Idiopathic Microscopic Colitis of Rhesus Macaques: Quantitative Assessment of Colonic Mucosa

Authors


Correspondence to: Amir Ardeshir, California National Primate Research Center, University of California Davis, Davis, CA 95616. Fax: (530) 752–2880. E-mail: aardeshir@ucdavis.edu

ABSTRACT

Idiopathic chronic diarrhea (ICD) is a common cause of morbidity and mortality among juvenile rhesus macaques. While lesions may be absent at colonoscopy, the histopathologic evaluation of the biopsy specimens is consistent with human macroscopic colitis (MC). In this study, we developed an isotropic uniform random sampling method to evaluate macroscopic and microscopic changes and applied it on proximal ascending colon in monkeys. Colonic tissue and peripheral blood specimens were collected from six MC and six control juvenile macaques at necropsy. Uniform random samples were collected from the colon using punch biopsy tools. The volume of epithelium and lamina propria were estimated in thick (25 µm) sections using point probes and normalized to the area of muscularis mucosae. Our data suggests a significant increase of the Vs of the lamina propria (1.9-fold, P = 0.02) and epithelium (1.4-fold, P = 0.05) in subjects with MC. The average colonic surface mucosa area in the MC monkeys increased 1.4-fold over the controls (P = 0.02). The volume of the proximal colon in animals with MC showed a 2.4-fold increase over the non-diarrhea control monkeys (P = 0.0001). Cytokine, chemokine, and growth factor levels in peripheral blood were found to be correlated with the volume estimate of the lamina propria and epithelium. We found that ICD in macaques has features which simulates human MC and can be used as a spontaneous animal model for human MC. Furthermore, this developed sampling method can be used for unbiased preclinical evaluation of therapeutics in this animal model. Anat Rec, 2013. © 2013 Wiley Periodicals, Inc.

INTRODUCTION

Microscopic Colitis in Rhesus Macaques

Diarrhea is a frequent health problem in rhesus macaque colonies (Hird et al., 1984; Elmore et al., 1992; Adler et al., 1993; Sestak et al., 2003; Blackwood et al., 2008). The incidence of diarrhea in NHP colonies varies but may involve up to 15–20% of the population annually. Shigella, Campylobacter, Yersinia, and Salmonella are common causes of diarrhea in these colonies (Brady and Morton, 1998; Blackwood et al., 2008). However, in a subset of these diarrhea cases no pathogenic bacteria and parasites or other common causes are found and defined as idiopathic chronic diarrhea (ICD) (Broadhurst et al., 2012).

At the California National Primate Research Center (CNPRC) ∼25% of the diarrhea cases are diagnosed as ICD (5% annual incidence). The incidence of ICD cases has been reported as high as 15% of the population in other breeding colonies. The histopathologic analysis of the colonic tissue specimens of ICD cases reveal characteristic changes including lymphocytic/plasmacytic colitis with crypt changes (hyperplasia, goblet cell depletion, crypt abscesses), surface epithelium alterations (attenuation, tufting, exocytosis and micro-ulceration) as well as lamina propria changes (inflammatory cell infiltrates, fibrosis, amyloid deposition). Cecum and proximal ascending colon in the large intestine are typically involved in ICD as evidenced by a marked thickening of the mucosa and possible inflammatory reflux into the terminal ileum.

The endoscopic appearance of the colonic lining is normal (or near normal) however colonic biopsy specimens are characterized by abnormal histopathology (Pardi et al., 2002; Olesen et al., 2004; Pardi, 2004; Zippi et al., 2010). These features of the ICD cases as well as its clinical complications (watery, nonbloody diarrhea that lasts for over a month period) are similar to those found in human microscopic colitis (MC), a subtype of human inflammatory bowel disease (Kingham et al., 1982; Bohr et al., 2000; Jaskiewicz et al., 2006).

