A novel animal model for external anal sphincter insufficiency

Abstract PURPOSE: Reliable animal models are essential to evaluate future therapeutic options like cell-based therapies for external anal sphincter insufficiency. The goal of our study was to describe the most reliable model for external sphincter muscle insufficiency by comparing three different methods to create sphincter muscle damage. METHODS: In an experimental animal study, female Lewis rats (200-250 g) were randomly assigned to three treatment groups (n = 5, each group). The external sphincter muscle was weakened in the left dorsal quadrant by microsurgical excision, cryosurgery, or electrocoagulation by diathermy. Functional evaluation included in vivo measurements of resting pressure, spontaneous muscle contraction, and contraction in response to electrical stimulation of the afferent nerve at baseline and at 2, 4, and 6 weeks after sphincter injury. Masson's trichrome staining and immunofluorescence for skeletal muscle markers was performed for morphological analysis. RESULTS: Peak contraction after electrical stimulation was significantly decreased after sphincter injury in all groups. Contraction forces recovered partially after cryosurgery and electrocoagulation but not after microsurgical excision. Morphological analysis revealed an incomplete destruction of the external sphincter muscle in the cryosurgery and electrocoagulation groups compared to the microsurgery group. CONCLUSIONS: For the first time, three different models of external sphincter muscle insufficiency were directly compared. The animal model using microsurgical sphincter destruction offers the highest level of consistency regarding tissue damage and sphincter insufficiency, and therefore represents the most reliable model to evaluate future therapeutic options. In addition, this study represents a novel model to specifically test the external sphincter muscle function.

Standardised animal models of host microbial mutualism.

An appreciation of the importance of interactions between microbes and multicellular organisms is currently driving research in biology and biomedicine. Many human diseases involve interactions between the host and the microbiota, so investigating the mechanisms involved is important for human health. Although microbial ecology measurements capture considerable diversity of the communities between individuals, this diversity is highly problematic for reproducible experimental animal models that seek to establish the mechanistic basis for interactions within the overall host-microbial superorganism. Conflicting experimental results may be explained away through unknown differences in the microbiota composition between vivaria or between the microenvironment of different isolated cages. In this position paper, we propose standardised criteria for stabilised and defined experimental animal microbiotas to generate reproducible models of human disease that are suitable for systematic experimentation and are reproducible across different institutions.

Orthodontic therapy and gingival recession: a systematic review

To perform a systematic review on the effect of changes in incisor inclination owing to orthodontic treatment and the occurrence of gingival recession. PubMed, EMBASE Excerpta Medica and CENTRAL of the Cochrane Library were searched and a hand search was performed. From 1925 articles identified, 17 articles were finally included: six experimental animal studies and 11 retrospective clinical studies in humans. More proclined teeth compared with less proclined teeth or untreated teeth had in most studies a higher occurrence or severity of gingival recession. Contradictory results were found regarding a possible statistically significant correlation between the extent of gingival recession and the amount of incisor proclination during treatment, width of attached gingiva, hygiene, periodontal condition or thickness of the symphysis. There are no high quality animal or clinical studies on this topic. Movement of the incisors out of the osseous envelope of the alveolar process may be associated with a higher tendency for developing gingival recessions. The amount of recession found in studies with statistically significant differences between proclined and non-proclined incisors is small and the clinical consequence questionable. Because of the low level of evidence of the included studies, the results should be considered with caution. Further randomized clinical studies including clinical examination of hygiene and gingival condition before, during and after treatment are needed to clarify the effect of orthodontic changes in incisor inclination and the occurrence of gingival recession.

Rapidly excised and cryofixed rat tissue

All preparation efforts of biological samples in electron microscopy are focused to preserve structures as close as possible to the native state. To achieve this goal with tissues, it is of advantage to have a very short time between excision and fixation. The most common approach is chemical fixation: cross-linking of the tissue samples with aldehydes followed by postfixation with osmium tetroxide. Here, the fastest approach for tissue samples is perfusion. However, the diffusion of the fixation solution from blood vessels into the depth of the tissue is still slow and does not allow an overall instant fixation of a single cell. As a result, osmotic effects become evident (swelling or shrinkage of cell organelles). Another possibility is to take a tissue sample from the experimental animal. Excision of tissue can last quite some time, which results in even more pronounced autolytic induced osmotic effects. Furthermore, the animal does not survive the procedure in most cases. Alternatively, microbiopsies are an elegant technique to rapidly excise small quantities of tissue. Some tissues, such as liver and muscle, may be obtained using a non-lethal approach. To avoid the artifacts introduced by chemical fixation, high-pressure freezing of microbiopsies (brain, liver, kidney, and muscle) is a powerful alternative to chemical fixation. Here, we describe the microbiopsy method, and high-pressure freezing/freeze-substitution (HPF/FS) as a follow-up procedure. Cryosectioning of high-pressure frozen samples is optimally preserving the ultrastructure; however, it is not considered to be a routine approach yet.

