JDDH

Introduction

Gut tissue is composed of several layers, including the enteric nervous system (ENS) and two muscle layers (the inner circular and the outer longitudinal layers). Impairments in the structure or function of the ENS or the SIP syncytium (constituted by Smooth muscle cells - SMC, Interstitial cells of Cajal - ICC, and Plateletderived growth factor receptor α-positive cells - PDGFRα+) affect gut motility, causing nutritional and digestive dysfunctions. Muscle tissue is composed of intestinal SMC, which are spindle-shaped myocytes, where myosin is the molecular motor that allows the acto-myosin complex to slide (through displacement of the myosin head on actin by ATP hydrolysis) and shorten during contractile activity [1].

Pediatric intestinal pseudo-obstructions (from now on referred to as ‘PIPO’) [2] and Hirschsprung disease (from now on referred to as ‘HSCR’) [2] are considered the most severe chronic primary intestinal dysmotility of the pediatric age. Citing Thapar et al. [2], “PIPO is a disorder characterized by the chronic inability of the gastrointestinal tract to propel its contents, mimicking mechanical obstruction in the absence of any lesion occluding the gut, with persistence of symptoms for 2 months from birth or at least 6 months thereafter. It is regarded as the most severe end of a spectrum of gut motility disorders comprising a heterogeneous group of conditions affecting the structure and/or function of components of the intestinal neuromusculature”. In particular, the present paper refers to genetic primary PIPO, an umbrella of rare conditions where chronic intestinal pseudo-obstruction is the primary symptom (not secondary to other pathologies, nor idiopathic) and is caused by an ascertained genetic driver. Defects in the enteric smooth muscle, enteric nervous system, mitochondria, or cells of Cajal can result in a dramatic reduction in peristalsis, which causes recurrent intestinal pseudo-obstruction and leads to several hospitalizations and surgical interventions. Depending on the affected component of the intestinal structure or on genetic information, primary PIPO can be classified as ‘myogenic’, ‘neurogenic’, ‘mitochondrial’ or ‘mesenchymal’ [3]. PIPO diagnostic process can be difficult, especially when no clear genetic driver associated with the clinical condition is identified in patients.

Moreover, the lack of ascertained causative molecular mechanisms underlying PIPO onset is preventing the identification of useful pharmacological therapies. Excluding intestinal transplant (which is now mostly discouraged), no treatment exists for PIPO: the only management strategy consists of the implementation of total parenteral nutrition and decompressing ostomies, exposing patients to the constant risk for infection, kidney failure and nutritional imbalance [3]. Hirschsprung disease (HSCR), also known as congenital aganglionic megacolon, is another rare congenital condition impacting on the gut. It is a neurocristopathy, characterized by the absence of enteric ganglion cells in both the submucosal (Meissner) and myenteric (Auerbach) plexuses in a distal segment of the gastrointestinal tract. This aganglionosis results in tonic contraction of the affected bowel, which is unable to relax and prevents the passage of stool, thus causing megacolon and functional intestinal obstruction. HSCR is a genetically complex disorder, with both monogenic and polygenic influences. The RET proto-oncogene is the principal contributor [4], whereas other possibly implicated genes include SOX10, EDNRB, EDN3, GDNF, NRTN, and L1CAM. HSCR classification, based on the extent of aganglionosis from the anus upward, includes ‘ultrashort segment’ [5], ‘short-segment’ [6], ‘long-segment’ [7], ‘total colonic aganglionosis’ [8], and ‘total intestinal aganglionosis [9]. Affected patients present with severe constipation and abdominal distension, which require surgical removal of the pathological segments of the distal bowel. This remains the only possible treatment currently available for HSCR disease.

