined. For tissue stained using NADPHd, a minimum of 50 neurons/whole mount for the stomach and a minimum of 80 neurons/whole mount for the small and large intestines were measured. For tissue stained using Cuprolinic Blue, a minimum of 100 neurons/whole mount for the stomach and

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a minimum of 150 neurons/whole mount for the small and large intestines were measured. The results are expressed as mean percentages of either the total number of NADPHdor Cuprolinic Blue-labeled neurons using, respectively, a bin width of 50 and 25 Am2. The mean percentages for each interval were obtained from the individual percentages of each rat. 2.8. Statistical analysis and display of the data Statistical analyses were generated using Statistica (version 6.0; Statsoft, Tulsa, OK) and GraphPad Prism (version 3.0; GraphPad Software, San Diego, CA); GraphPad Prism was also used to create graphs. For neuron counts, a mean density score was computed for each region of each animal, and an unpaired t-test was used to compare differences between the two ages for each region. The same was done for neuron size for each age and region. In addition to a statistical comparison of mean neuron sizes, we also fitted a curve that best illustrated the trend of the data. This was accomplished by computing a medium lowess curve for each age/region. A medium lowess curve is useful when one wants to accurately reflect the trend of the data without choosing a model or determining the expected best-fit values (Motulsky, 1999). For all statistical tests, a level of probability of 0.05 or less was considered significant. Photomicrographs were acquired using a Spot RT Slider camera (Diagnostic Instruments, Sterling Heights, MI). Photoshop software (version 6.0; Adobe Systems, Mountain View, CA) was used to: (1) apply text; (2) adjust brightness, contrast, and sharpness; and (3) organize the final layouts for printing. Images were printed using a Kodak 8670 thermal printer (Eastman Kodak, Rochester, NY). 2.9. Validation trials for the protocols In the process of developing the protocols just outlined, several validation checks were performed. They addressed four different issues: Test 1. For intestinal counts, does sampling by positioning the counting grid at seven equidistant sampling points around the circumference of an intestinal column Fig. 1. Validation trials for the protocols used in the current study. (A) At 3 months of age, there were no differences between the myenteric neuron densities, whether they were derived from counting total number of neurons or counting seven equidistant sampling points. This was true for both stains. (B) The findings reported in A were replicated in animals at 24 months of age in which neuron counts had been corrected for changes in tissue area. (C) For both stains, there were no differences between myenteric neuron densities, whether they were derived from adjacent tissue from the same rat (one group) or from different rats (two groups). (D) Neuronal densities from double-labeled tissue resulted in an underestimate of the NADPHd-labeled population of neurons, most likely a result of the Cuprolinic Blue stain masking lightly stained NADPHd-positive neurons. An asterisk indicates a level of probability of 0.05 or less.

accurately reflect the true neuronal density? For both the Cuprolinic Blue and the NADPHd stains, we determined as accurately as possible the neuronal density for two

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entire circumferential strips (1.0 mm in the axial dimension) by counting the total number of neurons contained within both strips, and compared these measures to the density scores determined by sampling from seven equidistant locations (jejunal whole mounts; 3-month-old rats; n = 8/stain) (see Fig. 1A). Separate ttests were run for the two stains. For both stains, there were no differences between the density scores determined using total neuron counts or derived from seven equidistant sampling points ( p values >0.05). Test 2. Does the use of a correction factor control for differences in tissue area that occur with age? Test 1 was repeated using 24-month-old rats (jejunum; n = 8/Cuprolinic Blue; n = 7/NADPHd) (see Fig. 1B). One difference, however, was that the total number of neurons was corrected for change in intestinal length, whereas counts from seven equidistant sampling points were corrected for changes in length and width. Separate t-tests were run for each stain. Once again, for both stains, there were no differences between the density scores determined using total neuron counts or derived from seven equidistant sampling points ( p values >0.05). Test 3. Is it accurate to compare between different stains from different groups of animals? The density of Cuprolinic Blue- and NADPHd-labeled neurons, using seven equidistant sampling points, were taken from adjacent pieces of jejunum from the same rats at 3 months of age (n = 6) and compared to previous counts taken from separate groups of rats (Test 1; 3-month-old rats; seven sampling points) (see Fig. 1C). Separate ttests were used for each stain. For both stains, there were no differences between the density scores derived from the same rat or from different groups of rats ( p values >0.05).

