Choline acetyltransferase and inducible nitric oxide synthase are increased in myenteric plexus of diabetic guinea pig

Autonomic Neuroscience: Basic and Clinical 118 (2005) 12 – 24 www.elsevier.com/locate/autneu

Choline acetyltransferase and inducible nitric oxide synthase are increased in myenteric plexus of diabetic guinea pig Kathy J. LePard* Department of Physiology, Chicago College of Osteopathic Medicine, Midwestern University, 555 West 31st Street, Downers Grove, IL 60515, United States Received 30 July 2004; received in revised form 8 December 2004; accepted 11 December 2004

Abstract Alterations in enzymes in myenteric neurons from ileum were investigated in guinea pigs treated with either the pancreatic beta cell toxin streptozotocin or vehicle. After 5–6 weeks, expressions of choline acetyltransferase, neuronal nitric oxide synthase and inducible nitric oxide synthase were determined in longitudinal and myenteric plexus preparations using indirect immunohistochemistry. In ileum from streptozotocin-treated animals, the density of choline acetyltransferase-immunoreactive nerve fibers within the tertiary plexus and the percent total myenteric neurons expressing inducible nitric oxide synthase were increased, but the percent total myenteric neurons expressing neuronal nitric oxide synthase was not changed. Diabetes resulted in selective alterations in myenteric neurons including an increased density of cholinergic tertiary fibers and percentage of neurons expressing the inducible isoform of nitric oxide synthase. These adaptive changes by myenteric neurons to diabetes may contribute to gastrointestinal dysfunctions associated with diabetes. D 2004 Elsevier B.V. All rights reserved. Keywords: Diabetes mellitus; Experimental; Streptozotocin; Myenteric plexus; Choline O-acetyltransferase; Nitric oxide synthase, Type I; Nitric oxide synthase, Type II

1. Introduction Gastrointestinal (GI) afflictions are not normally lifethreatening but do profoundly affect quality of life. Diabetic patients experience a wide range of GI discomforts including heartburn, nausea, vomiting, diarrhea, constipation, fecal incontinence and abdominal pain (Bytzer et al., 2001; Folwaczny et al., 1999). Some diabetic patients have abnormalities in small intestinal motility arising from identifiable conditions such as celiac sprue or bacterial overgrowth in the small intestine; but over 50% of GI complaints are idiopathic (Valdovinos et al., 1993). Autonomic neurons and hormones regulate gastrointestinal function. Enteric autonomic neurons respond to luminal contents and smooth muscle tension and initiate local reflexes to optimize motility and secretion. Extrinsic

* Tel.: +1 630 515 6391; fax: +1 630 971 6414. E-mail address: [email protected]. 1566-0702/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.autneu.2004.12.002

parasympathetic and sympathetic nerve fibers synapse on intrinsic enteric neurons to evoke alterations in motility and secretion according to the demands of the central nervous system. Therefore, alterations in gastrointestinal motility observed in diabetic patients may arise from dysfunction of extrinsic (parasympathetic and sympathetic) and/or intrinsic (enteric) neurons. Increased prevalence of GI complications in diabetic patients has been associated with autonomic neuropathy in some studies (Enck et al., 1994; Werth et al., 1992) but not others (Clouse and Lustman, 1989; Jebbink et al., 1993; Ko et al., 1999). Tests of autonomic neuropathy usually address the function of extrinsic sympathetic and parasympathetic nerves using noninvasive cardiovascular and/or sweating tests (Ewing et al., 1985), not gastrointestinal function. Few studies have investigated neuropathy of enteric neurons in diabetic patients with GI complaints because these require invasive full-thickness biopsies. In case studies of diabetic patients, alterations in enteric neurons have been documented. Colonic biopsies, taken

K.J. LePard / Autonomic Neuroscience: Basic and Clinical 118 (2005) 12–24

from diabetic patients with autonomic neuropathy, revealed ultrastructural damage to submucosal neurons, including degeneration of processes (Riemann and Schmidt, 1982). A jejunal biopsy from a diabetic patient with peripheral neuropathy and gastroparesis showed alterations in both enteric and sympathetic neurons. In the circular muscle layer, the volume density of enteric inhibitory nerve fibers positive for neuronal nitric oxide synthase (nNOS), vasoactive intestinal peptide (VIP) or PACAP was decreased, but the volume density of excitatory nerve fibers positive for substance P was increased (He et al., 2001). Therefore, neuropathy of enteric neurons may contribute to GI complications of diabetes. The role of enteric neurons in GI complications of diabetes is more easily isolated and completely addressed using animal models. The immunofluorescence, tissue content and nerve-evoked release of peptides, neuronal enzymes and neurotransmitters have been most extensively studied in rat models of diabetes (Belai et al., 1985, 1996; Belai and Burnstock, 1990; Buchan, 1990; Di Giulio et al., 1989; Lincoln et al., 1984; Riemann and Schmidt, 1982; Spa˚nge´us and el Salhy, 1998; Yu and Ouyang, 1999). The magnitude of these changes is specific to the plexus [myenteric or submucous (Belai and Burnstock, 1990; Buchan, 1990)], segment of gut (Belai et al., 1985; Belai and Burnstock, 1987; Lincoln et al., 1984; Yu and Ouyang, 1999), peptide and model of diabetes. Alterations in peptide, enzyme and neurotransmitter content are observed in alloxan-induced diabetic rats (Di Giulio et al., 1989), spontaneously diabetic BB Wistar rats (Buchan, 1990; Yu and Ouyang, 1999), streptozotocin (STZ)-induced diabetic rats (Belai et al., 1985, 1987, 1996; Belai and Burnstock, 1990; Wiklund et al., 1993; Yu and Ouyang, 1999) and BALB/CJ spontaneously diabetic mice (el-Salhy and Spa˚nge´us, 1998). These animal models of type I diabetes are characterized by low plasma insulin and high plasma glucose. These studies used an experimental model of diabetes in guinea pigs, a model of type I diabetes characterized by low plasma insulin and c-peptide, but transient hyperglycemia, to investigate the density of innervation of the longitudinal muscle by choline acetyltransferase (ChAT) immunoreactive (ir) motor neurons and the percent of nNOS-ir and inducible (i) NOS-ir myenteric neurons per total Hu-ir

13

myenteric neurons. This model will allow for the more selective investigation of the role of insulin in neuropathy of enteric nerves.