Strategies to prevent neuronal damage in paediatric bacterial meningitis

PURPOSE OF REVIEW: The mortality of bacterial meningitis can reach 30%, and up to 50% of survivors suffer from persisting neurological deficits as a consequence of the disease. The incidence of neurological sequelae of bacterial meningitis has not improved over the last decade. Adjunctive therapeutic options are limited, and ongoing research into the pathophysiology of brain damage in bacterial meningitis aims at providing the scientific basis for future development of more efficient adjunctive options. RECENT FINDINGS: In a population with good access to health care, dexamethasone given before or at the time of initiation of antibiotic therapy acts beneficially in paediatric pneumococcal meningitis, but not in meningococcal meningitis. In experimental animal models, brain-derived neurotrophic factor protected against brain injury and improved hearing while melatonin, which has antioxidant properties among other effects, reduced neuronal death. Transgene technology can be used to provide new insights into the pathophysiology of the disease and to identify potential therapeutic targets. SUMMARY: Although dexamethasone improves outcome of bacterial meningitis under defined circumstances, the morbidity of bacterial meningitis still remains unacceptably high. Experimental models may help to identify new therapeutic strategies to further improve the neurological outcome in young children suffering from bacterial meningitis.

ECG-triggered muscular counterpulsation for treatment of low cardiac output

BACKGROUND: Skeletal muscular counterpulsation (MCP) has been used as a new noninvasive technique for treatment of low cardiac output. The MCP method is based on ECG-triggered skeletal muscle stimulation. The purpose of the present study was to evaluate acute hemodynamic changes induced by MCP in the experimental animal. METHODS: Eight anaesthetized pigs (43+/-4 kg) were studied at rest and after IV â-blockade (10 mg propranolol) before and after MCP. Muscular counterpulsation was performed on both thighs using trains (75 ms duration) of multiple biphasic electrical impulses with a width of 1 ms and a frequency of 200 Hz at low (10 V) and high (30 V) amplitude. ECG-triggering was used to synchronize stimulation to a given time point. LV pressure-volume relations were determined using the conductance catheter. After baseline measurements, MCP was carried out for 10 minutes at low and high stimulation amplitude. The optimal time point for MCP was determined from LV pressure-volume loops using different stimulation time points during systole and diastole. Best results were observed during end-systole and, therefore, this time point was used for stimulation. RESULTS: Under control conditions, MCP was associated with a significant decrease in pulmonary vascular resistance (-18%), a decrease in systemic vascular resistance (-11%) and stroke work index (-4%), whereas cardiac index (+2%) and ejection fraction (+6%) increased slightly. Pressure-volume loops showed a leftward shift with a decrease in end-systolic volume. After â-blockade, cardiac function decreased (HR, MAP, EF, dP/dt max), but it improved with skeletal muscle stimulation (HR +10% and CI +17%, EF +5%). There was a significant decrease in pulmonary (-19%) and systemic vascular resistance (-29%). CONCLUSIONS: In the animal model, ECG-triggered skeletal muscular counterpulsation is associated with a significant improvement in cardiac function at baseline and after IV â-blockade. Thus, MCP represents a new, non-invasive technique which improves cardiac function by diastolic compression of the peripheral arteries and veins, with a decrease in systemic vascular resistance and increase in cardiac output.