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Test 4. Should double-labeled tissue be used to determine neuron density? Rats at 3 months of age were used. After 24 h post-fixation, whole mounts were processed according to the NADPHd protocol, rinsed in deionized water, and further stained according to the Cuprolinic Blue protocol. The tissue was mounted on gelatin-coated slides, air-dried overnight, and coverslipped using cytoseal. Density scores from doublelabeled tissue (jejunum; n = 6; seven equidistant sampling points) were compared to the neuron density derived from single-labeled tissue from two different groups of rats (Test 1; 3 month old rats; seven sampling points) (see Fig. 1D). Separate t-tests were used. In the double-labeled tissue, there was no difference ( p>0.05) between Cuprolinic Blue counts; however, the density of NADPHd-labeled neurons was significantly reduced by 23% ( P < 0.0001). Thus, underestimation of the NADPHd-positive population of myenteric neurons occurred in the double-labeled tissue, most likely because Cuprolinic Blue staining masked NADPHd reactions. 2.9.1. Conclusions from validation trials Taken together, these findings validate: (a) the use of seven equidistant sampling points per circumferential column to determine the density of either the entire population or subpopulation of neurons; (b) the use of a correction factor to correct for age- and stretch-induced changes in tissue area; and (c) the use of separate groups (different ages/stain) of animals to determine the proportion of subpopulations of neurons. In addition, counts from tissue double-stained with both Cuprolinic Blue and NADPHd underestimated the NADPHd-positive population of neurons, necessitating the use of single-stained

Fig. 2. NADPHd-positive neurons in the stomach at 3 and 24 months of age. There were no age-related changes in the densities of NADPHd-positive neurons for the regions sampled. Scale bar = 100 Am.

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Table 1 Density of NADPHd-positive neurons (neurons/cm2) and percentage of neurons NADPHd-positive Region

Stomach Forestomach Corpus Antrum Small intestine Duodenal bulb Duodenum Jejunum Ileum

a

% NADPHd-positive

3 months

24 months

3 months 24 months

2013 F 178 2618 F 80 2723 F 203

2130 F 52 2478 F 79 2204 F 244

46 36 30

Large intestine Colon 5019 F 107 Rectum 4644 F 67

6899 F 664 5159 F 319 6450 F 118 5107 F 208

5071 F 184 4418 F 139

39 34 53 34

34 38

Region

Neuron density (mean F S.E.M.)a

% Loss

b

Neuron density (mean F S.E.M.)

6757 F 289 4963 F 102 6595 F 97 4779 F 87

Table 2 Total neuronal density (neurons/cm2) and age-related neuron loss (%)

50 37 24

54 47 57 45

58 58

a

There were no age-related changes in the density of the NADPHdlabeled neurons in the stomach, small intestine, and large intestine (all p values >0.05). b Extrapolated from total neuronal density in Table 2.

tissue from different groups of rats to get accurate counts.

3. Results 3.1. Neuron counts 3.1.1. 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

3 months Stomach Forestomach Corpus Antrum

24 months

4403 F 466 7222 F 270 9109 F 581

4263 F 188 6724 F 248 9069 F 229

3 7 0

Small intestine Duodenal bulb Duodenum Jejunum Ileum

17,410 F 884 14,446 F 500 12,550 F 195 14,060 F 156

12,770 F 845 10,913 F 517 11,282 F 216 11,372 F 418

27 25 10 19

Large intestine Colon Rectum

14,625 F 458 12,154 F 178

8669 F 265 7602 F 277

41 37

a There were no age-related changes in the total neuronal density in the stomach (all p values >0.05); however, there were significant losses of neurons with age in the small intestine (all p values < 0.01) and large intestine (both p values < 0.0001).

>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) the neuronal counts (see below) were very similar for both ages, a correction factor for increased area with age was not used on stomach counts. There were no age-related changes in the density of the NADPHd-labeled neurons in the forestomach, corpus, and antrum ( p values >0.05) (see Fig. 2 and Table 1). Similarly, there were no age-related changes in the density of the

Fig. 3. Age-related changes in the myenteric plexus from representative regions of the stomach, small intestine, and large intestine at 3 and 24 months of age. The total population of myenteric neurons was stained using Cuprolinic Blue. No change in neuronal density in the corpus is evident at 24 months, whereas a reduction in the densities of neurons is clearly noticeable in the duodenum and colon. Scale bar = 100 Am.

