[Surgical reconstruction of chest wall instability : Indications, contraindications and timing of surgery]. / Operative Rekonstruktion der Thoraxwandinstabilität : Indikationen, Kontraindikationen und Operationszeitpunkt.

The impact of energy on the thorax can lead to serial rib fractures, sternal fractures, the combination of both and to injury of intrathoracic organs depending on the type, localization and intensity. Sometimes this results in chest wall instability with severe impairment of the respiratory mechanics. In the last decade the importance of surgical chest wall reconstruction in cases of chest wall instability has greatly increased. The evidence for a surgical approach has in the meantime been supported by prospective randomized multicenter studies, multiple retrospective data analyses and meta-analyses based on these studies, including a Cochrane review. The assessment of form and severity of the trauma and the degree of impairment of the respiratory mechanism are the basis for a structured decision on an extended conservative or surgical reconstructive strategy as well as the timing, type and extent of the operation. The morbidity (rate of pneumonia, duration of intensive care unit stay and mechanical ventilation) and fatality can be reduced by a timely surgery within 72 h after trauma. In this article the already established and evidence-based algorithms for surgical chest wall reconstruction are discussed in the context of the current evidence.

An initiative to assess and improve the resources and patient care processes used among Chest Wall Injury Society collaborative centers study (CWIS-CC2).

BACKGROUND: Over the last two decades, the acute management of rib fractures has changed significantly. In 2021, the Chest Wall injury Society (CWIS) began recognizing centers that epitomize their mission as CWIS Collaborative Centers. The primary aim of this study was to determine the resources, surgical expertise, access to care, and institutional support that are present among centers. METHODS: A survey was performed including all CWIS Collaborative Centers evaluating the resources available at their hospital for the treatment of patients with chest wall injury. Data about each chest wall injury center care process, availability of resources, institutional support, research support, and educational offerings were recorded. RESULTS: Data were collected from 20 trauma centers resulting in an 80% response rate. These trauma centers were made up of 5 international and 15 US-based trauma centers. Eighty percent (16 of 20) have dedicated care team members for the evaluation and management of rib fractures. Twenty-five percent (5 of 20) have a dedicated rib fracture service with a separate call schedule. Staffing for chest wall injury clinics consists of a multidisciplinary team: with attending surgeons in all clinics, 80% (8 of 10) with advanced practice providers and 70% (7 of 10) with care coordinators. Forty percent (8 of 20) of centers have dedicated rib fracture research support, and 35% (7 of 20) have surgical stabilization of rib fracture (SSRF)-related grants. Forty percent (8 of 20) of centers have marketing support, and 30% (8 of 20) have a web page support to bring awareness to their center. At these trauma centers, a median of 4 (1-9) surgeons perform SSRFs. In the majority of trauma centers, the trauma surgeons perform SSRF. CONCLUSION: Considerable similarities and differences exist within these CWIS collaborative centers. These differences in resources are hypothesis generating in determining the optimal chest wall injury center. These findings may generate several patient care and team process questions to optimize patient care, patient experience, provider satisfaction, research productivity, education, and outreach. LEVEL OF EVIDENCE: Therapeutic/Care Management; Level V.

Alterations in Lens Free Water Distribution Are Associated with Shape Deformation in Accommodation.

Objective: To investigate whether a redistribution of water within the crystalline lens is associated with the shape deformation that occurs during accommodation. Design: Observational, cross sectional study. Subjects: Eleven young adults without presbyopia (aged 18-39 years) and 9 middle-aged adults with presbyopia (aged 40-55 years). Methods: Magnetic resonance imaging (MRI) scans of the lens were acquired on a 3 Tesla clinical MRI scanner, without and with the presentation of a 3 Diopter accommodative stimulus. The MRIs were postprocessed using established methods to extract the geometric dimensions and spatial maps of water distribution of the lens. Main Outcome Measures: Accommodative changes in the full 3-dimensional description of lens shape, the lens total-water distribution profile, and the lens free-water distribution profile. Results: Viewing of an accommodative stimulus by young subjects elicited an elastic shape deformation of the lens consistent with accommodation that was associated with an elevated, smoother free-water distribution, primarily in the anterior region of the lens. In contrast, viewing of an accommodative stimulus by presbyopic subjects produced an atypical shape deformation of the lens that was instead associated with a lowered free-water distribution, primarily in the anterior region of the lens. No discernible changes to the lens total-water distribution were observed in response to the accommodative stimulus in either subject cohort. Conclusions: The present study suggests that protein-mediated alterations in the free-water distribution of the anterior region of the lens influence the shape deformation in accommodation, presenting pharmacological modulation of free-water distribution as an attractive novel approach for treating presbyopia. Financial Disclosures: The authors have no proprietary or commercial interest in any materials discussed in this article.