2. Material and methods 2.1. Animals Male Hartley guinea pigs (200–300 g, Charles River, Portage, MI) were given a single intraperitoneal (ip) injection of freshly prepared citrate buffer (5 M, pH 4.5, vehicle-treated group) or streptozotocin (STZ, pancreatic beta cell toxin, 50 mg/ml, 280 mg/kg, Sigma Chemical, St. Louis, MO) dissolved in citrate buffer. This dose of STZ induced diabetes in guinea pigs (Schlosser et al., 1984). Diabetes in STZ-treated animals was confirmed by assessing final body weight, urine glucose, and kidney weight at the time of euthanasia. These parameters were also determined for vehicle-treated animals to establish a normal baseline for statistical comparison. One group of age-matched animals (n=4) was not given any ip injection. Animals were maintained on normal guinea pig chow and H2O ad libitum. At 5–6 weeks after injection, animals were euthanized. A guinea pig was placed inside an enclosed chamber and lightly anesthetized with halothane, an inhalation anesthetic. After losing the righting reflex, the animal was stunned and the carotid artery and spinal cord were severed using sharp scissors. This method of euthanasia was approved by the Midwestern University Research and Animal Care Committee. A segment of ileum was placed in warmed, oxygenated Krebs solution of the following composition (mM): NaCl, 117; KCl, 4.7; CaCl2, 2.5; MgCl2, 1.2; NaH2PO4, 1.2; NaHCO3, 25; glucose, 11. A segment of ileum was tightly stretched mucosa-sidedown in a dish lined with sylgard then treated with fixative. LMMP preparations were peeled off the tissue and cut into small squares. At least two small squares for each antibody were placed in a 24 well plate lined with sylgard then treated with primary and secondary antibodies (Table 1). Tissues were mounted in buffered glycerol for fluorescence microscopy.

Table 1 Primary and secondary antibodies used in staining LMMP preparations Primary

Host

Dose

Cat no.

Company

Secondary

Dose

Cat no.

Company

ChAT nNOS iNOS PGP 9.5 HuC/HuD DBH

goat rabbit rabbit rabbit mouse mouse

1:100 1:500 1:200 1:250 1:200 1:400

AB144P N53130 N32030 AB1761 A21275 MAB308

CH BDTL BDTL CH MP CH

Donkey anti-goat FITC Sheep anti-rabbit CY3 Sheep anti-rabbit CY3 Sheep anti-rabbit CY3 Alexa fluor 488a Goat anti-mouse TRITC

1:100 1:100 1:100 1:100 NA 1:100

AP180F C2306 C2306 C2306 NA T5393

CH Sigma Sigma Sigma NA Sigma

a HuC/HuD was directly tagged with Alexa fluor 488. Abbreviations: BDTL: BD Transduction Labs, Lexington, KY; MP: Molecular Probes, Eugene, OR; CH: Chemicon International, Temecula, CA; Sigma: Sigma Chemical, St. Louis, MO; NA: not applicable.

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2.2. Choline acetyltransferase Ileum was processed for immunohistochemical localization of ChAT. Tissues were fixed for 4 h at room temperature in 0.2% picric acid and 2% paraformaldehyde in 0.1 M phosphate buffered saline (PBS, pH 7.40). Tissues were washed 310 min in 0.1 M PBS then permeabilized for 60 min in a solution of 0.1 M PBS containing Triton X100 (0.5%) and 4% rabbit serum (Sigma). Tissues were incubated for 12–24 h at room temperature in a dark, humidified chamber with primary antisera to ChAT (Table 1) then rinsed 310 min in 0.1 M PBS containing 0.5% Triton X-100 and 4% rabbit serum. Tissues were incubated for 2 h at room temperature in a dark, humidified chamber with secondary antisera (Table 1) then rinsed 310 min in 0.1 M PBS containing 0.5% Triton X-100 and 4% rabbit serum. The primary and secondary antibodies were diluted using 0.1 M PBS containing 0.5% Triton X-100 and 4% rabbit serum. 2.3. Neuronal nitric oxide synthase, inducible nitric oxide synthase, protein gene product 9.5, or HuC/HuD Tissues were fixed overnight at 4 8C in modified Zamboni’s fixative (15% saturated picric acid and 2% formaldehyde in 0.1 M PBS, pH 7.40). Tissues were washed 310 min in dimethyl sulphoxide and then in PBS (0.1 M, pH 7.40). Tissues were incubated overnight in a dark, humidified chamber with primary antisera to nNOS, iNOS, or protein gene product 9.5 (PGP) (Table 1) then rinsed 310 min in 0.1 M PBS. Tissues were incubated for 2 h at room temperature in a dark, humidified chamber with secondary antisera (Table 1) then washed 310 min in 0.1 M PBS. Some LMMP were double-stained with nNOS or iNOS and HuC/HuD (Hu), a neuronal marker that stains neuronal cell bodies but not processes (Table 1). For double staining, the primary nNOS or iNOS antibody and the Hu antibody were applied to the tissue at the same time at the final concentration listed in Table 1. The iNOS antibody does not stain the same population of neurons as the nNOS antibody (Podlasek et al., 2001). Myenteric neurons immunoreactive for iNOS were still observed after pretreatment of the tissue with the nNOS blocking peptide (BD Transduction Labs) at a concentration that prevented binding of the nNOS primary antibody (1:100) (personal observation). 2.4. Dopamine b-hydroxylase Tissues were fixed for 4 h at room temperature in fixative (0.4% saturated picric acid and 4% formaldehyde in 0.1 M PBS, pH 7.40). Tissues were washed 310 min in 0.1 M PBS then permeabilized for 60 min in a solution of 0.1 M PBS containing Triton X-100 (0.3%) and 4% rabbit serum. Tissues were incubated for 12–24 h at room temperature in a dark, humidified chamber with primary