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Fig. 4. NADPHd-positive neurons in the small intestine at 3 and 24 months of age. There were no age-related changes in the densities of NADPHd-positive neurons for the regions sampled. Scale bar = 100 Am.

Cuprolinic Blue-labeled neurons in the same regions ( p values >0.05) (see Fig. 3 and Table 2). 3.1.2. Small intestine The areas of the duodenum, jejunum, and ileum were compared to determine if unintentional stretch-induced

differences (from processing) existed between the 3month-old rats stained with either NADPHd or Cuprolinic Blue. Separate t-tests for each region were used. There were no differences in tissue surface areas between the two staining groups for any of the three regions ( p values >0.05). On the other hand, the small intestines of 24-

Fig. 5. NADPHd-positive neurons in the large intestine at 3 and 24 months of age. There were no age-related changes in the densities of NADPHd-positive neurons for the regions sampled. Scale bar = 100 Am.

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Table 3 Neuron size Region

Stain

Age (months)

N

Totala

Neuron sizeb,c

Forestomach

Cup. Blue

3 24 3 24 3 24 3 24 3 24 3 24 3 24 3 24

9 7 6 8 6 5 5 5 8 7 6 5 7 6 7 8

1003 806 362 474 1411 1056 557 466 1679 1227 729 491 1210 938 761 725

267 F 20 316 F 33 711 F 38 711 F 19 158 F 9 177 F 11 434 F 20 453 F 30 241 F 12 295 F 13 484 F 22 577 F 10 215 F 14 272 F 15 460 F 14 552 F 19

NADPHd Ileum

Cup. Blue NADPHd

Colon

Cup. Blue NADPHd

Rectum

Cup. Blue NADPHd

a

Total number of neurons measured. Mean F S.E.M (Am2). c There were no age-related differences in Cuprolinic Blue- or NADPHd-positive neuron sizes for either the forestomach or the ileum ( p values >0.05); however, significant age-associated increases in neuron size did occur in both the colon and the rectum for both stains ( p values < 0.01). b

month-old rats were significantly ( p < 0.0001) longer than those of 3-month-old rats (mean F S.E.M.; 110 F 1.1 and 97 F 0.8 cm, respectively). Thus, correction factors based on the difference in mean intestinal area (cf. Johnson et al., 1998; Cowen et al., 2000; Phillips and Powley, 2001) were applied to the cell counts of 24-month-old rats. Counts from 24-month-old rats stained with NADPHd were corrected using a correction factor derived from the areas of the 3month-old rats stained with NADPHd. Likewise, cell counts from 24-month-old rats stained with Cuprolinic Blue were corrected using a factor derived from the areas of the 3month-old rats stained with Cuprolinic Blue. The density of myenteric neurons has been shown to be higher in the duodenal bulb (i.e., the first 0.5 cm of small intestine adjacent to the pyloric sphincter) than in the rest of the small intestine (Jarvinen et al., 1999; Phillips and Powley, 2001), and for that reason, we analyzed the duodenum as two separate regions (i.e., the duodenal bulb and the more distal duodenum). There were no age-related losses of NADPHd-positive neurons in the four regions of the small intestine sampled ( p values >0.05) (see Fig. 4 and Table 1). There were, however, significant losses of CuproFig. 6. Age-related changes in NADPHd-positive neuron size from representative regions of the stomach, small intestine, and large intestine. Neuron sizes were taken from NADPHd-positive neurons, so they reflect age-related changes in the nitrergic subpopulation of myenteric neurons. Neuron size is reported in 50-Am2 bins. The mean percentages were obtained from the single percentages of each rat for every interval. The insets in each graph are medium lowess curves for both 3 and 24 months of age, and the curves are used to reflect the trend of the data. No age-related change in neuron size was evident for the forestomach (A; p>0.05) or ileum (B; p>0.05), whereas the NADPHd-positive neurons in the colon (C; p < 0.01) and rectum (D; p < 0.01) were significantly larger at 24 months of age.