Quantitative Pathology

Morphometry is the measurement of the structures in three dimensions and is often the goal of structural quantitation. Since much of the structural data in normal and pathologically altered tissues requires the use of tissue sections, stereology must be used (Elias and Hyde, 1980). Stereology is a well-defined set of techniques that provides quantitative information about three dimensional structures obtained from lower dimensions, e.g., from two dimensional sections (Elias and Hyde, 1980; Gundersen et al., 1988; Hyde et al., 1992). Stereology is used to estimate multiple structural parameters such as number of cells, length, surface area, and volume, in tissue and provides objective information about the third dimension (Hyde et al., 2007). The prerequisite for any stereological method is an accurate, unbiased sampling of the organ of interest (Gundersen and Jensen, 1987; Gundersen et al., 1988; West and Gundersen, 1990; Hyde et al., 1992; Gundersen et al., 1999; Cruz-Orive and Gual-Arnau, 2002). This unbiased tissue sampling may require more time but, but can be stored in digital image format and used for estimations of structural features to answer a variety of questions even if stored in digital image format (Hyde et al., 1991; Hyde et al., 1992).

MATERIALS AND METHODS

Animal Selection

Twelve Juvenile rhesus macaques, aged nine months to four years (Table 1), with a history of ICD were randomly selected. Control subjects were selected randomly from a list of nondiarrhea animals sent to necropsy for a variety of medical reasons, primarily trauma. Monkeys with at least one out of their last three stool examinations positive for ova, parasites, Salmonella, Shigella, Campylobacter, or Yersinia were excluded from the study pool. Likewise, subjects with a positive immunofluorescence assay (IFA) for Giardia spp. and Cryptosporidium spp. as well as those with other obvious causes of diarrhea were excluded from the study. All animals were identified as Simian immunodeficiency virus and Simian betaretrovirus free.

Table 1. Demographic information of the monkeys assigned to the study
Animal IDGroupNecropsy ReasonAge at Sampling
CNTRL1ControlTrauma3 yrs 10 mos
CNTRL2ControlArthritis3 yrs 2 mos
CNTRL3ControlTrauma3 yrs 9 mos
CNTRL4ControlTrauma3 yrs 10 mos
CNTRL5ControlArthritis3 yrs 2 mos
CNTRL6ControlArthritis2 yrs 1 mos
MC1ICDChronic Diarrhea4 yrs
MC2ICDChronic Diarrhea3 yrs 2 mos
MC3ICDChronic Diarrhea2 yrs 10 mos
MC4ICDChronic Diarrhea2 yrs 5 mos
MC5ICDChronic Diarrhea3 yrs 2 mos
MC6ICDChronic Diarrhea2 yrs 9 mos

Estimation of Organ Volume and Luminal Surface

The volume estimation of the entire organ enables morphologic changes to be quantified within the whole organ. The Cavalieri estimator is a simple, well known technique in volume estimation. For parenchymal organs such as lung, liver, and kidney, it involves cutting the organ into slabs of equal thickness and determining the cumulative area by point counting the organ surface and multiplying by the average slab thickness (Luciw et al., 2011). It is a very efficient estimator of volume (Hyde et al., 1992). Estimation of the volume of the tissue wall of the gastrointestinal tract does not require sectioning the organ into slabs. Rather, the opened tract can be photographed with a ruler in place and an average tissue thickness measured with calipers. Point counting from a grid composed of countable points with a known area per point overlying the digital image determines the surface area which is multiplied by the average tissue thickness to estimate volume.

Organ Sample Collection

The concept of unbiased sampling requires that each part of an organ of interest has been randomly selected and has an equal chance of being selected for analysis. This requirement is best met by introducing randomness into the sampling process and using efficient systematic uniform random sampling (SURS) (Braendgaard et al., 1990; Larsen, 1998). This technique has been used extensively in stereological analysis and was the basis of our sample selection at the gross and histologic level. Practical application of SURS required the selection of a random number from a random number table. Then starting with the random number, samples were collected at a set sampling interval throughout the tissue (Gundersen and Jensen, 1987; Braendgaard et al., 1990; Wreford, 1995; Luciw et al., 2011). SURS ensures that every point in the tissue has an equal chance of being selected for analysis. To start the sampling, the whole colon was collected at the necropsy and cut into three sections (Fig. 1A). The colonic tubular structure was opened (Fig. 1B,C). Opened colonic tissues were fixed overnight in 10% buffered formalin (Fig. 1D). The ultimate goal was to collect approximately 10 unbiased samples from the proximal colon which could be embedded in paraffin blocks. A Plexiglas® grid template containing evenly spaced 5 mm holes spaced at a known interval was randomly placed over the tissue. Using a random start, tissue underlying the holes, was sampled at a set interval using a 5-mm biopsy punch colonic (Fig. 1E,F). Holes that covered less than 50% of colonic tissue were excluded. Since the number of samples excluded were about 6%, we expect any bias due to exclusion to be minimal. Punch biopsies were cut in half using a random angle from 0 – 180 degrees (Fig. 1G) and were placed in cassettes on their cut surface and embedded in paraffin (Fig. 1H). Paraffin embedded tissues were cut (25 µm) and stained with hematoxylin and eosin (H&E). Digital images, 25 µm deep with a stack of 15 images at 1.67 µm intervals, were acquired with an automated slide scanner (Olympus VS110 whole slide scanner) for storage in a database (Fig. 1I). With a guard region of one image plane on the top and at the bottom the total depth of tissue used for analysis was 21.66 µm (West et al., 1991; Garman et al., 2001; Uranova et al., 2004). Six middle image planes out of total 13 were used for quantitation.