antisera to dopamine h-hydroxylase (DBH) (Table 1) then rinsed 310 min in 0.1 M PBS containing 0.3% Triton X100 and 4% rabbit serum. Tissues were incubated for 2 h at room temperature in a dark, humidified chamber with secondary antisera (Table 1) then rinsed 310 min in 0.1 M PBS containing 0.3% Triton X-100 and 4% rabbit serum. The primary and secondary antibodies were diluted with 0.1 M PBS containing 0.3% Triton X-100 and 4% rabbit serum. 2.5. Image analysis LMMP were visualized using a fluorescence microscope (Nikon Eclipse 400 or Leica DMIL) with attached digital camera (Diagnostics Instruments, or MagnaFire, Optronics). Images of sequential ganglia and/or tertiary plexus were obtained with no regard for ganglia size or fluorescence intensity. Optimal exposure times to capture the best image were determined for each protein, then did not vary. If needed, images for cell counts were sharpened using Microsoft Photo Editor. Digital images 766 by 510 pixels were 0.60 Am/pixel at 20, and 0.31 Am/pixel at 40. Digital images 1280 by 1024 pixels were 0.327 Am/pixel at 20, and 0.167 Am/pixel at 40. Image size varied between proteins but not between vehicle- and STZ-treated animals. Digital images were in 32 bit RGB color at an image resolution of 100 dpi and a computer screen resolution of 92 dpi. Image ProPlus software (ver. 4.5.0.29, Media Cybernetics, Sliver Spring, MD) was used to analyze all digital images. 2.6. Area occupied by immunoreactive neurons and/or processes For many images, the area occupied by all immunoreactive structures was determined. Within an area of interest (AOI), the software automatically counted and measured the area occupied by strongly fluorescent, immunoreactive structures. Individual areas were then summed. If needed, the minimum fluorescence intensity and/or the minimum area of a single bright object were defined to ensure inclusion of all immunoreactive structures and/or exclusion of all background within the AOI, otherwise that AOI was excluded from data analysis. 2.7. Size of myenteric ganglia Size of myenteric ganglia was determined using some of the nNOS and iNOS images used for cell counts. An AOI was loosely drawn around the ganglion to exclude primary and secondary fiber tracts. Area of the ganglion was determined as described above (Area occupied by immunoreactive neurons and/or processes) (Spa˚nge´us and el Salhy, 1998). Data were expressed as Am2 and compared between vehicle and STZ using Mann Whitney U-test with significance at pb0.05.

K.J. LePard / Autonomic Neuroscience: Basic and Clinical 118 (2005) 12–24

2.8. Area of the tertiary plexus The tertiary plexus is located between wide, primary fiber tracts connecting individual ganglion and other secondary fiber tracts. The tertiary plexus is composed of axons that innervate longitudinal muscle. The percent of the total image occupied by the tertiary plexus was determined using 20 images stained for PGP. Using the entire image, the area occupied by ganglia and primary and secondary fiber tracts was determined as described above (area occupied by immunoreactive neurons and/or processes) (Renzi et al., 2000). Tertiary area was calculated by subtracting the area occupied by ganglia and the primary and secondary fiber tracts from the total image area. Data were expressed as percent total area (meanFS.E.M.) and compared between vehicle and STZ using Student’s t-test with significance at pb0.05. 2.9. Density of choline acetyltransferase-immunoreactive or protein gene product 9.5-immunoreactive tertiary fiber tracts Using 20 images, densities of PGP-ir and ChAT-ir tertiary fibers were determined. The area occupied by ChATir or PGP-ir tertiary fiber tracts was determined as described above (Area occupied by immunoreactive neurons and/or processes). The selected AOI did not include ganglia or primary or secondary fiber tracts. In addition, the inclusive area of each AOI was measured. The percent total area of the AOI occupied by ChAT-ir or PGP-ir tertiary fibers was calculated for vehicle and STZ (Belai et al., 1997). Data were expressed as meanFS.E.M. and compared using Mann Whitney U-test with significance pb0.05. 2.10. Quantification of neuronal nitric oxide synthaseimmunoreactive and inducible nitric oxide synthase-immunoreactive neurons Ganglia for cell counting were selected based only on quality of Hu staining, not the presence or absence of iNOSir or nNOS-ir neurons or the size of ganglia. Ganglia were both larger and smaller than the size of the image at 40 (237 by 157 Am). If a ganglion was larger than the image, partial neurons were counted. The numbers of Hu-ir and nNOS-ir or iNOS-ir cells were counted at 40 for 8–40 images per animal (5–11 animals) by a blinded investigator. Images of Hu and NOS immunofluorescence were sometimes merged to better distinguish between overlapping Huir neurons. For some of these images, ganglia size was determined so the packing density of myenteric neurons could be calculated for each ganglion (total number of Hu-ir neurons/size of myenteric ganglion). Data were expressed as total number of Hu-ir, nNOS-ir or iNOS-ir neurons per ganglion, percent iNOS-ir or nNOS-ir neurons per total Huir neurons and packing density of Hu-ir neurons, then compared between vehicle and STZ using Mann–Whitney

15

U-test with significance at pb0.05. Data were expressed as meanFS.E.M. 2.11. Average fluorescence intensity Using a relative scale, a quantitative value, representing the fluorescence intensity of the fluorophore indirectly attached to the protein, was assigned to each pixel along a line placed either through the center of ganglia or in the adjacent tertiary plexus. The average fluorescence intensity was calculated over the distance of the line. An overall average was obtained for ganglia and tertiary plexus then compared between vehicle and STZ using Mann–Whitney U-test with significance at pb0.05. Data were expressed as meanFS.E.M. Other studies have validated that optical density or fluorescence intensity of stained proteins in tissues correlates well with neurotransmitter content as determined by Western blot analysis (Cellek et al., 2003; Chen et al., 1999; Friedmann et al., 1995). 2.12. Area of dopamine b-hydroxylase-immunoreactive varicosities Each image of myenteric ganglion (40) was converted to 2 tones of black (DBH-negative) and white (DBH-ir) every 0.62 Am (2 pixels) using the posterize command (Microsoft Photo Editor). The number of DBH-ir vesicles was counted and their individual areas were measured then summed. Data were averaged for vehicle and STZ and compared using Mann Whitney U-test with significance at pb0.05. Data were expressed as meanFS.E.M. 2.13. Frequency distributions Using all data from vehicle-treated animals, the 25th, 50th, 75th, and 100th percentiles of fluorescence intensities or areas were calculated. Using these quartiles, the frequency distributions for all data from STZ-treated animals were determined then compared to vehicle-treated animals using Chi Square test with significance at pb0.05.