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linic Blue-labeled neurons with age. The attrition occurred in all four regions of the small intestine sampled ( p values < 0.01) (see Fig. 3 and Table 2). The greatest age-related loss of neurons occurred in the duodenal bulb, with about 27% of the total population of neurons dying by 24 months of age. Since the Cuprolinic Blue-stained neuronal population consists of nitrergic and cholinergic neurons, the lack of change in number of nitrergic neurons indicates that agerelated cell losses were restricted to the cholinergic phenotype. The net effect was a proportionate increase in nitrergic neurons with age (see Table 1). 3.1.3. Large intestine At 3 months of age, there were no differences in tissue areas between the group stained with Cuprolinic Blue and the group stained with NADPHd for either the colon or the rectum ( p values >0.05). The large intestines of 24-monthold rats were significantly ( p < 0.0001) longer than the large intestines of 3-month-old rats (mean F S.E.M.; 21 F 0.3 and 19 F 0.4 cm, respectively); therefore, correction factors were applied to the cell counts from 24-month-old rats. Consistent with the findings in the small intestine, there were no age-related losses of NADPHd-positive neurons in either region of the large intestine sampled ( p values >0.05) (see Fig. 5 and Table 1). For both regions of the large intestine, however, there were significant age-related losses of Cuprolinic Blue-labeled neurons ( p values < 0.0001) (see Fig. 3 and Table 2). On average, about 39% of the total number of neurons had died by 24 months of age. The loss of Cuprolinic Blue-stained neurons (presumably owing to the loss of cholinergic neurons) with the concurrent stability of nitrergic neurons produced an age-related increase in the percentage of nitrergic neurons in the myenteric plexus (see Table 1). 3.2. Neuron sizes 3.2.1. NADPHd-labeled neurons There were no age-related changes in the mean neuron size of NADPHd-positive neurons in either the forestomach or ileum ( p values >0.05); however, significant age-related increases in mean neuron size occurred in both the colon and the rectum ( p values < 0.01) (see Table 3). This increase in nitrergic neuron size was reflected as a shift to the right when lowess curves were fitted to the frequency distribution of neuron sizes (see Fig. 6). Fig. 7. Age-related changes in neuron size from representative regions of the stomach, small intestine, and large intestine. Neuron sizes were taken from Cuprolinic Blue-labeled tissue, so they reflect changes in the total population of myenteric neurons. Neuron size is reported in 25-Am2 bins. The mean percentages were obtained from the single percentages of each rat for every interval. The insets in each graph are medium lowess curves for both 3 and 24 months of age. From the graphs and inserts, it is clear that there is no age-related change in neuron size for either the forestomach (A; p>0.05) or ileum (B; p>0.05); however, neurons in both the colon (C; p < 0.01) and rectum (D; p < 0.01) are significantly larger at 24 months of age as evidenced by the shift to the right in the curves shown in the insets.

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3.2.2. Cuprolinic Blue-labeled neurons Similar to the nitrergic pattern, there were no age-related changes in mean neuron size in either the forestomach or ileum ( p values >0.05); however, a significant age-related increase in mean neuron size did occur in both the colon and the rectum ( p values < 0.01) (see Table 3). When lowess curves were fitted to the frequency distributions of neuron sizes, the increase in neuron size was reflected as a shift to the right (see Fig. 7).

3.3. Additional morphological observations 3.3.1. Swollen axons During the quantification phase of the study, swollen NADPHd-positive axons were noted in some of the tissue; therefore, all pieces of tissue at each age were scanned by an experimenter who was blind to age, and, for each region, the number of whole mounts that contained swollen nitrergic axons were recorded. [Quinolinic phthalocyanine stains only

Fig. 8. Swollen NADPHd-positive axons were found in both smooth muscle (A – C, F – I) and myenteric ganglia (D, J) at 24 months of age. These swollen axons were not found in the stomach, but were regularly found in the small intestine (A – C) and frequently found in the large intestine (D – J). Scale bars in A, E, I, and J = 50 Am. Scale bars in B, C, D, F, G, and H = 25 Am.

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somata, so a comparable analysis could not be performed on neurons stained with Cuprolinic Blue.] At 3 and 24 months of age, there were no swollen axons in the stomach (n = 0/6 and 0/8 whole mounts, respectively). In fact, at 3 months of age, swollen axons were never found in either the small (duodenum, n = 0/8; jejunum, n = 0/8; ileum, n = 0/8) or large intestine (colon, n = 0/8; rectum, n = 0/7); however, this was not the case for tissue from 24-month-old rats. At 24 months of age, 38 – 88% of the small intestine whole mounts (duodenum, n = 7/9; jejunum, n = 3/8; ileum, n = 7/8) and 100% of the large intestine whole mounts (colon, n = 9/9; rectum, n = 8/8) contained swollen axons. The swollen axons were routinely found in the circular muscle; however, on occasion, they were located within a myenteric ganglion or in the secondary plexus between ganglia (see Fig. 8).