Abdominal magnetic resonance imaging in small rodents using a clinical 1.5 T MR scanner

Because of superior soft-tissue contrast compared to other imaging techniques, non-invasive abdominal magnetic resonance imaging (MRI) is ideal for monitoring organ regeneration, tissue repair, cancer stage, and treatment effects in a wide variety of experimental animal models. Currently, sophisticated MR protocols, including technically demanding procedures for motion artefact compensation, achieve an MRI resolution limit of < 100 microm under ideal conditions. However, such a high spatial resolution is not required for most experimental rodent studies. This article describes both a detailed imaging protocol for MR data acquisition in a ubiquitously and commercially available 1.5 T MR unit and 3-dimensional volumetry of organs, tissue components, or tumors. Future developments in MR technology will allow in vivo investigation of physiological and pathological processes at the cellular and even the molecular levels. Experimental MRI is crucial for non-invasive monitoring of a broad range of biological processes and will further our general understanding of physiology and disease.

The influence of dosing schedules and cerebrospinal fluid bactericidal activity on the therapy of bacterial meningitis

Bacterial meningitis represents an infection in an area of impaired host defence. Optimal therapy of meningitis requires attaining bactericidal activity within cerebrospinal fluid (CSF). Studies in experimental animal models of meningitis suggest that maximal rates of bacterial killing in vivo and optimal cure rates are achieved when CSF antibiotic concentrations exceed the MBC of the test strain by greater than or equal to ten-fold. The results of clinical trials support this conclusion. In addition, a variable post-antibiotic effect occurs in-vivo after short periods of exposure to antimicrobial activity, thus maintaining therapeutic efficacy with intermittent dosage regimens. These basic principles of therapy are outlined in this review and serve as a basis for rational treatment regimens. For most antibiotics, the optimal dose, dosage interval, and duration of therapy for bacterial meningitis remain to be established.

Microscopic wire guide-based orotracheal mouse intubation: description, evaluation and comparison with transillumination

Airway access is needed for a number of experimental animal models, and the majority of animal research is based on mouse models. Anatomical conditions in mice are small, and the narrow glottic opening allows intubation only with a subtle technique. We therefore developed a microscopic endotracheal intubation method with a wire guide technique in mice anaesthetized with halothane in oxygen. The mouse is hung perpendicularly with its incisors on a thread fixed on a vertical plate. The tongue is placed with a pair of forceps between the left hand's thumb and forefinger and slightly pulled, while the neck and thorax are positioned using the third and fourth fingers. By doing so, the neck can be slightly stretched, which allows optimal visualization of the larynx and the vocal cords. To ensure a safe intubation, a fine wire guide is placed under vision between the vocal cords and advanced about 5 mm into the trachea. An intravenous 22G x 1 in. plastic or Teflon catheter is guided over this wire. In a series of 41 mice, between 21 and 38 g, the success rate for the first intubation attempt was >95%. Certainty of the judgement procedure was 100% and success rate was higher using the described method when compared with a transillumination method in a further series. The technique is safe, less invasive than tracheostomy and suitable for controlled ventilation and pulmonary substance application.

T cell-mediated immunoregulation in the gastrointestinal tract

In the intestinal tract, only a single layer of epithelial cells separates innate and adaptive immune effector cells from a vast amount of antigens. Here, the immune system faces a considerable challenge in tolerating commensal flora and dietary antigens while preventing the dissemination of potential pathogens. Failure to tightly control immune reactions may result in detrimental inflammation. In this respect, 'conventional' regulatory CD4(+) T cells, including naturally occurring and adaptive CD4(+) CD25(+) Foxp3(+) T cells, Th3 and Tr1 cells, have recently been the focus of considerable attention. However, regulatory mechanisms in the intestinal mucosa are highly complex, including adaptations of nonhaematopoietic cells and innate immune cells as well as the presence of unconventional T cells with regulatory properties such as resident TCRgammadelta or TCRalphabeta CD8(+) intraepithelial lymphocytes. This review aims to summarize the currently available knowledge on conventional and unconventional regulatory T cell subsets (Tregs), with special emphasis on clinical data and the potential role or malfunctioning of Tregs in four major human gastrointestinal diseases, i.e. inflammatory bowel diseases, coeliac disease, food allergy and colorectal cancer. We conclude that the clinical data confirms some but not all of the findings derived from experimental animal models.