Recent Advances in Rib Plating.

Rib fractures are a common injury in blunt trauma and are associated with high morbidity and mortality. Recent advances in surgical stabilization of rib fractures (SSRF) have led to better patient outcomes for those with highly unstable complex rib fractures, as well as those with less severe injuries. This result has been due in part to the expansion of indications for repair, as well as the development of new hardware systems to address a variety of fracture patterns and injuries. This joint advancement of operator techniques, outcomes research, and industry development has brought SSRF to the forefront of rib fracture management and challenged non-operative paradigms. The future of repair is now shifting focus, as surgeons develop minimally invasive approaches and challenge manufacturers to develop new systems, instruments, and materials to address increasingly complex fracture patterns. These expansions promise to make SSRF an increasingly effective form of management for traumatic rib fractures.

Single-Cell RNA Sequencing Analysis of the Early Postnatal Mouse Lens Epithelium.

Purpose: The lens epithelium maintains the overall health of the organ. We used single-cell RNA sequencing (scRNA-seq) technology to assess transcriptional heterogeneity between cells in the postnatal day 2 (P2) epithelium and identify distinct epithelial cell subtypes. Analysis of these data was used to better understand lens growth, differentiation, and homeostasis on P2. Methods: scRNA-seq on P2 mouse lenses was performed using the 10x Genomics Chromium Single Cell 3' Kit (v3.1) and short-read Illumina sequencing. Sequence alignment and preprocessing of data were conducted using 10x Genomics Cell Ranger software. Seurat was employed for preprocessing, quality control, dimensionality reduction, and cell clustering, and Monocle was utilized for trajectory analysis to understand the developmental progression of the lens cells. CellChat and GO analyses were used to explore cell-cell communication networks and signaling interactions. Results: Lens epithelial cells (LECs) were divided into seven subclusters, classified by specific gene markers. The expression of crystallin, cell-cycle, and metabolic genes was not uniform, indicating distinct functional roles of LECs. Trajectory analysis predicted a bifurcation of differentiating and cycling cells from an Igfbp5+ progenitor pool. We also identified heterogeneity in signaling molecules and pathways, suggesting that cycling and progenitor subclusters have prominent roles in coordinating crosstalk. Conclusions: scRNA-seq corroborated many known markers of epithelial differentiation and proliferation while providing further insight into the pathways and genes directing these processes. Interestingly, we demonstrated that the developing epithelium can be divided into distinct subpopulations. These clusters reflect the transcriptionally diverse roles of the epithelium in proliferation, signaling, and maintenance.

Chest Wall Injury Society recommendation for surgical stabilization of nonunited rib fractures to decrease pain, reduce opiate use, and improve patient reported outcomes in patients with rib fracture nonunion after trauma.

BACKGROUND: Rib fractures are common injuries which can be associated with acute pain and chronic disability. While most rib fractures ultimately go on to achieve bony union, a subset of patients may go on to develop non-union. Management of these nonunited rib fractures can be challenging and variability in management exists. METHODS: The Chest Wall Injury Society's Publication Committee convened to develop recommendations for use of surgical stabilization of nonunited rib fractures (SSNURF) to treat traumatic rib fracture nonunions. PubMed, Embase, and the Cochrane database were searched for pertinent studies. Using a process of iterative consensus, all committee members voted to accept or reject the recommendation. RESULTS: No identified studies compared SSNURF to alternative therapy and the overall quality of the body of evidence was rated as low. Risk of bias was identified in all studies. Despite these limitations, there is lower-quality evidence suggesting that SSNURF may be beneficial for decreasing pain, reducing opiate use, and improving patient reported outcomes among patients with symptomatic rib nonunion. However, these benefits should be balanced against risk of symptomatic hardware failure and infection. CONCLUSION: This guideline document summarizes the current CWIS recommendations regarding use of SSNURF for management of rib nonunion. LEVEL OF EVIDENCE: Therapeutic/Care Management; Level IV.

Age-Dependent Changes in the Water Content and Optical Power of the In Vivo Mouse Lens Revealed by Multi-Parametric MRI and Optical Modeling.