In this scenario of extremely rare and orphan motility disorders, the identification of suitable models of pathology and functional assays can support advances in the understanding of the disease and improvements in diagnostic approaches, and foster the identification of new potentially effective therapies. In particular, affected gut tissues, which intrinsically recapitulate the 3D features of the disease, can represent useful models of pathology, especially if coupled to functional readouts, adequate for the disease. In the context of such rare dysmotility pathologies, the functional endpoint for tissue evaluation is the contraction ability.Examples of ex vivo gut muscle tissue contractility quantification exist in literature, regarding murine and human models. Bautista et al. [10] analyzed ileal segments of homozygote knockout Piezo1^ΔSMC mice mounted on a multi-wire myograph, revealing contractile patterns characterized by reduced amplitude, altered duration, and increased irregularity in intervals, compared to controls. In TβRIIΔk-fib transgenic mice, characterized by ligand-dependent upregulation of TGF-β signaling, colonic fibrosis is associated with diminished colonic strip contractility, in organ-bath experiments following carbachol stimulation [11]. Smooth muscle contraction analysis allowed to observe that in human urinary bladder age-dependent Rho kinase activity plays a key role in mediating carbachol-induced contraction [12]. Also, investigation of HSCR gut samples has been carried out [13], demonstrating that intestinal tissues from patients affected with HSCR exhibit significantly reduced acetylcholineinduced contractility compared with other gut malformations. Similar studies have assessed contractility in gut tissue segments from adult, generic – non genetically assessed, nor primary - CIPO [14], where colonic smooth muscle function was evaluated using classical isometric organ-bath assays, after equilibration under resting tension, and pharmacological stimulation with agents such as KCl and carbachol. Tests showed a markedly reduced contractile tissue ability in CIPO tissues compare to cancer controls.

These works highlight how ex vivo studies of gut segments can provide mechanistic insights into disease-related changes in motility, and provided the inspiration for profitably extending the ex vivo tissue contraction evaluation to the functional assessment of primary genetic PIPO, employing a dual-sensor system to provide the full characterization of the contraction activity. To this end, a low-cost custom dynamometric system, inspired by literature [15] and developed ad-hoc for this study, was implemented. Relying on this tool, the present pilot work aims to quantify force and displacement parameters during the contraction of ex vivo human intestinal tissues from pediatric/young adult patients affected with genetic primary PIPO or HSCR, compared to controls. The impact of the proposed pilot study is double. The primary aspect regards providing additional knowledge on this poorly known and often understudied group of diseases by quantifying the functional impairment on tissue contraction. Especially for PIPO, this study aims to contribute to fill the gap between knowledge about organ dysfunction (which is clinically recognized [2] and quantified through antroduodenal manometry (ADM) [16] and the impairment quantified at the cellular level [17]. This might be useful to reinforce the diagnostic path, on one side, particularly in the absence of a clear genetic driver of the disease. On the other side, given the lack of pharmacological therapies for such disorders, the proposed approach may support the future screening, in human pre-clinical samples, of promising new therapeutic approaches.

Methods

Experimental setup

A new setup was custom-built, drawing conceptual and technical inspiration from literature [15]. The dynamometric system included an isometric sensor (7004, Ugo Basile, Gemonio, VA, Italy), which is able to measure the force exerted by the tissue during its contractile activity (operating range: 0-10 g), and an isotonic sensor (7006, Ugo Basile, Gemonio, VA, Italy), which is able to measure tissue displacement (operating range: 15° about the centre). Data capsule Evo (17308, Ugo Basile), which converts analogical to digital data (resolution 16 bit per channel), was coupled to sensors and connected to a recording workstation equipped with Labscribe4 (Ugo Basile). The working principles and a schema of the whole setup are reported in (Fig 1A). A homemade polymethyl methacrylate (PMMA) bath chamber was developed to accommodate tissues (Fig 1B). Two connectors for each cylinder allow chamber filling: medium flux is regulated by two peristaltic pumps controlled by an ad hoc Python routine running on Raspberry PI. An oxygenator and a thermostatic bath (to maintain the water in the bath chamber at 37°C) are used to keep the tissue viable throughout the experimental phases.

Patient recruitment, tissue preparation and stimulation protocol

The cohort of patients affected with intestinal dysmotility (named ‘IntD’) includes paediatric and young adult patients affected with primary genetic PIPO (2 patients, ileum and colon) or HSCR (3 patients: for 2 patients, colon; for 1 patient, ileum and colon). The control cohort (named ‘Ctrl’) includes patients affected with pathologies different from primary genetic PIPO or HSCR, with no episodes of secondary intestinal pseudo-obstruction in their life. It consisted of 2 cases of necrotizing enterocolitis (for both, ileum and colon), 6 cases of inflammatory bowel diseases (for 3 subjects, ileum and colon; for 3 subjects, ileum), 2 cases of intestinal perforation (for 1 patient, ileum; for 1 patient, both ileum and colon), 1 case of peritonitis (ileum), 1 case of appendicitis (ileum), and 1 case of trisomy 19 (ileum). Human tissue samples were collected at the IRCCS Istituto Giannina Gaslini Pediatric Hospital in Genova (Italy). Sampling was performed when a surgical procedure was necessary for patients, without any detriment for donor subjects (approved ethics protocol: N. CET - Liguria: 244/2024 - DB id 13938). Most tissues are collected during elective surgery, zwhen performing ostomies, anastomoses or corrective/reconstructive interventions.