3. Results Animals treated with the pancreatic beta cell toxin streptozotocin (STZ) had decreased final body weight, elevated urine glucose and enlarged kidneys as compared to vehicle-treated animals (Table 2). The size of individual myenteric ganglion and number of Hu-ir neurons per ganglion were evaluated for ileum from vehicle- and STZ-treated animals. The size of myenteric ganglia was decreased by approximately 9% in LMMP from STZ-treated animals [Am2: Vehicle (10 animals, n=265) 7630F197; STZ (12 animals, n=220) 6961F165*; *pb0.05]. In addition, the number of Hu-ir neurons per ganglion was decreased by approximately 6% in STZ-

16

K.J. LePard / Autonomic Neuroscience: Basic and Clinical 118 (2005) 12–24

Table 2 Characteristics of vehicle-treated and STZ-treated guinea pigs Vehicle STZ

Initial weight (g)

Final weight (g)

Urine glucose (mg/dl)

Kidney weight (g)

Kidney weight/body weight (10 3)

274F3 (n=31) 273F3 (n=38)

567F13 (n=31) 322F12* (n=37)

0F0 (n=30) 1175F107* (n=36)

2.14F0.05 (n=30) 1.84F0.05* (n=37)

3.79F0.04 (n=30) 5.89F0.15* (n=37)

n=number of animals, *pb0.05 as compared to vehicle.

treated animals [Vehicle (15 animals, n=435), 33F1; STZ (19 animals, n=502), 31F1*; *pb0.05]. As a result of these two changes, the packing density of neurons was increased in myenteric ganglia from STZ-treated animals [Hu-ir neurons/ganglion area (Am2)*10,000: Vehicle (10 animals, n=254) 43F1, STZ (12 animals, n=220) 51F1*, *pb0.05). The density of all axons innervating the longitudinal muscle was assessed using PGP 9.5, an excellent marker for visualizing all neural processes of the tertiary plexus (Karaosmanoglu et al., 1996). The percent of the total image area that was occupied by the tertiary plexus was not significantly altered in LMMP from STZ-treated animals [Vehicle (7 animals, n=38) 82F1%, STZ (8 animals, n=36) 80F1%, NS]. In contrast, the density of PGP-ir fibers within the tertiary plexus was decreased in ileum from STZ-treated animals (Fig. 1E,F) [density: Vehicle (8 animals, n=55), 13F1%, STZ (6 animals, n=47), 11F1%*, *pb0.05]. 3.1. Choline acetyltransferase In LMMP from STZ- and vehicle-treated animals, ChAT immunoreactivity was observed in the cytoplasm and dendritic processes of neurons and in primary, secondary and tertiary fiber tracts. The high density of ChAT-ir varicose processes in ganglia obscured ChAT-ir cell bodies such that they could not be counted. The density of ChAT-ir fibers in the tertiary plexus appeared increased in LMMP from STZ-treated animals. In LMMP from STZ-treated animals, the fluorescence intensities of ganglia and tertiary plexus were not altered (Table 3). The frequency distributions of all fluorescence intensities of tertiary plexus (Fig. 1D) and ganglia (Fig. 1C) were not altered in LMMP from STZ-treated (Fig. 1B) as compared to vehicle-treated (Fig. 1A) animals. In contrast, the density of ChAT-ir fibers within the tertiary plexus was increased in STZ-treated animals [density: Vehicle (5 animals, n=107) 11.4F0.3%, STZ (5 animals, n=95) 15.1F0.3%*, *pb0.05] (Fig. 1A and B). 3.2. Neuronal nitric oxide synthase In LMMP from STZ- and vehicle-treated animals, nNOS immunoreactivity was observed in the cytoplasm and dendrites of neurons and in primary fiber tracts. Some nNOS-ir neurons had large cell bodies with club-like dendrites while others had small cell bodies with no visible dendrites. Neuronal NOS immunoreactivity in secondary and tertiary fiber tracts was sparse, weak and non-varicose. The fluorescence intensity of nNOS-ir cytoplasm appeared

weaker in LMMP from STZ-treated animals. Because of sparse nNOS-ir neuronal processes within ganglia, a weaker fluorescence intensity of nNOS-ir in ganglia would reflect either a reduced amount of enzyme in the neuronal cytoplasm or a fewer number of nNOS-ir neurons within ganglia. The fluorescence intensities of ganglia, but not tertiary plexus, were decreased in LMMP from STZ-treated animals (Table 3). The frequency distributions of all fluorescence intensities of ganglia (Fig. 2C) and tertiary plexus (Fig. 2D) were shifted to weaker quartiles in ileum from STZ-treated (Fig. 2B) as compared to vehicle-treated (Fig. 2A) animals. In STZ-treated as compared to vehicle-treated animals, there was no difference in the total number of nNOSpositive neurons per ganglion [Vehicle (5 animals, n=118), 8.0F0.4; STZ (7 animals, n=196), 7.4F0.3; NS] and the density of nNOS-positive neurons per ganglionic area [Vehicle (4 animals, n=62), 9F1; STZ (4 animals, n=62, 11F1, NS]. There was no change in the percent of nNOS-ir neurons per total Hu-ir neurons in LMMP from STZ-treated animals [Vehicle (5 animals, n=118), 25F1%; STZ (7 animals, n=196), 29F1%; p=0.08] (Fig. 2). Every nNOS-ir neuron was immunoreactive for HuC/HuD. Because nNOSir tertiary fibers were sparse with weak fluorescence, their density could not be determined. 3.3. Inducible nitric oxide synthase In LMMP from STZ- and vehicle-treated animals, iNOS immunoreactivity was observed in cytoplasm and dendrites of neurons, primary fiber tracts and longitudinal muscle. Some iNOS-ir neurons had large cell bodies with club-like dendrites while others had small cell bodies with no visible dendrites. Inducible NOS immunoreactivity in secondary and tertiary fiber tracts was sparse, weak and non-varicose. The fluorescence intensities of iNOS immunoreactivity within ganglia and longitudinal muscle appeared stronger in LMMP from STZ-treated animals. Because of sparse iNOSir neuronal processes within ganglia, stronger fluorescence intensities of iNOS immunoreactivity in ganglia would reflect either a greater amount of enzyme in neuronal cytoplasm or a higher number of iNOS-ir neurons within ganglia. Because iNOS-ir tertiary fibers were sparse, stronger fluorescence intensity of iNOS immunoreactivity in the tertiary plexus would reflect greater expression of iNOS in longitudinal muscle. The fluorescence intensities of both ganglion and tertiary plexus were increased in LMMP from STZ-treated animals (Table 3). The frequency distributions of all fluorescence intensities of ganglia (Fig. 3C)

K.J. LePard / Autonomic Neuroscience: Basic and Clinical 118 (2005) 12–24

17

B.