Figure 1.

Sample collection procedure. (A) Colon was cut into three segments (proximal, mid, and distal). (B, C) Each segment was cut and opened. (D) Opened colon was fixed overnight in 10% buffered formalin. (E) A Plexiglas® grid template containing evenly spaced 5-mm holes was randomly placed over the colon and a biopsy punch tool was used to collect the samples through the template. (F) Using stratified uniform sampling with a random start, samples were collected for histological analysis. (G) Fixed punch biopsies were cut in half. (H) Cut punch biopsies were placed in the cassette on their cut surface and embedded in paraffin. (I) Paraffin embedded tissues were cut in thick sections (25 1m) and stained using H&E stain and slides were scanned and digitized using robotic slide scanner.

Stereology

Different probes were applied to microscopic sections to quantify the structural features, and the intersection of the probe and the feature of interest was counted (Hsia et al., 2010b). Point hits were used to estimate volume, and line (Fakir probes) intersections were used for estimation of the surface area. In stereology, the measurements are commonly treated with the calculation of ratios (i.e. quantity of structure per unit volume of reference compartment). It is essential that the reference structure and the structure of interest be measured at the same time and that the reference space not significantly change between individuals (Gundersen et al., 1988; Amann et al., 1992; Hsia et al., 2010a). Stereological methods for the estimates are well established and there are several software programs available to assist in systematic uniform random sampling (URS) of microscopic fields for analysis and application of probes on the selected fields. We used Visiopharm™ MicroImager and NewCast to perform the URS.

The following is a brief overview of approaches for structural estimates:

Estimation of Volume Density

The volume density of the compartment of interest in the organ is the sum of the points hitting the compartment of interest divided by the sum of the points hitting the reference (Table 2):

display math(1)

where n is the number of the fields of view, P(Y) is the numbers of points hitting the compartment of interest (epithelium or lamina propria of the proximal colon), and P(ref) is the numbers of point hitting the colonic mucosa (reference volume).

Table 2. The link between the probe dimension and the dimension of the structure of interest
Dimension of the feature of interestProbe Type
Volume (3D)Point (0D)
Surface (2D)Linear (1D)
Length (1D)Surface (2D)
Number (0D)Volume (3D)

Surface Density

Surface estimation requires linear probes (Table 2). Intersections between line probes and the feature of interest are countable events. Specialized isotropic fakir probes, a module of the ELLIPSE® software package (version 2.0.8.1, ELLIPSE®, Kosice, Slovakia) were used to offset the bias of the tissue orientation at the time of collection (i.e., biopsies were all embedded in the same orientation). The fakir probes were randomly oriented in three-dimensions, thereby accounting for tissue orientation bias (Kubinova, 1998; Kubinova and Janacek, 1998, 2001; Kubinova et al., 2001; Kubinova et al., 2003; Janacek et al., 2011).

Surface density, Sv, is defined as:

display math(2)

where n is the number of the fields of view, Ii is the number of intersections of the feature of interest (muscularis mucosae) with the linear probe, l/p is the length of the line probe per grid point, and P(ref) is the number of points hitting the colonic mucosa (reference volume).