Each resected sample undergoes a standardized procedure. Sample is put in a tube (50 ml) containing 20 ml of RPMI culture medium with 10% FBS, 2 mM L-Glutamine, 2 ml Penicillin-Streptomycin solution (100x) and transferred to the laboratory, where the intestinal ring is placed in oxygenated Krebs solution (in mM: 117 NaCl, 4.6 KCl, 2.5 CaCl₂, 1.2 MgSO₄, 1 NaH₂PO₄, 25 NaHCO₃, 10 HEPES, 11 D-glucose) open vertically and epithelial and adipose layers are removed.  The tissue is cut longitudinally into pieces of selected sample size (length approximately 1 cm, thickness approximately 3 mm) and either processed immediately (fresh tissue) or stored at –80 °C in vials containing a solution of 90% serum and 10% dimethyl sulfoxide (DMSO) (frozen tissue). In the latter case, tissues are defrosted just before performing tests. Upon thawing, tissues are placed in oxygenated Krebs solution. Waiting for being tested, pieces are preserved in a petri dish containing cold, oxygenated Krebs solution, and stored in fridge. Tissues are processed and measured within 3 hours from their arrival in the laboratory.

By means of a hook puncturing the bottom side of the tissue and a spring clip to secure it at the upper end (Fig. 1C), each tissue was accommodated in the bath chamber in a vertical configuration, thus enabling the measurement of sample contraction along the longitudinal muscle. Surgical threads (HS6822 Ethicon; polypropylene; needle length: 26 mm, needle curvature 1/2, needle gauge: 3.0) were used to connect the tissues to the sensors. Polypropylene is a non-absorbable monofilament with minimal stretch, thus offering strength and stability.

Initially, the bath chambers are filled with Krebs solution to mimic the human body environment. and Length adaptation and preload was necessary to adjust isotonic and isometric sensors to each specimen, to keep the thread taut in the organ bath, thus ensuring minimal mechanical stress and maintaining the tissue close to its physiological resting state. Preload ranged from 1 to 1.5 g (depending on tissue weight), while the initial tissue length spanned between 5 and 10 mm (according to sample size). In this tissue relaxed condition, the baseline signal is recorded for 5 minutes (Fig.1D). Carbachol (CCh) 50  (prepared immediately before use) is added, to pharmacologically induce tissue contraction. Dose was selected as the saturation concentration, based on literature [12]. Tissue response is recorded for 20 minutes, inferred from the literature as an adequate time frame for tissue contraction [12] (Figure 1A-1D).

Figure 1: (A) Dynamometric setup schema. Setup components are described, together with the working principles of the sensors. (B) Prototype (realized with Fusion 360 software) of the developed homemade polymethyl methacrylate (PMMA) bath chamber, to accommodate tissues. Two connectors for each cylinder allow filling: medium flux is regulated by two peristaltic pumps controlled by an ad hoc Python routine running on Raspberry PI. An oxygenator and a thermostatic bath (to maintain the water in the bath chamber at 37°C) kept the tissue viable throughout the experimental phases. PMMA cylinders: tightly connected to the base via magnetic blockage and stabilizers; bottom cup: drilled on both sides facilitating fluid and oxygen passage connectors to the oxygenator; upper cup: integrated with magnets to stabilize the inox tissue support; and connectors to the thermostatic bath: to maintain a temperature of 37°C. (C) Anchoring system for anchoring the gut open-ring pieces, comprising a spring clip, bottom hook and surgical thread to maintain the tissue in a vertical configuration. (D) Typical acquired signal, including 5 mins of baseline (relaxation time), preload, starting experiment with carbachol (cch) administration and 20 mins of contraction activity.