Purpose: The purpose of this study was to utilize in vivo magnetic resonance imaging (MRI) and optical modeling to investigate how changes in water transport, lens curvature, and gradient refractive index (GRIN) alter the power of the mouse lens as a function of age. Methods: Lenses of male C57BL/6 wild-type mice aged between 3 weeks and 12 months (N = 4 mice per age group) were imaged using a 7T MRI scanner. Measurements of lens shape and the distribution of T2 (water-bound protein ratios) and T1 (free water content) values were extracted from MRI images. T2 values were converted into the refractive index (n) using an age-corrected calibration equation to calculate the GRIN at different ages. GRIN maps and shape parameters were inputted into an optical model to determine ageing effects on lens power and spherical aberration. Results: The mouse lens showed two growth phases. From 3 weeks to 3 months, T2 decreased, GRIN increased, and T1 decreased. This was accompanied by increased lens thickness, volume, and surface radii of curvatures. The refractive power of the lens also increased significantly, and a negative spherical aberration was developed and maintained. Between 6 and 12 months of age, all physiological, geometrical, and optical parameters remained constant, although the lens continued to grow. Conclusions: In the first 3 months, the mouse lens power increased as a result of changes in shape and in the GRIN, the latter driven by the decreased water content of the lens nucleus. Further research into the mechanisms regulating this decrease in mouse lens water could improve our understanding of how lens power changes during emmetropization in the developing human lens.

Charged multivesicular body protein 4b forms complexes with gap junction proteins during lens fiber cell differentiation.

Charged multivesicular body protein 4b (CHMP4B) is a core sub-unit of the endosomal sorting complex required for transport III (ESCRT-III) machinery that serves myriad remodeling and scission processes of biological membranes. Mutation of the human CHMP4B gene underlies rare forms of early-onset lens opacities or cataracts, and CHMP4B is required for lens growth and differentiation in mice. Here, we determine the sub-cellular distribution of CHMP4B in the lens and uncover a novel association with gap junction alpha-3 protein (GJA3) or connexin 46 (Cx46) and GJA8 or Cx50. Immunofluorescence confocal microscopy revealed that CHMP4B localized to cell membranes of elongated fiber cells in the outer cortex of the lens-where large gap junction plaques begin to form-particularly, on the broad faces of these flattened hexagon-like cells in cross-section. Dual immunofluorescence imaging showed that CHMP4B co-localized with gap junction plaques containing Cx46 and/or Cx50. When combined with the in situ proximity ligation assay, immunofluorescence confocal imaging indicated that CHMP4B lay in close physical proximity to Cx46 and Cx50. In Cx46-knockout (Cx46-KO) lenses, CHMP4B-membrane distribution was similar to that of wild-type, whereas, in Cx50-KO lenses, CHMP4B localization to fiber cell membranes was lost. Immunoprecipitation and immunoblotting analyses revealed that CHMP4B formed complexes with Cx46 and Cx50 in vitro. Collectively, our data suggest that CHMP4B forms plasma membrane complexes, either directly and/or indirectly, with gap junction proteins Cx46 and Cx50 that are often associated with "ball-and-socket" double-membrane junctions during lens fiber cell differentiation.

Increased Hemichannel Activity Displayed by a Connexin43 Mutation Causing a Familial Connexinopathy Exhibiting Hypotrichosis with Follicular Keratosis and Hyperostosis.

Mutations in the GJA1 gene that encodes connexin43 (Cx43) cause several rare genetic disorders, including diseases affecting the epidermis. Here, we examined the in vitro functional consequences of a Cx43 mutation, Cx43-G38E, linked to a novel human phenotype of hypotrichosis, follicular keratosis and hyperostosis. We found that Cx43-G38E was efficiently translated in Xenopus oocytes and localized to gap junction plaques in transfected HeLa cells. Cx43-G38E formed functional gap junction channels with the same efficiency as wild-type Cx43 in Xenopus oocytes, although voltage gating of the gap junction channels was altered. Notably, Cx43-G38E significantly increased membrane current flow through the formation of active hemichannels when compared to wild-type Cx43. These data demonstrate the association of increased hemichannel activity to a connexin mutation linked to a skeletal-cutaneous phenotype, suggesting that augmented hemichannel activity could play a role in skin and skeletal disorders caused by human Cx43 mutations.