Volume/Surface Estimation

All the counts were performed using the same image field of view (magnification and image field size) for each sample from the proximal colon; to normalize the volume count values, a volume/surface estimation was calculated for the volume of each compartment (lamina propria and epithelium), where the estimated surface used came from muscularis mucosae. Crypt and the surface epithelium were considered as epithelium. Lamina propria was measured as the thin layer of cellular infiltrations which lies beneath the epithelium and the muscularis mucosae.

display math(3)

or

display math(4)

when the same field of view is used to count the reference space for both the volume and surface estimates the value is identical for both and divide to one in the equation. So, Vv(Y,r)/Sv(Y,r) can be simplified as:

display math(5)

The simplified calculation reduces the time of the feature counting due to omitting the P(ref), where n is the number of the fields of view, Ii is the number of intersections of the feature of interest (muscularis mucosae) with the linear probe, l/p is the length of the line per grid point (density in measurable units), and P(Y) is the numbers of points hitting the epithelium and lamina propria.

Tissue Shrinkage

A juvenile nondiarrhea and a MC macaques were selected age matched with the study cohort animals. Fresh colonic tissue samples (proximal section) were collected and the width and length were measured at the necropsy and seven days post fixation. Punch biopsy tool was used to cut the tissues samples and the diameter of the punches was measured before and after embedding in the paraffin. The shrinkage percentage in each step was calculated and used to determine the total shrinkage. The total shrinkage (in percentage) was calculated as the product of two steps shrinkage numbers multiplied by 100. The Volume /surface estimation for each compartment was divided by the total shrinkage to adjust for the shrinkage factor. Vs presented in this manuscript is adjusted for shrinkage (Dorph-Petersen et al., 2001).

Multiplex Analyses of Inflammatory Mediators in Peripheral Blood

Cytokine, chemokine, and growth factor levels were measured using commercially available xMAP® (Luminex Corporation, Austin, TX) technology based microbead immunoassay reagents from Life Technologies (Carlsbad, CA). The assay is formatted to simultaneously detect and quantitate levels of 28 monkey specific analytes. The cytokines used in this assay were IL-1b, IL-1RA, IL-2, IL-4, IL-5, IL-6, IL-10, IL12, IL-15, IL-17, G-CSF, CM-CSF, IFN-γ, and TNF-α. The chemokines were eotaxin, IL-8, MCP-1, MDC, MIF, MIG, MIP-1a, MIP-1b, I-TAC, and RANTES. The growth factors were EGF, FGF-basic, HGF, and VEGF. In these solid phase sandwich immunoassays, microbeads of defined spectral properties coated with analyte specific antibodies were incubated with serum samples to capture the specific analytes. After washing, the beads were then reacted with analyte-specific biotinylated detector antibodies that bound to the appropriate immobilized analytes. After additional washing, the beads were reacted with streptavidin-R-phycoerythrin that bound to the biotinylated detector antibodies associated with the immune complexes on the beads, forming a four-member solid phase sandwich. After a final washing to remove all unbound material, the beads were analyzed in a Luminex instrument. By monitoring the spectral properties of the beads and amount of associated R-phycoerythrin fluorescence for each serum sample and comparing to known standards, the concentration of each analyte was determined (Teigler et al., 2012).

Statistics

Statistical analysis has been performed in R Version 2.15.2 (http://www.r-project.org/). We calculated the CE for ratio estimators (Vs, volume of colonic compartment to the surface of the muscularis mucosa) according to the following formula (Cruzorive, 1980):

display math(6)

Where k = number of fields.

RESULTS

Sampling Method

Our goal in sampling of the colon was to collect isotropic uniform random (IUR) samples. However, the texture of the colonic tissues does not have the rigidity of some the parenchymal organs such as kidney, or even lung. It is difficult to randomly position the tissue even after it is fixed. One way to fix the anisotropy of the punch biopsy samples would be to use the Isector, which is a small rubber sphere mold that contains 5 mm diameter holes to hold small specimens and subsequently allows rolling in random directions on the table to randomly orient the tissue specimens and provide isotropic sections (Mayhew, 1992; Nyengaard and Gundersen, 1992; Lokkegaard et al., 2001; Bruel and Nyengaard, 2005; Eisele et al., 2008; Dougherty et al., 2010). Although, this is a relatively easy solution it may be difficult to differentiate epithelium and muscularis mucosae in isotropic sections. Another method that can be applied to the collected samples to resolve anisotropy is based on randomizing the test probe in the appropriate dimension. In the current study we used this technique on the collected samples to produce isotropic test systems. The surface compartment of interest, muscularis mucosae of the colon was used to normalize the volume estimate values (epithelium and lamina propria). Line probes (Table 2) were used to estimate the surface area. The intersections of the lines and the outer edge of the muscularis mucosae in six middle focal planes of the thick colonic sections were recorded (Fig. 2). As a general rule, the probe density was adjusted to count at least 200 points in a total of six focal planes (Fig. 3) from at least 10 punch biopsy sections (Cruz-Orive and Weibel, 1990). This helped the sampler to collect randomly distributed tissue samples. The duration of each sampling was recorded (at the time of start of the procedure to the time of capping the collection tubes). The process time ranged from an hour to an hour and a half, depending on the size and disease status of the animal. Our data suggests that animals with MC require less time to collect the samples (p<0.05) (Fig. 3) due to fact that almost no fecal matter was seen in the colon thus requiring almost no cleaning. In contrast, control animals (animals with no diarrhea at the time of sample collection) usually have impacted proximal ascending and transverse colon. Also, our data demonstrates that older animals require more time to collect the samples due to the increased size of the colon.