Histomorphometric and Radiographical Changes After Lumbar Implantation of the PEEK Nonfusion Interspinous Device in the BB.4S Rat Model

Study Design. An experimental animal study. Objective. To investigate histomorphometric and radiographical changes in the BB.4S rat model after PEEK (polyetheretherketone) nonfusion interspinous device implantation. Summary of Background Data. Clinical effectiveness of the PEEK nonfusion spine implant Wallis (Abbott, Bordeaux, France; now Zimmer, Warsaw, IN) is well documented. However, there is a lack of evidence on the long-term effects of this implant on bone, in particular its influence on structural changes of bone elements of the lumbar spine. Methods. Twenty-four male BB.4S rats aged 11 weeks underwent surgery for implantation of a PEEK nonfusion interspinous device or for a sham procedure in 3 groups of 8 animals each: 1) implantation at level L4–L5; 2) implantation at level L5–L6; and 3) sham surgery. Eleven weeks postoperatively osteolyses at the implant-bone interface were measured via radiograph, bone mineral density of vertebral bodies was analyzed using osteodensitometry, and bone mineral content as well as resorption of the spinous processes were examined by histomorphometry. Results. Resorption of the spinous processes at the site of the interspinous implant was found in all treated segments. There was no significant difference in either bone density of vertebral bodies or histomorphometric structure of the spinous processes between adjacent vertebral bodies, between treated and untreated segments and between groups. Conclusion. These findings indicate that resorption of spinous processes because of a result of implant loosening, inhibit the targeted load redistribution through the PEEK nonfusion interspinous device in the lumbar spinal segment of the rat. This leads to reduced long-term stability of the implant in the animal model. These results suggest that PEEK nonfusion interspinous devices like the Wallis implants may have time-limited effects and should only be used for specified indications.

Pulsed versus continuous wave CO2 laser excisions of 100 oral fibrous hyperplasias: a randomized controlled clinical and histopathological study

BACKGROUND In experimental animal studies, pulsing the CO2 laser beam has been shown to reduce the thermal damage zone of excised oral mucosal tissue. However, there is still controversy over whether this is borne out under clinical conditions. OBJECTIVE To compare the outcome following excisional biopsies of fibrous hyperplasias using a pulsed (cf) versus a continuous wave (cw) CO2 laser mode regarding the thermal damage zone, duration of surgeries, intra- and postoperative complications, postoperative pain sensation, scarring and/or relapse during the initial 6 months. MATERIALS AND METHODS One hundred Swiss-resident patients with a fibrous hyperplasia in their buccal mucosa were randomly assigned to the cw mode (5 W) or the cf mode (140 Hz, 400 microseconds, 33 mJ, 4.62 W) group. All excisions were performed by one single oral surgeon. Postoperative pain (2 weeks) was recorded by visual analogue scale (VAS; ranging from 0 to 100). Intake of analgesics and postoperative complications were recorded in a standardized study form. The maximum width of the collateral thermal damage zone was measured (µm) in excision specimens by one pathologist. Intraoral photographs at 6-month follow-up examinations were evaluated regarding scarring (yes/no). RESULTS Median duration of the excision was 65 seconds in the cw and 81 seconds in the cf group (P = 0.13). Intraoperative bleeding occurred in 16.3% of the patients in the cw and 17.7% of the cf group. The median value of the thermal damage zone was 161(±228) μm in the cw and 152(± 105) μm in the cf group (P = 0.68). The reported postoperative complications included swelling in 19% and minor bleeding in 6% without significant differences between the two laser modes. When comparing each day separately or the combined mean VAS scores of both groups between Days 1-3, 1-7, and 1-15, there were no significant differences. However, more patients of the cw group (25%) took analgesics than patients of the cf group (9.8%) resulting in a borderline significance (P = 0.04). Scarring at the excision site was found in 50.6% of 77 patients after 6 months, and more scars were identified in cases treated with the cf mode (P = 0.03). CONCLUSIONS Excision of fibrous hyperplasias performed with a CO2 laser demonstrated a good clinical outcome and long-term predictability with a low risk of recurrence regardless of the laser mode (cf or cw) used. Scarring after 6 months was only seen in 50.6% of the cases and was slightly more frequent in the cf mode group. Based on the findings of the present study, a safety border of 1 mm appears sufficient for both laser modes especially when performing a biopsy of a suspicious soft tissue lesion to ensure a proper histopathological examination.

