Equine model for soft-tissue regeneration.

Soft-tissue regeneration methods currently yield suboptimal clinical outcomes due to loss of tissue volume and a lack of functional tissue regeneration. Grafted tissues and natural biomaterials often degrade or resorb too quickly, while most synthetic materials do not degrade. In previous research we demonstrated that soft-tissue regeneration can be supported using silk porous biomaterials for at least 18 months in vivo in a rodent model. In the present study, we scaled the system to a survival study using a large animal model and demonstrated the feasibility of these biomaterials for soft-tissue regeneration in adult horses. Both slow and rapidly degrading silk matrices were evaluated in subcutaneous pocket and intramuscular defect depots. We showed that we can effectively employ an equine model over 6 months to simultaneously evaluate many different implants, reducing the number of animals needed. Furthermore, we were able to tailor matrix degradation by varying the initial format of the implanted silk. Finally, we demonstrate ultrasound imaging of implants to be an effective means for tracking tissue regeneration and implant degradation.

Owner-reported coughing and nasal discharge are associated with clinical findings, arterial oxygen tension, mucus score and bronchoprovocation in horses with recurrent airway obstruction in a field setting.

REASONS FOR PERFORMING STUDY: In clinical practice, veterinarians often depend on owner-reported signs to assess the clinical course of horses with recurrent airway obstruction (RAO). OBJECTIVES: To test whether owner-reported information on frequency of coughing and observation of nasal discharge are associated with clinical, cytological and bronchoprovocation findings in RAO-affected horses in nonstandardised field conditions. STUDY DESIGN: Cross-sectional study comparing healthy and RAO-affected horses. METHODS: Twenty-eight healthy and 34 RAO-affected Swiss Warmblood horses were grouped according to owner-reported 'coughing frequency' and 'nasal discharge'. Differences between these groups were examined using clinical examination, blood gas analyses, endoscopic mucus scores, cytology of tracheobronchial secretion and bronchoalveolar lavage fluid, and airway hyperresponsiveness determined by plethysmography with histamine bronchoprovocation. RESULTS: Frequently coughing horses differed most markedly from healthy control animals. Histamine bronchoprovocation-derived parameters were significantly different between the healthy control group and all RAO groups. Mucus grades and tracheobronchial secretion and bronchoalveolar lavage fluid neutrophil percentages had particularly high variability, with overlap of findings between groups. Owner satisfaction with the clinical status of the horse was high, even in severely affected horses. CONCLUSIONS: Owner-reported coughing and nasal discharge are associated with specific clinical and diagnostic findings in RAO-affected horses in field settings. While airway hyperresponsiveness differentiates best between healthy horses and asymptomatic RAO-affected horses, the absence of coughing and nasal discharge does not rule out significant neutrophilic airway inflammation. Owner satisfaction with the clinical status of the horse was uninformative.

Age effects on blood gas, spirometry, airway reactivity, and bronchoalveolar lavage fluid cytology in clinically healthy horses.

BACKGROUND: Despite the increasing number of geriatric horses attended by veterinarians, there is a lack of understanding of aging-related changes on the respiratory system of horses. OBJECTIVE: To identify aging-related changes on the respiratory function and bronchoalveolar lavage fluid (BALF) cytology of horses. ANIMALS: Fifteen healthy young adult (2-11 years) and 16 healthy aged (≥20 years) horses. METHODS: The respiratory system was examined by measurement of arterial blood gases (ABG), use of respiratory inductive plethysmography (RIP) for assessment of breathing pattern and ventilatory parameters, histamine bronchoprovocation, and BALF cytology. RESULTS: No significant differences were detected with regard to values obtained by ABG or bronchoprovocation of young adult and aged healthy horses. In aged horses, there were significant differences in mean ± SD of the following parameters when compared to young horses: prolonged expiratory time (Te) measured by RIP (3.9 ± 1.5 s versus 3.0 ± 0.6 s), decreased percentage of alveolar macrophages (40.6 ± 11.3% versus 53.5 ± 9.6%), and increased percentage of lymphocytes (53.4 ± 9.5% versus 43.9 ± 11.0%). No correlations between airway reactivity and ventilatory parameters, ABG, or BALF cytology were found in this asymptomatic population. CONCLUSIONS: These results suggest that aging does not cause changes in the results obtained by ABG, most RIP-derived variables, and bronchoprovocation in the horse. A decreased percentage of macrophage and an increased percentage of lymphocytes in the BALF cytology may be expected in the asymptomatic geriatric horse and may be a result of aging.