Antibody gene transfer treatment drastically improves epidermal pathology in a keratitis ichthyosis deafness syndrome model using male mice.

BACKGROUND: Keratitis ichthyosis deafness (KID) syndrome is a rare disorder caused by hemichannel (HC) activating gain-of-function mutations in the GJB2 gene encoding connexin (Cx) 26, for which there is no cure, or current treatments based upon the mechanism of disease causation. METHODS: We applied Adeno Associated Virus (AAV) mediated mAb gene transfer (AAVmAb) to treat the epidermal features of KID syndrome with a well-characterized HC blocking antibody using male mice of a murine model that replicates the skin pathology of the human disease. FINDINGS: We demonstrate that in vivo AAVmAb treatment significantly reduced the size and thickness of KID lesions, in addition to blocking activity of mutant HCs in the epidermis in vivo. We also show that AAVmAb treatment eliminated abnormal keratinocyte proliferation and enlarged cell size, decreased apoptosis, and restored the normal distribution of keratin expression. INTERPRETATION: Our findings reinforce the critical role played by increased HC activity in the skin pathology associated with KID syndrome. They also underscore the clinical potential of anti-HC mAbs coupled with genetic based delivery systems for treating the underlying mechanistic basis of this disorder. Inhibition of HC activity is an ideal therapeutic target in KID syndrome, and the genetic delivery of mAbs targeted against mutant HCs could form the basis of new therapeutic interventions to treat this incurable disease. FUNDING: Fondazione Telethon grant GGP19148 and University of Padova grant Prot. BIRD187130 to FM; Foundation for Ichthyosis and Related Skin Types (FIRST) and National Institutes of Health grant EY 026911 to TWW.

Double Deletion of PI3K and PTEN Modifies Lens Postnatal Growth and Homeostasis.

We have previously shown that the conditional deletion of either the p110α catalytic subunit of phosphatidylinositol 3-kinase (PI3K), or its opposing phosphatase, phosphatase and tensin homolog (PTEN), had distinct effects on lens growth and homeostasis. The deletion of p110α reduced the levels of phosphorylated Akt and equatorial epithelial cell proliferation, and resulted in smaller transparent lenses in adult mice. The deletion of PTEN increased levels of phosphorylated Akt, altered lens sodium transport, and caused lens rupture and cataract. Here, we have generated conditional p110α/PTEN double-knockout mice, and evaluated epithelial cell proliferation and lens homeostasis. The double deletion of p110α and PTEN rescued the defect in lens size seen after the single knockout of p110α, but accelerated the lens rupture phenotype seen in PTEN single-knockout mice. Levels of phosphorylated Akt in double-knockout lenses were significantly higher than in wild-type lenses, but not as elevated as those reported for PTEN single-knockout lenses. These results showed that the double deletion of the p110α catalytic subunit of PI3K and its opposing phosphatase, PTEN, exacerbated the rupture defect seen in the single PTEN knockout and alleviated the growth defect observed in the single p110α knockout. Thus, the integrity of the PI3K signaling pathway was absolutely essential for proper lens homeostasis, but not for lens growth.

Connexin hemichannel inhibition ameliorates epidermal pathology in a mouse model of keratitis ichthyosis deafness syndrome.

Mutations in five different genes encoding connexin channels cause eleven clinically defined human skin diseases. Keratitis ichthyosis deafness (KID) syndrome is caused by point mutations in the GJB2 gene encoding Connexin 26 (Cx26) which result in aberrant activation of connexin hemichannels. KID syndrome has no cure and is associated with bilateral hearing loss, blinding keratitis, palmoplantar keratoderma, ichthyosiform erythroderma and a high incidence of childhood mortality. Here, we have tested whether a topically applied hemichhanel inhibitor (flufenamic acid, FFA) could ameliorate the skin pathology associated with KID syndrome in a transgenic mouse model expressing the lethal Cx26-G45E mutation. We found that FFA blocked the hemichannel activity of Cx26-G45E in vitro, and substantially reduced epidermal pathology in vivo, compared to untreated, or vehicle treated control animals. FFA did not reduce the expression of mutant connexin hemichannel protein, and cessation of FFA treatment allowed disease progression to continue. These results suggested that aberrant hemichannel activity is a major driver of skin disease in KID syndrome, and that the inhibition of mutant hemichannel activity could provide an attractive target to develop novel therapeutic interventions to treat this incurable disease.

Readmission for pleural space complications after chest wall injury: Who is at risk?