Experimental design

To evaluate gut tissue contraction, force, displacement, and speed were retrieved from the isometric and isotonic sensors of the system. The first set of evaluations aimed to assess the homogeneity of the control population. In particular, patients’ sex (female/male) and age (infant-child: 0-8 years; adolescent: 8-17 years), sample preservation mode (frozen/fresh), and intestinal tract (ileum/colon, given the known difference in their stiffness [18, 19]) were considered. Results from this analysis guided the successive comparison between Ctrl and IntD data distributions.

Data acquisition, analysis and statistics

Each sample was characterized by the following metadata: subjects’ clinical and demographic features (age, sex and disease), intestinal segment under analysis, and sample preservation method (fresh or frozen). Isotonic or isometric responses were recorded for each sample as voltage output signals and translated into displacement (D) and force (F) values respectively, through a twopoint calibration process. In brief, the isometric sensor employs two weights (0 and 5 g), which are associated with the respective voltage outputs in the acquisition system; in the isotonic case, the sensor displacements (0 and 5 mm) are leveraged and associated with the voltage outputs. Starting from the known values, the calibration process establishes a linear relationship between the dependent variables y (F and D) and the independent variable x (the voltage output, V_out) following the relation y=a*x+b. Calibration is performed once before a set of acquisitions. Data conditioning and plotting were performed in the MATLAB environment. To minimize noise, data were smoothed via a Gaussian window moving along the signal (width of 700 samples). Signal was shifted to a baseline, where the minimum value was 0. Under these conditions, the following parameters were extracted during sample contraction: force, F=F_max, from the isometric sensor; displacement, calculated in mm and plotted as the percentage of variation of tissue length, L: D%=D*100/L; and contraction speed, S=ΔD/ΔT, obtained by processing data from the isotonic sensor (T = time). The Kolmogorov‒Smirnov test was applied to verify the normality of the datasets, and the statistical significance of nonparametric distributions was calculated via the Mann‒ Whitney test. Nonparametric data are presented as medians and interquartile ranges (IQRs). In the plots, ‘N’ indicates the number of independent patients, and ‘R’ represents the total number of analysed samples.

Results

Recruited patients

Overall, 13 subjects were included in the Ctrl population. The ‘IntD’ cohort included 1 myogenic (ACTG2^R257C) and 1 mitochondrial (LIG3) PIPO case, and 3 HSCR patients (Table 1). Samples were collected when patients underwent ‘ostomy’ surgery in the presence of PIPO, and ‘corrective surgery’ in the presence of HSCR disease. Samples from PIPO patients come from the bowel and colonic tract. Samples from HSCR cases derive from the aganglionic gut segment. Images of tissue staining from the recruited patients provide information about the studied specimen (Supplemental Figs. S1–S15).

Samples composing the Ctrl group were collected in conjunction with necessary surgery, in particular: NEC and

intestinal perforation affected patients underwent anastomosis; IBD and trisomy 19 affected patients underwent standard resection/ ostomy focused on the impaired intestinal tract; immunodeficiency, peritonitis, and appendicitis affected patients underwent ileostomy (Table 1).