Alveolar epithelial stem and progenitor cells: emerging evidence for their role in lung regeneration.

Lung injuries that impact the alveolus, such as emphysema, pulmonary fibrosis, and acute lung injury, are costly and prevalent problems. Moreover, the extent of alveolar injury and impairment of gas exchange is strongly associated with prognosis and survival. Thus, mechanisms of repair and regeneration of the lung alveolar compartment have received mounting attention as newer approaches to the study of stem and progenitor cells in this region unfold. The role of type II alveolar epithelial as the sole source of type I (AECI) and II (AECII) alveolar epithelial cells following lung injury has been recently challenged; recently, investigators have described stemprogenitor cells that function like precursors to AECII either in vitro or in vivo, both in mice and humans. Techniques to explore selfrenewal and multipotency have been rigorously applied to these putative stem-progenitor cell populations and the data thus far is compelling. This review provides background to the study of alveolar regeneration with the aim to provide context to the recent discoveries of putative stem-progenitor cells that may contribute to this process.

Pregnancy-associated progenitor cells: an under-recognized potential source of stem cells in maternal lung.

Novel therapies are needed for the treatment of acute and chronic lung diseases, many of which are incurable. The use of exogenous stem cells has shown promise in both animal models and clinical trials. However, to date, the stem cell literature has under-recognized naturally acquired pregnancy-associated progenitor cells (PAPCs). These cells are found at sites of injury or disease in female tissues. They persist for decades after parturition in maternal blood and organs, with the largest number being found in the maternal lungs. Their presence there may be one explanation for the sex differences observed in the prevalence and prognosis of some lung diseases. Although the clinical significance of these cells is as yet unknown, the literature suggests that some of the PAPCs are stem cells or have stem cell-like properties. PAPCs harvested from the blood or organs of parous women could potentially be used as an alternate source of cells with regenerative properties for the woman herself or her children. Because PAPCs preferentially traffic to the maternal lung they may play a significant role in recovery or protection from lung disease. In this review article, we discuss ongoing research investigating the administration of both adult and placenta-derived stem cells to treat lung disease, and how PAPCs may also play an important future therapeutic role.

Matrix modulation of compensatory lung regrowth and progenitor cell proliferation in mice.

Mechanical stress is an important modulator of lung morphogenesis, postnatal lung development, and compensatory lung regrowth. The effect of mechanical stress on stem or progenitor cells is unclear. We examined whether proliferative responses of epithelial progenitor cells, including dually immunoreactive (CCSP and proSP-C) progenitor cells (CCSP+/SP-C+) and type II alveolar epithelial cells (ATII), are affected by physical factors found in the lung of emphysematics, including loss of elastic recoil, reduced elastin content, and alveolar destruction. Mice underwent single lung pneumonectomy (PNY) to modulate transpulmonary pressure (mechanical stress) and to stimulate lung regeneration. Control mice underwent sham thoracotomy. Plombage of different levels was employed to partially or completely abolish this mechanical stress. Responses to graded changes in transpulmonary pressure were assessed in elastin-insufficient mice (elastin +/-, ELN+/-) and elastase-treated mice with elastase-induced emphysema. Physiological regrowth, morphometry (linear mean intercept; Lmi), and the proliferative responses of CCSP+/SP-C+, Clara cells, and ATII were evaluated. Plombage following PNY significantly reduced transpulmonary pressure, regrowth, and CCSP+/SP-C+, Clara cell, and ATII proliferation following PNY. In the ELN+/- group, CCSP+/SP-C+ and ATII proliferation responses were completely abolished, although compensatory lung regrowth was not significantly altered. In contrast, in elastase-injured mice, compensatory lung regrowth was significantly reduced, and ATII but not CCSP+/SP-C+ proliferation responses were impaired. Elastase injury also reduced the baseline abundance of CCSP+/SP-C+, and CCSP+/SP-C+ were found to be displaced from the bronchioalveolar duct junction. These data suggest that qualities of the extracellular matrix including elastin content, mechanical stress, and alveolar integrity strongly influence the regenerative capacity of the lung, and the patterns of cell proliferation in the lungs of adult mice.