Figure 2.

Fakir probe application. The intersections (arrows) of random line probes (green lines) with the crypts are counted to estimate the surface of the muscularis mucosae and will be used as the reference surface to normalize the volume estimation of epithelium and lamina propria (Vs).

Figure 3.

Sample collection duration. The time spent to measure the macroscopic features (length, width, and height) and take samples from the colon show an increase in animals with MC.

The surface area of the proximal colonic segment was calculated using the recorded length and the average of the width of the two ends (proximal and distal). The data suggested that the average surface area in the MC monkeys increased 1.4-fold over the control animals (P = 0.02) (Fig. 4). We also estimated the volume of the colonic compartment using the recorded length, average width, and the height of the segment at the time sampling. Data indicate that the volume of the proximal colon in animals with MC has a 2.4-fold increase over the control monkeys (nondiarrhea) cases (P = 0.0001) (Fig. 5).

Figure 4.

Surface area estimation of the mucosa macroscopically. Surface area of the proximal colon show increase in MC animals (P = 0.02). Data suggested that the inflamed colons were dilated.

Figure 5.

Volume estimation of the colon. The volume of the proximal colon was estimated with the recorded length, average width and the height of the organ at sampling. MC animals reveal a 2.4-fold increase in their proximal colonic volume (P = 0001).

Figure 6.

The number of point hits on the tissue with the test probes. Different test probes were used to estimate the volume of the lamina propria (LP), epithelium (Epith) and muscularis mucosae (MM). The probe density was adjusted to achieve at least 200 counts from at least 10 fields of views (half of the number of punch biopsies).

Stereological Analysis

Thick (25 µm) proximal ascending colonic sections were optically dissected and the six middle sections were used to count the colonic mucosa features. We used the muscularis mucosae to normalize the volume estimates. The estimated Vv of the features of interest (epithelium and lamina propria) was normalized to the estimated Sv of the muscularis mucosae. The V/S (mm3/mm2) estimate was corrected for shrinkage. The total one dimensional shrinkage (the product of post fixation- and post embedding-shrinkage) was calculated as 76.81% in normal colon and 74.49% in colon with MC. The results suggest an average increase in this ratio for both compartments in MC animals compared to control animals. The average fold increase for Vs of epithelium and lamina propria was estimated 1.4 and 1.9, respectively. Data suggested a marginally significant increase of the Vs of the surface epithelium (P = 0.05) and a statistically significant increase of the Vs of the lamina propria in subjects with MC (P = 0.02) (Fig. 6, 7).

Figure 7.

Volume estimation of the colonic compartment per muscularis mucosae surface. The volume of the colonic compartments (epithelium and lamina propria) was normalized to the estimated surface of the muscularis mucosae. The estimated Vv/Sv (Vs) was also adjusted for the shrinkage. The average fold increase for Vs (mm3/mm2) of epithelium and lamina propria was estimated 1.4 and 1.9, respectively. Data suggested a marginally significant increase of the Vs of the surface epithelium (P = 0.05) and a statistically significant increase of the Vs of the lamina propria in subjects with MC (P = 0.02).

Coefficient of Error and Coefficient of Variation

The coefficient of error (CE) of the ratio estimate, estCE(Vs) of epithelium and lamina propria were calculated using the method developed by Cruz-Orive (Cruzorive, 1980). The relatively small estCE(Vs) of epithelium compartment (0.089) and estCE(Vs) of lamina propria (0.139) suggests that the effort put into the sampling cascade (tissue blocks per individual, percentage of sampling area of each block) provided a precise estimate of the Vs per compartment of interest.