4. 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, in the large intestine, some NADPHdpositive cells hypertrophy. These findings and their implications are discussed more fully below.

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Table 4 Estimated density of NADPHd-negative neurons (neurons/cm2) and agerelated cell loss (%) Region

Neuron densitya 3 months

Stomach Forestomach Corpus Antrum

% Loss 24 months

2378 4622 6376

2132 4236 6892

10 8 +8

Small intestine Duodenal bulb Duodenum Jejunum Ileum

10,620 9535 5899 9280

5874 5784 4851 6255

45 39 18 33

Large intestine Colon Rectum

9653 7536

3641 3193

62 58

a Density scores for NADPHd-negative neurons (i.e., presumptive cholinergic neurons) were derived from the percentages and density scores reported, respectively, in Tables 1 and 2.

Conceivably, though such analyses are not yet available, an entire functional/chemical subtype(s) might be lost due to age-related cell death. Finally, one additional caveat that we would like to clarify is that a small population of neurons is potentially both nitrergic and cholinergic. This has been shown mostly in species other than the rat (mouse: Sang and Young, 1998; guinea pig: Lomax and Furness, 2000; Chiocchetti et al., 2003), but nonetheless, this subpopulation raises the possibility that not all cholinergic neurons are susceptible to cell death.

5. Age-related changes in myenteric neuron density 6. Additional observations on 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 inflicted entirely on the cholinergic subpopulation of neurons, the effect is proportionately even more dramatic. For example, in the colon, 41% of all neurons are lost at 24 months of age; however, when this loss is suffered entirely by cholinergic neurons, over 62% of the phenotype is lost by 24 months. The fact that age-related cell loss is restricted to a particular phenotype also raises another obvious question. Cholinergic neurons can be broken down into subclasses based on co-localization with other transmitters (e.g., calbindin and calretinin; Vanden Berghe et al., 1999; Brookes, 2001; Schemann et al., 2001; Chiocchetti et al., 2003), so it is possible that cell loss is still more restricted, and limited to one or more of these subclasses of cholinergic neurons.

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 accommodation and the proximity of the duodenal bulb to the pyloric sphincter make the two regions likely candidates for some functional specificity; the finding of a high density of nitrergic neurons in the jejunum, however, is harder to explain. Therefore, more areas from the jejunum need to be sampled in future studies to determine if the high percentage of nitrergic cells in the region sampled (on average, the jejunum is 77 cm in length and we only sampled from 10 cm of the mid-jejunum) reflects either a uniformly high percentage for the entire region or if we inadvertently sampled from a functionally specialized region (e.g., a flexure in the intestine).

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The densities of Cuprolinic Blue-labeled myenteric neurons in the GI tracts of 3- and 24-month-old Fischer 344 rats were similar to the counts previously reported for the same age groups (Phillips and Powley, 2001), and are consistent with the earlier conclusions that: (a) the pattern of cell loss varies by organ (i.e., no cell loss in the stomach, whereas significant cell loss occurs in the small and large intestines), (b) an oral-to-anal gradient in cell loss (i.e., cell loss in the stomach < cell loss in the small intestine < cell loss in the large intestine), and finally, (c) within organ, the regional degree of loss is not uniform (e.g., 27% cell loss in the duodenal bulb and only 10% cell loss in the jejunum).