Reproducibility of airway responsiveness in horses using flowmetric plethysmography and histamine bronchoprovocation.

BACKGROUND: Inflammatory airway disease has a high prevalence in horses, but is often a diagnostic challenge. Flowmetric plethysmography and histamine bronchoprovocation (FP/HBP) is a simple and effective tool for diagnosis, but reproducibility of these measurements made over time has not been established. HYPOTHESIS: We hypothesize that the measurement of airway responsiveness in horses using FP/HBP is consistent over both short and long periods of time. ANIMALS: Twenty-nine healthy adult horses from 2 university herds. METHODS: In this prospective experimental study, airway responsiveness was determined in each horse at day 0 (baseline [BL]) with FP/ HBP, using PC35 (provocative concentration of histamine needed to increase Delta(flow) by 35%) as a measure of airway responsiveness. Each horse was re-tested 1-4 weeks after BL (short-term [ST]) and again at 3-12 months after BL (long-term [LT]). RESULTS: In the ST period, 23/27 (85%) of the horses had a PC35 that was within 1 doubling concentration of histamine of their BL value, with a mean change of 0.52 doubling concentrations (95% CI 0.26-0.79, range 0-2.06). For the LT data, 19/26 (73%) of horses were within 1 doubling concentration of their BL value, with a mean change of 0.81 doubling concentrations (95% CI 0.45-1.17, range 0.14-3.10). There was no significant difference in reproducibility between the 2 groups of subjects. CONCLUSIONS AND CLINICAL IMPORTANCE: Repeated measurements of airway responsiveness obtained with FP/HBP show acceptable reproducibility over time periods up to a year. However, caution must be used when testing horses when ambient air temperature is low.

Association between cough and cytology of bronchoalveolar lavage fluid and pulmonary function in horses diagnosed with inflammatory airway disease.

BACKGROUND: The objective of this study was to examine the relationship between chronic cough, pulmonary mechanical function, bronchial hyper-responsiveness, and peripheral airway inflammation in adult performance horses with nonseptic inflammatory airway disease (IAD). HYPOTHESIS: We hypothesized that horses with a presenting complaint of cough have a higher percentage of inflammatory cells on bronchoalveolar lavage (BAL), greater airway obstruction, and airway hyper-responsiveness (AHR) than do horses without cough. ANIMALS/SAMPLE POPULATION: Adult performance horses (n = 137) referred for evaluation of signs of IAD including cough or exercise intolerance (university hospital patients) and BAL samples (n = 142) taken in first opinion practice. METHODS: A descriptive, retrospective cohort analysis was performed to evaluate the association between chronic cough and pulmonary mechanical function, AHR, BAL cytology, patient signalment, and comorbid features (multivariable logistic regression). RESULTS: Cough was significantly more prevalent in horses >7 years, and best characterized by a high BAL neutrophil count (>5%) and nasal discharge. Lung function mechanics, abnormal thoracic auscultation, and exercise intolerance did not retain statistical significance in the logistical regression analysis of cough. Although AHR was not related to neutrophilic airway inflammation (BAL neutrophils >5%), it was significantly associated with BAL mast cells >2%. CONCLUSIONS AND CLINICAL IMPORTANCE: Our data support that neutrophilic airway inflammation may potentiate cough without further changing respiratory mechanical function in IAD. In contrast, mast cell release increased AHR without affecting the incidence of cough. Cough may be used as an indicator of neutrophilic airway inflammation in the presence of low-grade nonseptic respiratory disease.

Cellular kinetics and modeling of bronchioalveolar stem cell response during lung regeneration.