Development and characterization of chemical cochleostomy in the Guinea pig

OBJECTIVES Creation of an atraumatic, hearing-preservation cochleostomy is integral to the future of minimally invasive inner ear surgery. The goal of this study was to develop and characterize a novel chemical approach to cochleostomy. STUDY DESIGN Prospective animal study. SETTING Laboratory. METHODS Experimental animal study in which phosphoric acid gel (PAG) was used to decalcify the otic capsule in 25 Hartley guinea pigs. Five animals in each of 5 surgical groups were studied: (1) mechanically opening the auditory bulla alone, (2) PAG thinning of the basal turn otic capsule, leaving endosteum covered by a layer of bone, (3) micro-pick manual cochleostomy, (4) PAG chemical cochleostomy, exposing the endosteum, and (5) combined PAG/micro-pick cochleostomy, with initial chemical thinning and subsequent manual removal of the last osseous layer. Preoperative and postoperative auditory brainstem responses and otoacoustic emissions were obtained at 2, 6, 10, and 16 kHz. Hematoxylin and eosin-stained paraffin sections were compared. RESULTS Surgical and histologic findings confirmed that application of PAG provided reproducible local bone removal, and cochlear access was enabled. Statistically significant auditory threshold shifts were observed at 10 kHz (P = .048) and 16 kHz (P = .0013) following cochleostomy using PAG alone (group 4) and at 16 kHz using manual cochleostomy (group 3) (P = .028). No statistically significant, postoperative auditory threshold shifts were observed in the other groups, including PAG thinning with manual completion cochleostomy (group 5). CONCLUSION Hearing preservation cochleostomy can be performed in an animal model using a novel technique of thinning cochlear bone with PAG and manually completing cochleostomy.

ICAM1 depletion reduces spinal metastasis formation in vivo and improves neurological outcome

INTRODUCTION Clinical treatment of spinal metastasis is gaining in complexity while the underlying biology remains unknown. Insufficient biological understanding is due to a lack of suitable experimental animal models. Intercellular adhesion molecule-1 (ICAM1) has been implicated in metastasis formation. Its role in spinal metastasis remains unclear. It was the aim to generate a reliable spinal metastasis model in mice and to investigate metastasis formation under ICAM1 depletion. MATERIAL AND METHODS B16 melanoma cells were infected with a lentivirus containing firefly luciferase (B16-luc). Stable cell clones (B16-luc) were injected retrogradely into the distal aortic arch. Spinal metastasis formation was monitored using in vivo bioluminescence imaging/MRI. Neurological deficits were monitored daily. In vivo selected, metastasized tumor cells were isolated (mB16-luc) and reinjected intraarterially. mB16-luc cells were injected intraarterially in ICAM1 KO mice. Metastasis distribution was analyzed using organ-specific fluorescence analysis. RESULTS Intraarterial injection of B16-luc and metastatic mB16-luc reliably induced spinal metastasis formation with neurological deficits (B16-luc:26.5, mB16-luc:21 days, p<0.05). In vivo selection increased the metastatic aggressiveness and led to a bone specific homing phenotype. Thus, mB16-luc cells demonstrated higher number (B16-luc: 1.2±0.447, mB16-luc:3.2±1.643) and increased total metastasis volume (B16-luc:2.87±2.453 mm3, mB16-luc:11.19±3.898 mm3, p<0.05) in the spine. ICAM1 depletion leads to a significantly reduced number of spinal metastasis (mB16-luc:1.2±0.84) with improved neurological outcome (29 days). General metastatic burden was significantly reduced under ICAM1 depletion (control: 3.47×10(7)±1.66×10(7); ICAM-1-/-: 5.20×10(4)±4.44×10(4), p<0.05 vs. control) CONCLUSION Applying a reliable animal model for spinal metastasis, ICAM1 depletion reduces spinal metastasis formation due to an organ-unspecific reduction of metastasis development.

Refinement of experimental design and conduct in laboratory animal research.

The scientific literature of laboratory animal research is replete with papers reporting poor reproducibility of results as well as failure to translate results to clinical trials in humans. This may stem in part from poor experimental design and conduct of animal experiments. Despite widespread recognition of these problems and implementation of guidelines to attenuate them, a review of the literature suggests that experimental design and conduct of laboratory animal research are still in need of refinement. This paper will review and discuss possible sources of biases, highlight advantages and limitations of strategies proposed to alleviate them, and provide a conceptual framework for improving the reproducibility of laboratory animal research.