BACKGROUND: Little is known about patient characteristics predicting postdischarge pleural space complications (PDPSCs) after thoracic trauma. We sought to analyze the patient population who required unplanned hospital readmission for PDPSC. METHODS: Retrospective review of adult patients admitted to a Level I Trauma Center with a chest Abbreviated Injury Scale (AIS) score of 2 or greater between January 2015 and August 2020. Those readmitted within 30 days of index hospitalization discharge for PDPSC were compared with those not readmitted. Demographics, injury characteristics, surgical procedures, imaging, and readmission data were retrieved. RESULTS: Out of 17,192 trauma evaluations, 3,412 (19.8%) suffered a chest AIS score of 2 or greater injury and 155 experienced an unplanned 30-day hospital readmission. Of those, 49 (1.4%) were readmitted for the management of PDPSC (readmit PDPSC) and were compared with patients who were not readmitted (no readmit, n = 3,257). The readmit PDPSC group was significantly older age, heavier, comprised of fewer men, and suffered a higher mean chest AIS score. The readmit PDPSC group had a significantly higher incidence of rib fractures, flail chest, pneumothorax, hemothorax, scapula fractures, and a higher rate of tube thoracostomy placement during index admission. The discharge chest X-ray in the readmit PDPSC group demonstrated a pleural space abnormality in 36 (73%) of patients. Mean time to readmission was 10.2 (7.2) days, and hospital length of stay on readmission was 5.8 (3.7) days. Pleural effusion was the most common readmission diagnosis (44 [90%]), and 42 (86%) required tube thoracostomy. CONCLUSION: We describe the subset of chest wall injury patients who require hospital readmission for PDPSC. Characteristics from index hospitalization associated with PDPSC include older age, female sex, heavier weight, presence of rib fractures, pleural space abnormality, scapular fracture, and chest tube placement. Further studies are needed to characterize this at-risk chest wall injury population, and to determine what interventions can facilitate outpatient management of postdischarge pleural space complications and mitigate readmission risk. LEVEL OF EVIDENCE: Prognostic and epidemiologic, Level IV; Care management, Level V.

Age-Dependent Changes in Total and Free Water Content of In Vivo Human Lenses Measured by Magnetic Resonance Imaging.

Purpose: To use magnetic resonance imaging (MRI) to measure age-dependent changes in total and free water in human lenses in vivo. Methods: Sixty-four healthy adults aged 18 to 86 years were recruited, fitted with a 32-channel head receiver coil, and placed in a 3 Tesla clinical MR scanner. Scans of the crystalline lens were obtained using a volumetric interpolated breath-hold examination sequence with dual flip angles, which were corrected for field inhomogeneity post-acquisition using a B1-map obtained using a turbo-FLASH sequence. The spatial distribution and content of corrected total (ρlens) and free (T1) water along the lens optical axis were extracted using custom-written code. Results: Lens total water distribution and content did not change with age (all P > 0.05). In contrast to total water, a gradient in free water content that was highest in the periphery relative to the center was present in lenses across all ages. However, this initially parabolic free water gradient gradually developed an enhanced central plateau, as indicated by increasing profile shape parameter values (anterior: 0.067/y, P = 0.004; posterior: 0.050/y, P = 0.020) and central free water content (1.932 ms/y, P = 0.022) with age. Conclusions: MRI can obtain repeatable total and free water measurements of in vivo human lenses. The observation that the lens steady-state free, but not total, water gradient is abolished with age raises the possibility that alterations in protein-water interactions are an underlying cause of the degradation in lens optics and overall vision observed with aging.

The Chest Wall Injury Society Recommendations for Reporting Studies of Surgical Stabilization of Rib Fractures.

BACKGROUND: Publications investigating the efficacy of surgical stabilization of rib fractures (SSRF) have increased exponentially. However, there is currently no standardized reporting structure for these studies, rendering both comparisons and extrapolation problematic. METHODS: A subject matter expert group was formed by the Chest Wall Injury Society. This group conducted a review of the SSRF investigational literature and identified variable reporting within several general categories of relevant parameters. A compliment of guidelines was then generated. RESULTS: The reporting guidelines consist of 26 recommendations in the categories of: (1) study type, (2) patient and injury characteristics, (3) patient treatments, (4) outcomes, and (5) statistical considerations. CONCLUSION: Our review identified inconsistencies in reporting within the investigational SSRF literature. In response to these inconsistencies, we propose a set of recommendations to standardize reporting of original investigations into the efficacy of SSRF.