Serologic Assay

Foreground fluorescence intensities were subtracted from background (ΔΔFI) and the values less than the minimum estimated concentration were treated as zeros. No analytes were detected for nine cytokines (GM-CSF, IL-2, IL-5, IL-6, IL-10, IL-15, IL-17), one chemokine (I-TAC) and one growth factor (VEGF). Peripheral blood pro-inflammatory cytokines were detected at higher levels in MC as compared to controls. The average values of serum INF-γ, TNF-α, IL-8, and IL-12 were elevated in monkeys with MC. The average INF-γ value increased two-fold (P = 0.009), TNF-α 1.4-fold (P = 0.004), IL-8 2.9-fold (P = 0.021) and IL-12 2.4-fold (P = 0.012) demonstrating an inflammatory response in the peripheral blood (Fig. 11). Chemokine were detected at higher levels for MIG, with average increase of 1.5 (P = 0.030) and lower MIF (P = 0.048) for MC subjects as compared with controls. No significant differences were found in several cytokines (G-CSF, IL-1b, IL-1RA, IL-4), chemokines (eotaxin, MCP-1, MDC, MIP-1a, MIP-1b, RANTES) as well as growth factors (Eotaxin, MCP-1, MDC, MIP-1a, MIP-1b, RANTES) between the two groups. The r square values were calculated using the square of the sample correlation coefficient and were analyzed to describe the relative amount of variance of the Vs differences for colonic compartments (epithelium and lamina propria) as explained or accounted for by the serum analytes. Circulatory MIG (P = 0.01) and IL-1β (P = 0.028) were correlated with the volume estimates of the proximal colon (Table 3). A positive correlation between the lamina propria volume estimates (Vs) and values for circulatory INF-γ (P = 0.042), IL-1β (P = 0.020), TNF-α (P = 0.021), and MIG (P = 0.004) were found (Table 3).

Figure 8.

Serum cytokine profiles show elevations in inflammatory markers in circulation. Peripheral blood were taken at the time of sampling and the cytokine responses were evaluated using a xMAP® technology based microbead immunoassay. Pro-inflammatory cytokines such as (INF-γ, TNF-α, IL-8, and IL-12) were increased in MC cases as compared with controls.

Figure 9.

Serum chemokines profile. Chemokine response in the peripheral blood (taken at the time of sampling) were evaluated using a xMAP® technology based microbead immunoassay. MC cases show an increase in macrophage migration inhibitory factor (MIF) (P = 0.048) and monokine induced by IFN-γ (MIG) (P = 0.03) as compared with controls.

Figure 10.

Serum growth factors profile. No growth factor analytes were statistically different between the two groups in their peripheral circulation.

Figure 11.

Circulatory cytokine and chemokine values correlate with epithelium volume estimates (Vs). Circulatory MIG (P = 0.01) and IL-1β (P = 0.028) were correlated with the volume estimates of the proximal colon (Table 3). Regression line is depicted using solid line and 95% CI of the best fit line is demonstrated with dotted line.

Figure 12.

Circulatory cytokine and chemokine values correlate with lamina propria volume estimates (Vs). Correlations between the lamina propria volume estimates (Vs) and traced values for circulatory INF-γ (P = 0.042), IL-1β (P = 0.020), TNF-α (P = 0.021), and MIG (P = 0.004) were found (Table 3). Regression line is depicted using solid line.

Table 3. The r square values (square of the sample correlation coefficient) were calculated to describe the relative amount of variance of the Vs differences for colonic compartments explained for by the serum analytes
 INF-γIL-1βIL-1RAIL-8IL-12MIFMIGTNF-α
  1. a

    P-value < 0.05.

Epithelium (Vs)Pearson r0.4950.5630.2160.3480.279−0.1140.6590.398
 95% confidence interval(−0.111 − 0.832)(−0.0156 − 0.859)(−0.409 − 0.703)(−0.283 − 0.768)(−0.351 − 0.735)(−0.646 − 0.492)(0.136 − 0.894)(−0.228 − 0.791)
 R square0.2450.3170.04670.1210.07790.01310.4340.159
 p−value0.0511a0.02820.24990.1340.18990.3618a0.00990.0998
          