Organ regeneration in mammals is hypothesized to require a functional pool of stem or progenitor cells, but the role of these cells in lung regeneration is unknown. Whereas postnatal regeneration of alveolar tissue has been attributed to type II alveolar epithelial cells (AECII), we reasoned that bronchioalveolar stem cells (BASCs) have the potential to contribute substantially to this process. To test this hypothesis, unilateral pneumonectomy (PNX) was performed on adult female C57/BL6 mice to stimulate compensatory lung regrowth. The density of BASCs and AECII, and morphometric and physiological measurements, were recorded on days 1, 3, 7, 14, 28, and 45 after surgery. Vital capacity was restored by day 7 after PNX. BASC numbers increased by day 3, peaked to 220% of controls (P<0.05) by day 14, and then returned to baseline after active lung regrowth was complete, whereas AECII cell densities increased to 124% of baseline (N/S). Proliferation studies revealed significant BrdU uptake in BASCs and AECII within the first 7 days after PNX. Quantitative analysis using a systems biology model was used to evaluate the potential contribution of BASCs and AECII. The model demonstrated that BASC proliferation and differentiation contributes between 0 and 25% of compensatory alveolar epithelial (type I and II cell) regrowth, demonstrating that regeneration requires a substantial contribution from AECII. The observed cell kinetic profiles can be reconciled using a dual-compartment (BASC and AECII) proliferation model assuming a linear hierarchy of BASCs, AECII, and AECI cells to achieve lung regrowth.

Impact of positive end-expiratory pressure during heterogeneous lung injury: insights from computed tomographic image functional modeling.

Image Functional Modeling (IFM) synthesizes three dimensional airway networks with imaging and mechanics data to relate structure to function. The goal of this study was to advance IFM to establish a method of exploring how heterogeneous alveolar flooding and collapse during lung injury would impact regional respiratory mechanics and flow distributions within the lung at distinct positive end-expiratory pressure (PEEP) levels. We estimated regional respiratory system elastance from computed tomography (CT) scans taken in 5 saline-lavaged sheep at PEEP levels from 7.5 to 20 cmH(2)O. These data were anatomically mapped into a computational sheep lung model, which was used to predict the corresponding impact of PEEP on dynamic flow distribution. Under pre-injury conditions and during lung injury, respiratory system elastance was determined to be spatially heterogeneous and the values were distributed with a hyperbolic distribution in the range of measured values. Increases in PEEP appear to modulate the heterogeneity of the flow distribution throughout the injured lung. Moderate increases in PEEP decreased the heterogeneity of elastance and predicted flow distribution, although heterogeneity began to increase for PEEP levels above 12.5-15 cmH(2)O. By combining regional respiratory system elastance estimated from CT with our computational lung model, we can potentially predict the dynamic distribution of the tidal volume during mechanical ventilation and thus identify specific areas of the lung at risk of being overdistended.

Behavioural lateralisation in the Australian magpie (Gymnorhina tibicen).

In many vertebrates, the brain is lateralised such that each hemisphere is specialised to serve specific functions. This may translate into lateralisation in behaviour through preferential use of receptors or appendages associated with a particular hemisphere (e.g., handedness) or in differential responses to stimuli perceived on the animal's left or right side. In this study, we investigated behavioural laterality in the Australian magpie, Gymnorhina tibicen. We found that, while the birds did not have a population bias for one antipredator behaviour (visual inspection of an approaching human), there were biases for another (alarm calling); those birds that used their left eye more relative to their right eye gave alarm calls more frequently. We also observed that juvenile birds begged for food on the right side of parents significantly more frequently than on the left side. These trends are consistent with trends in behavioural laterality that have been recorded in captive and lab-reared species. Ours is one of the few studies to observe patterns of laterality in a wild species.

Canine awake head-out plethysmography (HOP): characterization of external resistive loading and spontaneous laryngeal paralysis.