A.

C.

D. 50

50

Percent Total

40 30 20 10

Vehicle (9 animals, n=171) STZ

40

Percent Total

Vehicle (9 animals, n=261) STZ (11 animals, n=240) Chi test NS

(11 animals, n=164)

30

Chi test NS

20 10

0

0 0

50

100

150

200

250

0

20

40

60

80

100

120

Fluorescence Intensity

Fluorescence Intensity

E.

F.

Fig. 1. ChAT and PGP immunoreactivity in LMMP preparations from vehicle- and STZ-treated animals. The density of ChAT-ir fibers in the tertiary plexus was increased in LMMP from STZ-treated animals (B) as compared to vehicle-treated animals (A). The frequency distributions of ChAT fluorescence intensities in ganglia (C) and tertiary plexus (D) were not altered in STZ-treated as compared to vehicle-treated animals. In contrast, the density of PGP-ir fibers in the tertiary plexus was decreased in LMMP from STZ-treated (F) as compared to vehicle-treated animals (E). Bar is 10 Am. NS: not significant.

and tertiary plexus (Fig. 3D) were shifted to stronger quartiles in ileum from STZ-treated (Fig. 3B) as compared to vehicle-treated (Fig. 3A) animals.

In STZ-treated as compared to vehicle-treated animals, there was an increase in both the total number of iNOSpositive neurons per ganglion [Vehicle (10 animals, n=317),

Table 3 Relative intensity of fluorescence of enzymes in longitudinal muscle and myenteric plexus preparations ChAT

Ganglia Tertiary Plexus

nNOS

iNOS

Vehicle (9)

STZ (11)

Vehicle (4)

STZ (4)

Vehicle (7)

STZ (6)

110F2 (n=261) 42F1 (n=171)

106F2 (n=240) 44F2 (n=164)

92F3 (n=81) 40F2 (n=78)

81F3* (n=86) 39F2 (n=72)

148F3 (n=70) 71F3 (n=70)

160F3* (n=55) 92F3* (n=55)

For Vehicle or STZ, the number of animals is shown in parentheses. n=number of ganglion or tertiary plexus values obtained for each enzyme, *pb0.05 as compared to vehicle.

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K.J. LePard / Autonomic Neuroscience: Basic and Clinical 118 (2005) 12–24

A.

B.

C.

D. 50 Vehicle (4 animals, n=81) STZ (4 animals, n=86) Chi test p<0.05

40 30 20 10 0 0

50

100

150

Vehicle (4 animals, n=78) STZ (4 animals, n=72) Chi test p<0.05

40

Percent Total

Percent Total

50

200

Fluorescence Intensity

30 20 10 0

0

20

40

60

80

100

Fluorescence Intensity

Fig. 2. nNOS immunoreactivity in LMMP preparations from vehicle- and STZ-treated animals. The percent of nNOS-ir neurons per total Hu-ir neurons in myenteric ganglia was not altered in ileum from STZ-treated (B) as compared to vehicle-treated (A) animals. The frequency distributions of nNOS fluorescence intensities in ganglia (C) and tertiary plexus (D) were shifted to weaker quartiles in STZ-treated as compared to vehicle-treated animals. Bar is 10 Am.

7.9F0.2; STZ (11 animals, n=308), 8.7F0.3*; *pb0.05] and the number of iNOS-positive neurons per ganglionic area [Vehicle (6 animals, n=192, 10.9F0.4; STZ (8 animals, n=158), 12.5F0.5*; *pb0.05]. The percent of iNOS-ir neurons per total Hu-ir neurons was significantly increased in myenteric ganglia from STZ-treated animals [Vehicle (10 animals, n=317), 24F1%, STZ (11 animals, n=306), 26F1%*, *pb0.05] (Fig. 3). The percent iNOS-ir neurons per total Hu-ir neurons was also greater in vehicle-treated animals as compared to a separate group of animals receiving no ip injection (4 animals, n=218: 18F1%*, *pb0.05). Every iNOS-ir neuron was immunoreactive for HuC/HuD. 3.4. Dopamine b-hydroxylase In LMMP from STZ- and vehicle-treated animals, punctate DBH immunoreactivity was observed as vesicles within myenteric ganglia. The area occupied by DBH-ir vesicles within myenteric ganglia was not altered [Vehicle (7 animals, n=58) 4842F564 (range: 282– 20,994); STZ (5 animals, n=44) 4010F557 (range 356– 14,683); NS], but the frequency distribution of all areas was significantly altered (Fig. 4C) in ileum from STZtreated (Fig. 4B) as compared to vehicle-treated (Fig. 4A) animals.

4. Discussion In ileum from STZ-treated animals, specific changes occurred in myenteric neurons expressing the enzymes ChAT, nNOS and iNOS. In ileum from STZ-treated as compared to vehicle-treated guinea pigs, the density of ChAT-ir tertiary fibers and the percent of iNOS-ir myenteric neurons were increased, but the percent of nNOS-ir myenteric neurons was not altered. This guinea pig model of type I diabetes is characterized by insulin and C-peptide deficiencies and glycosuria (DiMattio, 1992; Gorray et al., 1986; Schlosser et al., 1987). In contrast to STZ-treated rat models, hyperglycemia was not a consistent feature (Gorray et al., 1986; Hootman et al., 1998; Schlosser et al., 1984, 1987; Wehner and Majorek, 1975). Two major factors contribute to diabetes-induced neuropathy: 1) hyperglycemia, and 2) insulin and C-peptide deficiencies (Sima, 2003). There is a strong link between insulin deficiency and neuropathy. Scavenging of intrathecal endogenous insulin with antiinsulin antibodies in normal animals did not alter plasma glucose, but resulted in atrophy of sciatic myelinated fibers and slowing of nerve conduction velocity (Brussee et al., 2004). In addition, neuroaxonal dystrophy was present in STZ-induced diabetic animals (Cellek et al., 2003; Lee et al., 2001; Schmidt et al., 1981, 1983b, 2003; Schmidt and

K.J. LePard / Autonomic Neuroscience: Basic and Clinical 118 (2005) 12–24

A.