Lamina Propria (Vs)Pearson r0.5190.597−0.02140.4960.232−0.2430.7160.594
 95% confidence interval(−0.0790 − 0.842)(0.0345 − 0.872)(−0.588 − 0.559)(−0.110 − 0.833)(−0.394 − 0.711)(−0.717 − 0.384)(0.241 − 0.914)(0.0310 − 0.871)
 r square0.2690.3560.0004590.2460.05390.05930.5120.353
 P−valuea0.0421a0.02030.47370.05070.23390.2229a0.0044a0.0208

DISCUSSION

MC is a chronic nonbloody diarrhea which accounts for about 10% of chronic diarrhea in humans (Fernandez-Banares et al., 1999; Fine and Seidel, 2000; Shah et al., 2001; Pardi et al., 2002; Pardi et al., 2007). MC is a less commonly reported form of human inflammatory bowel disease (IBD). The two major forms of human IBD are lymphocytic colitis and collagenous colitis (Fernandez-Banares et al., 1999; Fine et al., 2000; Shah et al., 2001; Olesen et al., 2004; Pardi, 2004; Limsui et al., 2009). It has been suggested that the prevalence of MC is underestimated and the true prevalence of this disease is increasing in humans (Olesen et al., 2004; Limsui et al., 2009; Zippi et al., 2010; Yen and Pardi, 2011). The cause(s) is uncertain (Pardi et al., 2007) but it has been suggested that infectious agents or some drugs may trigger the problem (Pardi et al., 2002; Olesen et al., 2004; Beaugerie and Pardi, 2005; Fernandez-Banares et al., 2007).

In rhesus macaques, idiopathic chronic diarrhea (ICD) is a common cause of morbidity and mortality among juvenile animals (Hird et al., 1984; Adler et al., 1993; Sestak et al., 2003; Fox et al., 2007; Blackwood et al., 2008; McKenna et al., 2008). This condition is characterized histologically as colitis with distinct plasmacytic and lymphocytic infiltrates. The clinical diagnosis consists of multiple negative cultures for common pathogens, as well as no pathogenic parasite. Broad spectrum antibiotic therapy as well as anti-parasitic therapy is usually part of the workup for these cases. While no lesions may be seen at the time of total colonoscopy, the histopathologic evaluation demonstrates crypt changes (hyperplasia, goblet cell depletion, crypt abscesses), surface epithelial changes (attenuation, tufting, exocytosis and micro-ulceration) and expansion of the lamina propria (inflammatory cell infiltrates, fibrosis, and occasional amyloid deposition) (Hird et al., 1984; Adler et al., 1993; Blackwood et al., 2008; Wilk et al., 2008). The disease primarily involves the cecum and proximal ascending colon with a marked thickening of the mucosa and may involve inflammatory reflux into the terminal ileum. Colonic lymph nodes are usually enlarged. In more severe cases, the inflammation can extend from the proximal ascending colon to more distal regions or even the rectum.

Clinically these monkeys show watery, nonbloody diarrhea that leads to weight loss, dehydration, and no response to common therapies and will ultimately need to be euthanized for ethical considerations. In breeding colonies of the rhesus macaques this condition is a common cause for euthanasia among juvenile macaques and accounts for about half of the medical culls which are not related to research. Although several viral and bacterial agents have been associated with this syndrome, no published study was found to support the causal relationship between these agents and the pathophysiology of MC in macaques (Duhamel et al., 1997; Fox et al., 2001; Fox et al., 2007; Farkas et al., 2012). It has been suggested that animals reared in a nursery and male monkeys are more likely than breast-fed and female monkeys to develop this condition (Elmore et al., 1992).

In the current study, we applied stereological techniques to collect samples and to quantitatively and unbiasedly evaluate the histopathologic changes in the colon of MC animals compared with control macaques. In the process of sampling we used a plastic template to collect the samples in a stratified random manner. Although stratified random sampling method has been used extensively in parenchymal organs (lung lobes, liver lobes, heart, brain, kidney), colon is not amenable to standard sampling techniques. The hollow, nonrigid structure of the colon makes the process of sampling challenging because sampling is only feasible in a transmural orientation. Thus, we used punch biopsies from the mucosa through the smooth muscle wall creating a biased sample orientation. Our initial goal was to obtain vertical uniform random (VUR) samples with the circular transmural cut samples that were cut in half at a random angle prior to embedding. Even though we pinned the colon to a cork board prior to fixation, there was curling and distortion of the mucosal surface from the true vertical direction in the sample once the tissue was dehydrated, embedded, sectioned and stained on the slide. As a result, we employed randomized linear probes (“fakir probes”) in the reference space to overcome this orientation bias for determination of the surface area of the muscularis mucosa.