We applied a novel head-out plethysmographic (HOP) method to study awake canine responses to external resistive loading and natural laryngeal paralysis. Measurements of inspiratory and expiratory specific airway resistance (sRaw(insp), sRaw(exp)) were obtained before and after uni- and bidirectional loading (R(add) = 5 cmH(2)O/L/s) in large-breed dogs (n = 9). Mean sRaw(insp) after inspiratory, and sRaw(exp) after expiratory loading were 31.4 and 33.3 cmH(2)Os, respectively. Bidirectional loads induced a significantly greater rise in both sRaw(insp) and sRaw(exp) (55.1 and 61.3 cmH(2)Os) compared to unidirectional loading (P < 0.001). Yet, type of loading did not affect flow-volume indices. The mean R(aw) of dogs was 4.81 cmH(2)O/L/s. Expiratory loading resulted in a significant 8.8% increase in functional-residual-capacity (FRC), compared to FRC(baseline) (76.7 ml/kg). Dogs (n = 5) with laryngeal paralysis demonstrated a significant increase in sR(aw) and R(aw) compared to controls without changes in FRC. In conclusion, HOP precisely characterized sR(aw) in response to external resistive loading. Hence, we could accurately quantify airway obstruction in awake dogs with laryngeal paralysis.

Radiographic scoring lacks predictive value in inflammatory airway disease.

REASONS FOR PERFORMING STUDY: Diagnosis of inflammatory airway disease (IAD) currently rests upon the results of bronchoalveolar lavage (BAL) cytology, lung function testing and histamine bronchoprovocation (HBP), none of which provides direct information about structural change in the lung. HYPOTHESIS: That thoracic radiography might better portray structural change in the lungs and therefore offer a good clinical assessment of IAD. METHODS: A radiographic scoring system was developed to assess the extent of bronchial and interstitial pattern on thoracic radiographs in the dorsocaudal, dorsocranial and caudoventral regions in 16 control horses and 33 horses with IAD. Mean scores were compared to ascertain whether thoracic radiographs could distinguish between the 2 groups. In order to determine whether independent observers reliably scored radiographs similarly, an inter-rater reliability score was employed for each radiographic observation. Correlations between radiographic scores, BAL cytology, lung function testing using the forced oscillatory technique and HBP were examined. RESULTS: Inter-rater reliability was only moderate. Radiographic scoring demonstrated no differences between the 2 groups. There were no correlations among BAL cytology, lung function testing, HBP and radiographic scores. CONCLUSIONS AND POTENTIAL RELEVANCE: Thoracic radiographs are a low-yield diagnostic modality in horses with a clinical history compatible with IAD. In the absence of clinical evidence of more extensive, infectious disease, thoracic radiographs neither refine nor improve diagnosis of IAD, but increase diagnostic costs.

Effects of aerosolized albuterol on physiologic responses to exercise in standardbreds.

OBJECTIVE: To examine the effects of an aerosolized beta2-adrenoreceptor agonist, albuterol, on performance during a standardized incremental exercise test in clinically normal horses. ANIMALS: 8 Standardbred pacing mares. PROCEDURE: Clinically normal horses, as judged by use of physical examination, hematologic findings, serum biochemical analysis, and airway endoscopy, were randomly assigned to 2 groups and were given 900 microg of albuterol via a metered-dose inhaler 30 minutes before beginning a standardized incremental exercise test in a crossover design with a 7-day minimum washout. Further examination included measurement of baseline lung mechanics, response to histamine bronchoprovocation, and bronchoalveolar lavage. RESULTS: No significant differences (albuterol vs placebo) were seen for any incremental exercise test variables (ie, maximum oxygen consumption, maximum carbon dioxide consumption, respiratory quotient, treadmill speed at heart rate of 200 beats/min, or number of steps completed during an incremental exercise protocol). Mast cell percentage was significantly (r = -0.84) associated with the concentration of aerosolized histamine that evoked a 100% increase in total respiratory system resistance. No other direct correlations between bronchoalveolar lavage fluid cell types and any indices of exercise capacity or airway reactivity were found. CONCLUSIONS AND CLINICAL RELEVANCE: Although no horse had exercise intolerance, 4 horses had airway hyperreactivity with bronchoalveolar lavage fluid mastocytosis; these horses may have been subclinically affected with inflammatory airway disease. In our study, albuterol did not enhance performance in 8 clinically normal racing-fit Standardbreds.

Vestibular signs associated with suspected lightning strike in two horses.

Two previously healthy 14-year-old horses developed right-sided unilateral vestibular signs after they had possibly been struck by lightning. Repeated radiographic and endoscopic evaluations did not reveal any significant changes. A brainstem auditory evoked response test indicated a subtle left to right interaural latency difference of waves I, III and V in the more severely affected pony, but its central conduction time remained normal.