#	Type	Age	Sex	Disease	Segment	Surgery	Type
	Cases (IntD population)
1	HSCR	6   months	M	Hirschsprung’s Disease, negative to RET,  short-segment HSCR form aganglionosis extended to rectum-sigmoid colon	Colon	Elective surgery	Ileocolectomy and ileostomy revision
2		1   year	F	Hirschsprung’s Disease,negative to RET  long-segment HSCR form aganglionosis extended to transverse colon	Colon	Elective Surgery	subtotal colectomy and ileosigmoid anastomosis
3		3   years	F	Hirschsprung’s Disease, negative to RET, short-segment HSCR form, aganglionosis extended to rectum-sigmoid colon	Colon	Elective surgery	Ileocecal resection and ileocolic anastomosis
4	CIPO	3   years	F	VSCM ACTG2R257C	Ileum & Colon	Emergency surgery	lleocolectomy and ileostomy re-creation
5		22 years	M	Mitochondrial CIPO LIG3	Ileum & Colon	Elective surgery	en Bloc Ileocecal resection
	Controls (Ctrl population)
6		3 months	M	Necrotizing enterocolitis	Ileum &   Colon	Elective Surgery	Ileo-sigmoid anastomosis   with near-total colectomy
7		3 months	M	Necrotizing enterocolitis	Ileum &   Colon	Elective surgery	en Bloc Ileocecal resection
8		4   months	F	Immunodeficiency	Ileum &   Colon	Elective surgery (ileum) Emergency surgery (colon)	ileostomy
9		7   years	M	Crohn’s Disease	Ileum &   Colon	Elective surgery	resection of the ileocecal block
10		15  years	F	Crohn’s Disease	Ileum	Elective surgery	ileo-sigmoid anastomosis
11		16  years	M	Crohn’s Disease	Ileum	Elective surgery	resection of the ileocecal block
12		17  years	M	Crohn’s Disease	Ileum	Elective surgery	ileo-sigmoid anastomosis
13		15  years	F	Ulcerative Colitis	Ileum &  Colon	Elective surgery	colectomy and ileostomy
14		2   years	F	Trisomy 19	Ileum	Elective surgery	ileal resection
15		2   years	M	Intestinal perforation	Ileum	Emergency surgery	ileo-sigmoid anastomosis
16		3   years	M	Intestinal perforation	Ileum &   Colon	Elective surgery	ileo-sigmoid anastomosis
17		12  years	F	Peritonitis	Ileum	Emergency surgery	ileostomy
18		12  years	F	Appendicitis	Ileum	Elective surgery	ileostomy

Table 1: Recruited subjects; Metadata of considered subjects, including control and intestinal dysmotility populations.

For each patient, the number of samples tested ranged from 2-5, depending on the dimension of the initial available tissue.

Homogeneity of the control population

Ctrl population was tested to evaluate its homogeneity and to assess whether sample-specific parameters impacted the tissue contractile outcome. The comparison between fresh and frozen control samples revealed no statistically significant differences in tissue performance in both isometric (pValue_isom=0.62) and isotonic (pValue_isot=0.071) evaluations (Fig. 2A,B). Analogously, no influence of patient sex (Fig. 2C,D) and age (Fig. 2E,F) on sample contraction emerged (sex: pValue_isom=0.63; pValue_isot=0.22; age: pValue_isom=0.78; pValue_isot=0.058). The intestinal tract of the sample source (either ileum or colon) did not seem to influence contractile behavior when the overall dataset was evaluated (pValue_isom=0.11; pValue_isot=0.067) (Fig. 3A,B). Not enough data from a single patient were available to clearly elucidate if a substantial difference exists between the colon and the ileum derived from the same individual and in the same pathophysiological condition. To provide additional information in this direction, all data of coupled ileum-colon were pooled together, and different colors were associated to identify each subject (Fig. 3C, D) (Figure 2A-F), (Figure 3A-D).

Figure 2: Analysis of the distributions of F and D considering different features of the Ctrl population. Force (F expressed as mN), displacement (calculated as mm and plotted as the percentage of variation in tissue length, L: D_%=D*100/L) and signal speed (expressed as mm/s) preservation mode: (A) Isometric data. N_fresh=6, R_fresh=23, N_frozen=6, R_frozen=15. (B) Isotonic data. N_fresh=6, R_fresh=13, Nfrozen=6, Rfrozen=28. Sex: (C) Isometric data. NFemale=5, RFemale=18, NMale=6, RMale=19. (D) Isotonic data. NFemale=5, RFemale=20, NMale=7, R_Male=22. Age: (E) Isometric data. N_Age<8=7, R_Age<8=21,  N_Age>8=6, R_Age>8=16. (F) Isotonic data. N_Age<8=7, R_Age<8=12, N_Age>8=6, RAge>8=32.

Figure 3: Analysis of the distributions of F and D considering different features of the Ctrl population. Sample origin from the intestinal segment: (A) Isometric data. N__ileum=10, R__ileum=29, N__colon=4, R__colon=8. (B) Isotonic data. N__ileum=9, R__ileum=28, N__colon=5, R__colon=12. Ileum & Colon subjects force (C) Isometric data. N__ileum=4, R__ileum=7, N__colon=4, R_{_colon}=9, different colors indicate different subjects (purple dots: subject 6, green dots: subject 7, orange dots: subject 16, blue dots: subject 8). Ileum & Colon subjects displacement: (D) Isotonic data. N_{_ileum}=4, R_{_ileum}=5, N__colon=4, R_{_colon}=5, different colors indicate different subjects as before.