B.

C.

D. 70

70 Vehicle

60 50

(6 animals, n=55)

40

Vehicle (7 animals, n=70) STZ (6 animals, n=55)

60

(7 animals, n=70) STZ

Percent Total

Percent Total

19

Chi test p<0.05

30 20

50

Chi test p<0.05

40 30 20 10

10

0

0 0120

140

160

180

200

220

Fluorescence Intensity

0

20

40

60

80

100 120 140

Fluorescence Intensity

Fig. 3. Photomicrographs of iNOS-ir in LMMP preparations from vehicle- and STZ-treated animals. The percent of iNOS-ir neurons per total Hu-ir neurons in myenteric ganglia was increased in ileum from STZ-treated (B) as compared to vehicle-treated (A) animals. The frequency distributions of iNOS fluorescence intensities in ganglia (C) and tertiary plexus (D) were shifted to stronger quartiles in STZ-treated as compared to vehicle-treated animals. Bar is 10 Am.

Cogswell, 1989) and genetic models of type I diabetes (Schmidt et al., 2004; Sima et al., 2000; Stevens et al., 1994), both characterized by hyperglycemia and hypoinsulinemia, but was not present in genetic models of type II diabetes (Schmidt et al., 2003, 2004; Sima et al., 2000) characterized by hyperglycemia and hyperinsulinemia. Therefore, the role of insulin deficiency in neuropathy was studied in a type I model of diabetes using the STZtreated guinea pig. This model allowed for the role of insulin and C-peptide deficiencies in neuropathy to be more directly evaluated without the confounding variable of persistent and extreme hyperglycemia. Type I diabetes induced by STZ treatment, rather than neurotoxic effects of STZ itself, caused the alterations in enteric nerves observed in this study. Previous studies using STZ-induced animal models of type I diabetes demonstrated that neuropathy was prevented or reversed by nicotinamide or insulin treatment. Treatment of animals with nicotinamide along with STZ preserved pancreatic beta cell histology and function (Hu et al., 1996) and prevented axonopathy in mesenteric nerves (Schmidt et al., 1983b) and elevation of tyrosine hydroxylase activity in prevertebral ganglia (Schmidt and Cogswell, 1989). Treatment of animals with STZ, then later normalization of plasma glucose levels by exogenous insulin or pancreatic islet transplantation, pre-

vented the delayed conduction velocity in sciatic nerve (Stevens et al., 1994) and axonopathy of mesenteric nerves (Schmidt et al., 1981, 1983b). Insulin treatment also reversed established neuropathy and reduction in nerve fiber number (Cellek et al., 2003; Schmidt and Cogswell, 1989). In STZ diabetic rats, expressions of different neuropeptides and neurochemicals were altered in submucous plexus versus myenteric plexus (Belai and Burnstock, 1990; Buchan, 1990) and in proximal colon versus ileum (Belai et al., 1985; Belai and Burnstock, 1987; Lincoln et al., 1984; Yu and Ouyang, 1999) arguing against a general toxic effect of STZ. These studies suggest that pancreatic dysfunction, not STZ itself, was required for neuropathy. Analysis of digital images relied on measurements of length or area. Variations in tissue compliance may have prevented comparable stretch of ileum from STZ- and vehicle-treated animals, thereby distorting quantitative measurements. To compare the degree of stretch of LMMP from STZ- and vehicle-treated animals, the size of individual ganglion, the number of neurons in each ganglion, the packing density of neurons per ganglion area, and the density of nerve fibers in the tertiary plexus were determined. According to Karaosmanoglu et al., when jejunal/ileal tissue from normal guinea pigs was stretched by 32%, the

20

K.J. LePard / Autonomic Neuroscience: Basic and Clinical 118 (2005) 12–24

A.

B.

C. 50 Vehicle (7 animals, n=58) STZ (5 animals, n=44) Chi test p<0.05

Percent Total

40 30 20 10 0 1542

4061

6069

20994

Total Area of DBH-ir Vesicles Fig. 4. Photomicrographs of DBH-ir in LMMP preparations from vehicle-treated (A) and STZ-treated (B) animals. (C) The frequency distribution of total area occupied by DBH-ir varicosities was shifted to smaller quartiles in STZ-treated as compared to vehicle-treated animals. Bar is 10 Am.

number of neurons/cm2 serosal area was decreased by 31% without significantly altering the size of ganglia (Karaosmanoglu et al., 1996). In ileum from STZ-treated animals, there was a slight, but significant (9%), reduction in the size of myenteric ganglia as compared to vehicle-treated animals. The image analysis method used to determine ganglia size in these studies resulted in low standard errors (2–3% of mean). A large pixel array at 40 (0.31 Am/pixel) was used and the image analysis program determined the boundaries, then measured the inclusive area of each ganglion based on fluorescence intensity (see Material and methods). Other studies used lower density pixel arrays and measured size by manually tracing the outline of ganglia with a cursor (Karaosmanoglu et al., 1996; Spa˚nge´us and el Salhy, 1998). This latter method would not likely detect a slight change in ganglion size. Even though slight, the reduction in ganglion size in STZ-treated as compared to vehicle-treated animals corresponded with a slight reduction in total number of myenteric neurons per ganglion (6%) observed in these studies. Using histological stains (Nissl, Giemsa), most studies (Buttow et al., 1997; Zanoni et al., 1997), but not all (Hernandes et al., 2000), reported a reduction in the number of myenteric neurons in intestine from STZ-treated rats. Using neuronal markers (myosin V, PGP 9.5), the number of myenteric neurons was reduced in small intestine from STZ-treated

