Three-Dimensional Imaging of the Enteric Nervous System in Human Pediatric Colon Reveals New Features of Hirschsprung's Disease.

BACKGROUND & AIMS: Hirschsprung's disease is defined by the absence of the enteric nervous system (ENS) from the distal bowel. Primary treatment is "pull-through" surgery to remove bowel that lacks ENS, with reanastomosis of "normal" bowel near the anal verge. Problems after pull-through are common, and some may be due to retained hypoganglionic bowel (ie, low ENS density). Testing this hypothesis has been difficult because counting enteric neurons in tissue sections is unreliable, even for experts. Tissue clearing and 3-dimensional imaging provide better data about ENS structure than sectioning. METHODS: Regions from 11 human colons and 1 ileal specimen resected during Hirschsprung's disease pull-through surgery were cleared, stained with antibodies to visualize the ENS, and imaged by confocal microscopy. Control distal colon from people with no known bowel problems were similarly cleared, stained, and imaged. RESULTS: Quantitative analyses of human colon, ranging from 3 days to 60 years old, suggest age-dependent changes in the myenteric plexus area, ENS ganglion area, percentage of myenteric plexus occupied by ganglia, neurons/mm2, and neuron Feret's diameter. Neuron counting using 3-dimensional images was highly reproducible. High ENS density in neonatal colon allowed reliable neuron counts using 500-µm2 × 500-µm2 regions (36-fold smaller than in adults). Hirschsprung's samples varied 8-fold in proximal margin enteric neuron density and had diverse ENS architecture in resected bowel. CONCLUSIONS: Tissue clearing and 3-dimensional imaging provide more reliable information about ENS structure than tissue sections. ENS structure changes during childhood. Three-dimensional ENS anatomy may provide new insight into human bowel motility disorders, including Hirschsprung's disease.

Robust, 3-Dimensional Visualization of Human Colon Enteric Nervous System Without Tissue Sectioning.

BACKGROUND & AIMS: Small, 2-dimensional sections routinely used for human pathology analysis provide limited information about bowel innervation. We developed a technique to image human enteric nervous system (ENS) and other intramural cells in 3 dimensions. METHODS: Using mouse and human colon tissues, we developed a method that combines tissue clearing, immunohistochemistry, confocal microscopy, and quantitative analysis of full-thickness bowel without sectioning to quantify ENS and other intramural cells in 3 dimensions. RESULTS: We provided 280 adult human colon confocal Z-stacks from persons without known bowel motility disorders. Most of our images were of myenteric ganglia, captured using a 20× objective lens. Full-thickness colon images, viewed with a 10× objective lens, were as large as 4 × 5 mm2. Colon from 2 pediatric patients with Hirschsprung disease was used to show distal colon without enteric ganglia, as well as a transition zone and proximal pull-through resection margin where ENS was present. After testing a panel of antibodies with our method, we identified 16 antibodies that bind to molecules in neurons, glia, interstitial cells of Cajal, and muscularis macrophages. Quantitative analyses demonstrated myenteric plexus in 24.5% ± 2.4% of flattened colon Z-stack area. Myenteric ganglia occupied 34% ± 4% of myenteric plexus. Single myenteric ganglion volume averaged 3,527,678 ± 573,832 mm3 with 38,706 ± 5763 neuron/mm3 and 129,321 ± 25,356 glia/mm3. Images of large areas provided insight into why published values of ENS density vary up to 150-fold-ENS density varies greatly, across millimeters, so analyses of small numbers of thin sections from the same bowel region can produce varying results. Neuron subtype analysis revealed that approximately 56% of myenteric neurons stained with neuronal nitric oxide synthase antibody and approximately 33% of neurons produce and store acetylcholine. Transition zone regions from colon tissues of patients with Hirschsprung disease had ganglia in multiple layers and thick nerve fiber bundles without neurons. Submucosal neuron distribution varied among imaged colon regions. CONCLUSIONS: We developed a 3-dimensional imaging method for colon that provides more information about ENS structure than tissue sectioning. This approach could improve diagnosis for human bowel motility disorders and may be useful for other bowel diseases as well.

Caring for a child with a disability in a Zambian community: A study using photo-elicitation.

BACKGROUND: Over the past 25 years, caregivers of children with disabilities (CWD) in low- and middle-income countries (LMIC) have received limited attention from researchers. Previous research identified the physical, emotional, and social challenges of caregiving in resource-limited areas. Researchers also identified protective factors, such as problem-focused coping and emotion-focused coping. This study seeks to explore experiences of caregivers of CWD through participant-driven photo-elicitation, in a low-income subdistrict of Lusaka, Zambia. METHODS: Researchers considered the experiences of caregivers providing support to their children with disabilities (CWD). Ten parent participants completed this qualitative study using photo-elicitation. Parent participants completed an individual interview to describe their experiences reflected in the pictures they took. The research team used qualitative content analysis to identify the themes. RESULTS: Five general themes emerged from the data. The last two themes, "Also, a human being," and "The community should learn," are unique in research of CWD caregivers in LMIC settings. It indicates ways that these parents sought to encourage more inclusion and acceptance of their CWD in the community and to speak to other parents who might be struggling with providing care for their CWD. CONCLUSIONS: This study expanded disability research to include perspectives from a low-income area in Lusaka, Zambia. In doing so, we acquire a new viewpoint and enhanced understanding of their community experiences. Practice considerations include encouraging disability advocates to continue their work of educating the community about the inherent dignity and worth of people with disabilities and supporting CWD's rights.

Empowering Caregivers of Children With Disabilities in Zambia: A Photovoice Study.

IMPORTANCE: Families with a child or children with a disability (CWD) living in low- and middle-income countries (LMICs) are a marginalized population experiencing multiple contextual factors within a social model of disability that contribute to decreased occupational participation. OBJECTIVE: To understand the advocacy messages that caregivers of CWD, living in an LMIC, wanted their community to learn about CWD. DESIGN: Photovoice methodology was used with 10 parents of a CWD. Photographs, accompanying narratives, and focus group session transcripts were analyzed using Wang and Burris's (1997) original Photovoice technique. SETTING: Urban subdistrict of Lusaka, Zambia. A community venue was used for the focus group, and the community photograph exhibition occurred in the subdistrict. PARTICIPANTS: Ten parents of a CWD living in an urban subdistrict of Zambia who were currently participating in a community-based program, Kusamala+, which addressed stigma in the community and supported parents of children with disabilities through training. RESULTS: Three themes were revealed: (1) "our children with disabilities should be shown love," (2) "our children with disabilities should be well taken care of," and (3) "our children with disabilities need help because they have different needs." CONCLUSIONS AND RELEVANCE: The findings indicate that a social disability model lens and participatory action research can be used to reveal occupational injustices that hinder parents' engagement in caregiving for their CWD in a marginalized community. Use of visual methodologies can be a powerful advocacy tool for marginalized populations. What This Article Adds: Our findings suggest that occupational therapists can better understand, and provide services to, families living in marginalized communities by using a social model of disability as a framework for evaluation and service provision. By highlighting issues of occupational injustice, occupational therapists can advocate for and empower communities of people with disabilities who face stigma and discrimination.