Respiratory applications for the future: one perspective.

Respiratory diagnostic instruments previously available only to referral centers should be converted into miniature, portable, wireless medical devices for use in practice. Internet and wireless connections of these instruments should expedite the process of seeking second opinions and in general information sharing. We are moving into a world of greater objectivity that should free us from speculation in some areas, yet open new avenues for research and innovation in others.

Development of a standardized nomenclature for bronchoscopy of the respiratory system of harbor porpoises (Phocoena phocoena).

Respiratory disease is common in captive and wild cetaceans. Bronchoscopy may permit early diagnosis of respiratory disease in dolphins and porpoises. Refinement of cetacean bronchoscopy requires development of a nomenclature system to facilitate description of the anatomic site at which lesions occur. A standard bronchoscopic nomenclature also permits serial evaluations of lesions and enhances communication between veterinarians. In this project, we adapted the bronchoscopic nomenclature devised by Amis and McKiernan for the dog and horse to the harbor porpoise (Phocoena phocoena). Silastic and air-dried models of the bronchial tree of the harbor porpoise were made to illustrate the anatomy and devise the nomenclature. Bronchial anatomy was consistent among the four porpoise lungs studied. The Amis and McKiernan nomenclature was readily adaptable to the harbor porpoise lung with minor modifications and may be useful for cetacean bronchoscopy.

Thoracoabdominal asynchrony failed to grade airway obstructions in foals.

Respiratory inductive plethysmography (RIP) can be used to obtain a valid measure of tidal volume in humans. This device also compares the contributions to ventilation of the thorax and abdomen. Although thoracoabdominal asynchrony is a prominent clinical feature for patients with airway obstruction, the accuracy of the RIP device to assess the severity of obstruction is unclear. This study analyzes how well RIP variables reflect the degree of a fixed external inspiratory plus expiratory resistive load in foals. Foals were employed because the species and age group are commonly afflicted with respiratory disease. Eight conscious, sedated (xylazine 1.25 mg/kg body wt) foals were subjected to randomly ordered resistive loads at the airway opening and, on a separate day, to histamine aerosol challenge. During resistive loading, phase angle changed significantly, as did phase relation (P < or = 0.05). However, no significant correlation was found between the degree of change in resistive load and the degree to which phase angle or relation was altered (r(s) = 0.41 and 0.25, respectively). In addition, neither phase angle nor relation changed significantly with histamine challenge. We conclude that, although RIP variables changed markedly with fixed upper airway resistive loading, the degree to which they changed was erratic and therefore not useful for grading these obstructions. Furthermore, RIP variables were insensitive measures of histamine-induced bronchoconstriction.

Airway responses to histamine aerosol in clinically normal foods.

OBJECTIVE: To describe the spectrum of nonspecific airway reactivity in a group of clinically normal foals. ANIMALS: 12 clinically normal mixed-breed foals, 48 to 92 days old, without history of clinical lung disease. PROCEDURE: Nonspecific airway reactivity was determined by measuring the extent of changes in dynamic compliance during nebulization of incrementally increasing concentrations of histamine aerosol. Degree of airway reactivity was expressed as the dose of histamine that evoked a decrease in dynamic compliance (Cdyn) to 65% of the after saline nebulization value (PC65Cdyn) or increase in pulmonary resistance (R(L)) to 135% of baseline (PC135R(L)). RESULTS: In all foals, it was possible to induce a decrease in Cdyn in dose-dependent manner to < or = 65% of baseline. Response of foals in terms of R(L) was more erratic, and, in 1 foal, R(L) decreased after histamine exposure. Mean+/-SD PC65Cdyn was 5.43+/-1.74 (range, 0.77 to 19.56) mg/ml, and mean PC135R(L) was 3.34+/-1.52 (range, -0.749 to 17.35) mg/ml. Body weight was not correlated to baseline Cdyn, R(L), PC65Cdyn, or PC135R(L). CONCLUSIONS AND CLINICAL RELEVANCE: Clinically normal foals had a wide range of airway reactivity, which may contribute to variation in clinical responses of foals to otherwise similar stimuli, such as infection, inflammation, and challenge exposure with environmental irritants.