rats (Zanoni et al., 2003) and non-obese diabetic mice (Spa˚nge´us and el Salhy, 1998). A reduction in neuron number was observed as early as 1 week after STZ injection (Buttow et al., 1997) and persisted for up to 8 months after STZ injection (Zanoni et al., 1997). In colon from STZtreated rats, DNA damage was observed in approximately 5% of myenteric neurons 4–8 weeks after STZ injection (Guo et al., 2004) suggesting that some neurons may undergo apoptotic cell death. In this study, 2 weeks of insulin-mediated euglycemia reversed myenteric DNA cell damage (Guo et al., 2004). Consistent with other models of diabetes, specific subpopulations of myenteric neurons were reduced in ileum of diabetic guinea pigs at 5–6 weeks after STZ treatment supporting the role of insulin deficiency in promoting cell loss. This population of myenteric neurons does not reflect a decrease in nNOS-ir or iNOS-ir neurons, but may include neurons expressing inhibitory peptide neurotransmitters as observed in other studies (He et al., 2001; Spangeus and El Salhy, 2001). Because the total number of Hu-ir neurons per ganglion was decreased, and the size of myenteric ganglion was also decreased, the packing density of myenteric neurons was increased by 19% in ileum from STZ-treated as compared to vehicle-treated animals. The packing density of myenteric neurons (neurons/cm2 ganglionic area) was reported to be very consistent among segments of intestine from rats,

K.J. LePard / Autonomic Neuroscience: Basic and Clinical 118 (2005) 12–24

21

despite large differences in degree of stretch before fixation (Karaosmanoglu et al., 1996). It may be inferred that, since stretch did not alter ganglionic area (Karaosmanoglu et al., 1996), area occupied by the tertiary plexus may be expanded by stretch, resulting in a greater distance between ganglia and primary fiber tracts, and a reduced density of PGP-ir fibers within the tertiary plexus. In ileum from STZ-treated animals, the percent of the total image occupied by the tertiary plexus was not altered, but there was a reduction in the density of PGP-ir fibers within the tertiary plexus. Other studies have reported a reduced volume density of PGP-ir nerve fibers in duodenal mucosa from pre-diabetic and non-obese diabetic mice (model of insulin-dependent diabetes) (Spa˚nge´us and el Salhy, 1998) but not obese diabetic mice (model of noninsulin-dependent diabetes) (Spangeus and El Salhy, 2001). Therefore, a diabetes-induced change in nerve fiber density was not species dependent, but may be a result of insufficient plasma insulin. Therefore, these alterations in ganglion size, packing density of myenteric neurons and density of PGP-ir tertiary fibers observed in LMMPs from STZ- versus vehicle-treated animals were not consistent with those observed in unstretched versus stretched LMMPs from normal animals. LMMPs from vehicle-treated and STZ-treated animals were under the same degree of tension before fixation. Taken together, these differences were not due to variability in stretch of the tissue, but instead, were due to diabetes. A reduction in cell number, ganglion size and nerve fiber density in tertiary plexus suggest a loss of enteric motor neurons projecting to longitudinal muscle in STZ-treated guinea pigs.

gastrointestinal tract (Schmidt et al., 1983a). In these studies, sympathetic innervation of the myenteric plexus was evaluated by DBH immunoreactivity of LMMP. Dopamine h-hydroxylase is the rate-limiting enzyme in the synthesis of norepinephrine, a neurotransmitter of sympathetic, but not parasympathetic or enteric, neurons. In STZ-treated guinea pigs, the mean area occupied by DBH-ir vesicles was not altered, but there was a shift from the third to the second quartile in total area occupied by DBH-ir varicosities. This shift in distribution may indicate very early degeneration of adrenergic nerve fibers by decreased expression of DBH. Though obviously swollen DBH-ir varicosities were not observed in this study as in later time periods (Cuervas-Mons et al., 1990), the fact that the fourth quartile was not altered may reflect the initial stages of varicosity swelling. At later time periods, 8–52 weeks after STZ injection in rats, sympathetic nerve fibers to the GI tract degenerated (Belai et al., 1991; CuervasMons et al., 1990a,b; Kniel et al., 1986; Lincoln et al., 1984; Schmidt et al., 1981). As a result of this degeneration, the amount of norepinephrine, the major neurotransmitter of postganglionic sympathetic nerve fibers innervating enteric ganglia, in the tissue was reduced (Belai et al., 1991; Cuervas-Mons et al., 1990b; Lincoln et al., 1984; Schmidt et al., 1981). In addition, the activity of the norepinephrinespecific enzyme, dopamine-h-hydroxylase, was decreased in non-obese diabetic mice (Spa˚nge´us and el Salhy, 1998). At this early time period (5–6 weeks after STZ injection), evidence of degeneration of extrinsic sympathetic nerve fibers was inconsistent.

4.1. Choline acetyltransferase

The enzyme nitric oxide synthase has at least three isoforms: neuronal (n or type I), endothelial (e or type III) and inducible (i or type II). The neuronal isoform, but not the inducible isoform, is constitutively expressed in myenteric neurons of the guinea pig (Forstermann et al., 1998). Nitric oxide is a neurotransmitter of nonadrenergic and noncholinergic (NANC) relaxations of intestinal smooth muscle (Crist et al., 1991; Wiklund et al., 1993) from the guinea pig. Inhibitory motor neurons that project to the smooth muscle and descending interneurons of the myenteric plexus contain nNOS, the rate-limiting enzyme for production of the gas nitric oxide (Costa et al., 1992). In ileum from STZ-treated as compared to vehicle-treated guinea pigs, the number of myenteric neurons expressing nNOS was not altered. The percent nNOS-ir neurons in myenteric plexus from vehicle-treated animals (25%) was in agreement with published data (22%) (Furness, 2000). In ileum from spontaneously diabetic BB Wistar rats (Takahashi et al., 1997) or STZ-treated rats (Wrzos et al., 1997; Zanoni et al., 2003), the number of myenteric neurons containing nNOS was not altered. Extrinsic, autonomic nerves alter NOS expression by myenteric neurons. In normal animals, extrinsic parasym-