7. 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 Am2 (we found a mean neuron size of 267 and 215 Am2, respectively), and in the small intestine, the peak neuron size was between 100 and 175 Am2 (we found a mean neuron size of 158 Am2 in the ileum). Furthermore, the distributions of cells sizes, reported in the present study, for the small and large intestines are similar to those reported for 6-month-old rats by Santer and Baker (1988) using NADH-tetrazolium reductase. Finally, for NADPHd-stained neurons, Cracco and Filogamo (1994) reported a mean somatic size of 446 Am2 in the ileum of 6month-old rats, and we found the mean neuron size to be 434 Am2 in 3-month-old rats. Direct size comparisons between Cuprolinic Blue- and NADPHd-labeled neurons are not practical because of the nature of the stains; Cuprolinic Blue labels the cytoplasm of the cell soma, while NADPHd labels the cytoplasm and processes (i.e., dendrites and axons) of the neurons. It is possible, however, to compare Cuprolinic Blue- or NADPHd-stained profiles across different time points. For 3 and 24 months of age, there was complete overlap of neuron size distributions for the Cuprolinic Blue samples, and, again, for the NADPHd samples in the forestomach and ileum. In contrast, in the large intestine, neurons stained with both markers were significantly larger at 24 months compared to 3 months. The lack of change in mean neuron sizes and the overlapping frequency distributions between 3 and 24 months for both stains in the forestomach and ileum argues against continued neuron growth over the lifespan of the animal (Cowen et al., 2000) as an explanation for increased neuron size in the large intestines of older rats. The increase in neuron size in the large intestine reflects, most likely, hypertrophy analogous to that which occurs in the stressed intestine (Earlam, 1971; Gabella, 1975; Brehmer et al., 2000). Specifically, the extensive loss of

cholinergic neurons presumably results in disruption of normal GI function in the large intestine (McDougal et al., 1984; Smits and Lefebvre, 1996b; Wade et al., 2002a), and forces the surviving neurons (the nitrergic neurons, as well as potentially the surviving cholinergic neurons) to respond by compensating to maintain function (e.g., peristalsis, secretion, and absorption). This increased stress and functional demand on the neurons may then cause them to hypertrophy. Consistent with our finding of hypertrophy of nitrergic neurons, Brehmer et al. (2000) recently demonstrated that neuron hypertrophy in the partially obstructed intestine was specific to the nitrergic phenotype. Because we did not determine if increased neuron size occurred in the cholinergic subpopulation, however, it remains to be seen if neuronal hypertrophy in the aged large intestine is cell-phenotype specific.

8. 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 diameter. If these enlarged axons are analogous to the sympathetic pathology, then aged nitrergic axons might be undergoing a process of retraction or dying back. Since swollen axons and terminals were observed in both the smooth muscle and myenteric ganglia, then it must be that both nitrergic inhibitory smooth muscle motor neurons and, to a lesser extent, inhibitory interneurons (Furness et al., 1994) are compromised in the aged gut. From these observations, it appears that even though the nitrergic neurons survive in the aged rat, the smooth muscle innervation may become progressively compromised as the animal ages (if the assumption is made that the deterioration of function occurs in a linear manner). An age-related decrease in smooth muscle innervation by inhibitory motor neurons over the lifespan of the rodent is consistent with a previously reported decrease in nitrergic contribution to nonadrenergic noncholinergic smooth muscle relaxation of the rat ileum (Smits and Lefebvre, 1996a).

9. 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

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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 loss of a specific chemical phenotype, (b) an increase in neuronal size, an apparent compensatory hypertrophy, in regions of the gut with the greatest amount of cell death, and (c) the presence of swollen axons throughout the intestines, are all consistent with age-related cell loss in the myenteric plexus being detrimental to the normal functioning of the gut.

10. 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; however, why cholinergic neurons in the stomach survive while neurons of the same phenotype die in the small and large intestines. Whether selective survival is due to intrinsic factors such as co-localization with other transmitters, the presence of trophic factors in the target tissue, or innervation by extrinsic nerves remains to be determined.

11. 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, 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 innervate (e.g., Brookes et al., 1991; Schemann et al., 2001), and stimulation of this ascending pathway evokes a strong contractile response (Neunlist et al., 1999; Furness, 2000; Schemann et al., 2001). Therefore, these descending inhibitory and ascending excitatory pathways (i.e., nitrergic and cholinergic neurons, respectively) work in concert to mediate relaxation in front and contraction behind a bolus of food or feces during peristalsis. One would speculate that in the aged gut, where cholinergic neurons die and nitrergic neurons survive, smooth muscle motility would be in turmoil. Specifically, the coordination and force of contraction would be disrupted or diminished and would result in motility related disorders. In fact, in the aged rodent (Smits and Lefebvre, 1996b; Wade et al.,

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2002a) and human (e.g., Merkel et al., 1993; Samal and Ramakrishna, 1997; Camilleri et al., 2000), this appears to be the case.

12. 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) of cholinergic neurons makes them vulnerable to the affects of age. Conversely, questions also need to be answered about the neurons that remain: (a) how do nitrergic neurons survive, and (b) how are these surviving neurons affected by the loss of their functional counterparts?

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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