Another possible method to unbiasedly sample colon would be to fix the colon in its natural tubular state filled with agar, and using a random start to cut and perform the sampling. This potentially would save time and resources. In some tissue sections with a distinct vertical axis thick vertical sections probed with cyloids or sine-weighted probes may be the easiest method to address the orientation (Gundersen et al., 1988; West, 2013). Unfortunately, due to specific characteristics of the colonic biopsy tissues these methods were not applicable here. Small colonic punch biopsies curl up after fixation and make it almost impossible to orient the tissues correctly. Alternatively, due to the small size of the tissue blocks, isector molds (Nyengaard and Gundersen, 1992) could be a practical method in this study. Isectors are small rubber molds with spherical cavities that can be used for small specimens embedding. Rolling the embedded tissue in a sphere randomly on the bench top provides the isotropy.

Using a slide scanner to scan a 15 stack of images was tremendously helpful to decrease the amount of time. The relatively large number of stacks of high resolution images provided a great amount of flexibility in choosing the guard region but on the other hand required great amount of disc storage. The high resolution quality may not be critical when estimating the larger tissue compartments (such as surface epithelium or lamina propria) however may act as an imperative step in the process, when the cell types or smaller compartments is of an interest.

To estimate the macroscopic volume of the proximal colon the length and width (proximal and distal) were measured. The average width, length and the average thickness (measured at multiple locations using calipers) were used to estimate the volume. Alternatively, the volume displacement or buoyant weight of the organ and assuming a specific gravity of one could be used. But due to the attachment of the stool particles to the surface we preferred using the direct measurement technique.

Our data demonstrates that the macroscopic volume of the colon in MC cases was increased 2.5-fold over controls (P = 0.0001) accounted for by an increase in the average Vs of lamina propria (P = 0.02) and increase in the epithelium Vs compartments (crypt and the surface epithelium) (P = 0.05) (Fig. 7). The increase in volume in the lamina propria is likely due to a significant inflammatory reaction possibly associated with some fibrosis. The increase in the epithelial compartment is likely secondary to a hyperplastic epithelial response associated with the inflammatory reaction (i.e., crypt hyperplasia). Goblet cell hyperplasia could also be a contributing factor as these cells are larger than most enterocytes. These results were supported by the observed microscopic changes and compartment estimations. The volume of the colonic compartments (epithelium and lamina propria) was normalized to the estimated surface of the muscularis mucosae. The estimated Vv/Sv (Vs) was also adjusted for the tissue shrinkage throughout the process of fixation and tissue embedding in paraffin. Further studies are necessary to characterize the subcomponents of the lamina propria and epithelium and to investigate cellular changes that account for the increase volume of these microenvironments.

The circulatory cytokine and chemokine profile was correlated with the stereological estimates (Vs) of the epithelium and lamina propria (Figs. 9, Table 3). Although this correlation may be used in therapeutic response evaluation and the progress of the inflammatory changes, more subjects are needed to assess this association.

Overall, features of ICD mimic MC (specifically lymphocytic colitis) in humans. Clinical symptoms (watery, nonbloody diarrhea for more than a month), colonic endoscopy appearance (normal colonic mucosa or with minimal changes), and histopathologic analysis of the colonic samples (lymphocystes and plasma cells infiltrates in lamina propria) with damaged surface epithelium (damage to surface epithelium, attenuation, tufting, exocytosis and micro-ulceration of epithelial cells and sometimes detachment) are seen in macaques with ICD. It also has been suggested that intraepithelial lymphocytes in the colonic mucosa are increased in lymphocytic form of microscopic in human patients (Kingham et al., 1982; Fernandez-Banares et al., 1999; Fraser et al., 2002; Pardi et al., 2002; Warren et al., 2002; Abdo et al., 2003; Olesen et al., 2004; Pardi, 2004; Brown and Lambie, 2008; Tangri and Chande, 2009; Zippi et al., 2010; Fernandez-Banares et al., 2011). This needs to be investigated to better characterize the inflammation.

ACKNOWLEDGEMENT

Authors would like to thank CNPRC Pathology Unit and Clinical Laboratories.

Ancillary