The density of cholinergic innervation of longitudinal muscle was increased in ileum from STZ-treated animals. In these studies, tissues from STZ- and vehicle-treated animals were under the same degree of tension before fixation, so the increased density of cholinergic fibers in the tertiary plexus was not an artifact of stretch. An increased volume density of cholinergic nerve fibers was observed in muscularis propria of duodenum from non-obese diabetic mice (Spangeus et al., 2000), but not obese diabetic mice (Spangeus and El Salhy, 2001). Six weeks after STZ injection, regenerating neuronal processes were observed in jejunal myenteric plexus from rats (Monckton and Pehowich, 1980). Therefore, a greater density of cholinergic motor nerve fibers in the tertiary plexus was more closely linked to type I diabetes and insulin deficiency, rather than type II diabetes. 4.2. Dopamine b-hydroxylase Diabetic neuropathy may involve a reduction in extrinsic parasympathetic and/or sympathetic innervation of the

4.3. Neuronal nitric oxide synthase

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pathetic and sympathetic nerves differentially regulate nNOS expression by myenteric neurons depending on the segment of gut and species of animal. The distal segments of the intestine (jejunum and ileum) are not densely innervated by the vagus nerve (Kirchgessner and Gershon, 1989). In the guinea pig ileum, systemic capsaicin treatment or surgical extrinsic denervation resulted in an increased number of nNOS-ir neurons in the myenteric plexus (Yunker and Galligan, 1998) suggesting regulation of nNOS by sympathetic afferent nerves. In the rat jejunum, truncal vagotomy did not alter the number of nNOS-ir neurons in myenteric ganglion. Rather, destruction of splanchnic efferent, but not afferent, nerves increased in the number of nNOS-ir neurons in myenteric ganglion (Nakao et al., 1998). Therefore, in both rat and guinea pig, sympathetic, not vagal, nerves modulated nNOS expression in distal small intestine with afferent nerves playing a more important role in guinea pigs and efferent nerves in rats. Taken together, at 5–6 weeks after STZ injection, neuropathy of sympathetic nerves innervating myenteric neurons was not extensive enough to cause a significant increase in nNOS cell number. In STZ-treated animals, there was a decrease in average fluorescence intensity of nNOS in myenteric ganglion. This could reflect either a decrease in the number of nNOS-ir cells in ganglia or in the amount of nNOS enzyme in the cytoplasm of individual neurons. In STZ-treated as compared to vehicle-treated guinea pigs, the percent of nNOS-ir myenteric neurons was not reduced. Therefore, the weaker fluorescence intensities of ganglia best reflected a decrease in the amount of enzyme in individual myenteric neurons. In rat models of diabetes, both increases (Adeghate et al., 2003) and decreases (Cellek et al., 2003; Takahashi et al., 1997) in nNOS protein have been reported. The average fluorescence intensity of nNOS-ir in the tertiary plexus was not altered, but a greater proportion of fluorescence intensities were weaker in tertiary plexus from STZ-treated animals. This could reflect either a decrease in enzyme concentration within individual motor nerve fibers or in density of nNOS-ir tertiary fibers. Because of the paucity of nNOS-ir fibers in the tertiary plexus, and their weak fluorescence intensity, their density could not be analyzed. In normal guinea pigs, the density of nNOS-ir fibers innervating longitudinal muscle was sparse (Furness et al., 1994) and only about 2% of myenteric neurons innervating the longitudinal muscle express nNOS (Furness, 2000). In animal models of diabetes (Belai et al., 1991; Cellek et al., 2003; Spangeus and El Salhy, 2001) and in diabetic patients (He et al., 2001) the density of inhibitory motor neurons innervating small intestinal smooth muscle was decreased. The reduction in immunostaining density of nNOS in gastric pylorus paralleled the decreased nNOS protein by Western blot (Cellek et al., 2003). Hence, the data suggest that the density of nNOS-ir tertiary fibers was slightly decreased in LMMP from STZ-treated animals. Because the number of nNOS expressing neurons was not

decreased in STZ-treated animals, the data may reflect early degeneration of nNOS-ir motor nerve fibers to the longitudinal muscle. This was similar to data from STZ diabetic rats where downregulation of nNOS protein in individual enteric fibers occurred by the first 12 weeks of diabetes followed by later loss of nNOS-ir fibers (Cellek et al., 2003). 4.4. Inducible nitric oxide synthase In ileum from STZ-treated as compared to vehicle-treated guinea pigs, the number of myenteric neurons expressing iNOS was increased. Vehicle-treated animals and animals that received no ip injection also expressed iNOS in myenteric neurons. In contrast, the inducible isoform of NOS was expressed in myenteric neurons only after induction of TNBS-colitis in rats (Miampamba and Sharkey, 1999) and ileitis in guinea pigs (Miller et al., 1995). In those studies, iNOS-ir myenteric neurons may not have been observed because tissue cross-sections were used, rather than whole mounts which allow for evaluation of multiple ganglia. Production of nitric oxide by the iNOS isoform has not been confirmed in ileum from STZ-treated guinea pigs. The inducible isoform of NOS can generate large quantities of nitric oxide which can damage cells by the formation of peroxynitrite (Miller et al., 1995) and nitrotyrosine (Miampamba and Sharkey, 1999). 4.5. Summary In ileum from STZ-treated guinea pigs, there were specific alterations in enzymes expressed by myenteric neurons. Similar to other animal models of diabetes, there was a reduction in number of myenteric neurons, ganglion size and density of enteric nerve processes, with an increased density of ChAT-ir motor nerve fibers, to the longitudinal muscle. Early evidence of sympathetic neuropathy was emerging at 5–6 weeks after STZ injection. The percent of iNOS-ir, but not nNOS-ir, per total Hu-ir neurons was increased in myenteric ganglia. The alterations observed in this animal model of diabetes suggest a primary role for insulin deficiency in enteric neuropathy.

Acknowledgements The authors wish to thank Walt Prozialeck, PhD for use of the Nikon microscope and camera. The authors wish to thank Richard L. Porter D.O., Joseph Kovacic D.O., Jonathan Patterson D.O. and Karyn DiNovo for expert technical assistance. RLP was supported by Chicago College of Osteopathic Medicine Summer Research Fellowship provided by the Office of Research and Sponsored Programs at Midwestern University.

K.J. LePard / Autonomic Neuroscience: Basic and Clinical 118 (2005) 12–24

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