Physiological Mechanisms Regulating Lens Transport.

The transparency and refractive properties of the lens are maintained by the cellular physiology provided by an internal microcirculation system that utilizes spatial differences in ion channels, transporters and gap junctions to establish standing electrochemical and hydrostatic pressure gradients that drive the transport of ions, water and nutrients through this avascular tissue. Aging has negative effects on lens transport, degrading ion and water homeostasis, and producing changes in lens water content. This alters the properties of the lens, causing changes in optical quality and accommodative amplitude that initially result in presbyopia in middle age and ultimately manifest as cataract in the elderly. Recent advances have highlighted that the lens hydrostatic pressure gradient responds to tension transmitted to the lens through the Zonules of Zinn through a mechanism utilizing mechanosensitive channels, multiple sodium transporters respond to changes in hydrostatic pressure to restore equilibrium, and that connexin hemichannels and diverse intracellular signaling cascades play a critical role in these responses. The mechanistic insight gained from these studies has advanced our understanding of lens transport and how it responds and adapts to different inputs both from within the lens, and from surrounding ocular structures.

Lens Connexin Channels Show Differential Permeability to Signaling Molecules.

Gap junction channels mediate the direct intercellular passage of small ions as well as larger solutes such as second messengers. A family of proteins called connexins make up the subunits of gap junction channels in chordate animals. Each individual connexin forms channels that exhibit distinct permeability to molecules that influence cellular signaling, such as calcium ions, cyclic nucleotides, or inositol phosphates. In this review, we examine the permeability of connexin channels containing Cx43, Cx46, and Cx50 to signaling molecules and attempt to relate the observed differences in permeability to possible in vivo consequences that were revealed by studies of transgenic animals where these connexin genes have been manipulated. Taken together, these data suggest that differences in the permeability of individual connexin channels to larger solutes like 3',5'-cyclic adenosine monophosphate (cAMP) and inositol 1,4,5-trisphosphate (IP3) could play a role in regulating epithelial cell division, differentiation, and homeostasis in organs like the ocular lens.

Using the Lens Paradox to Optimize an In Vivo MRI-Based Optical Model of the Aging Human Crystalline Lens.

Purpose: To optimize our in vivo magnetic resonance imaging (MRI)-based optical model of the human crystalline lens, developed with a small group of young adults, for a larger cohort spanning a wider age range. Methods: Subjective refraction and ocular biometry were measured in 57 healthy adults ages 18 to 86 years who were then scanned using 3T clinical magnetic resonance imaging (MRI) to obtain lens gradient of refractive index (GRIN) and geometry measurements. These parameters were combined with ocular biometric measurements to construct individualized Zemax eye models from which ocular refractive errors and lens powers were determined. Models were optimized by adding an age-dependent factor to the transverse relaxation time (T2)-refractive index (n) calibration to match model-calculated refractive errors with subjective refractions. Results: In our subject cohort, subjective refraction shifted toward hyperopia by 0.029 diopter/year as the lens grew larger and developed flatter GRINs with advancing age. Without model optimization, lens powers did not reproduce this clinically observed decrease, the so-called lens paradox, instead increasing by 0.055 diopter/year. However, modifying the T2-n calibration by including an age-dependent factor reproduced the decrease in lens power associated with the lens paradox. Conclusions: After accounting for age-related changes in lens physiology in the T2-n calibration, our model was capable of accurately measuring in vivo lens power across a wide age range. This study highlights the need for a better understanding of how age-dependent changes to the GRIN impact the refractive properties of the lens. Translational Relevance: MRI is applied clinically to calculate the effect of age-related refractive index changes in the lens paradox.

Signaling Between TRPV1/TRPV4 and Intracellular Hydrostatic Pressure in the Mouse Lens.

Purpose: The lens uses feedback to maintain zero pressure in its surface cells. Positive pressures are detected by transient receptor potential vanilloid (TRPV4), which initiates a cascade that reduces surface cell osmolarity. The first step is opening of gap junction hemichannels. One purpose of the current study was to identify the connexin(s) in the hemichannels. Negative pressures are detected by TRPV1, which initiates a cascade that increases surface osmolarity. The increase in osmolarity was initially reported to be through inhibition of Na/K ATPase activity, but a recent study reported it was through stimulation of Na/K/2Cl (NKCC) cotransport. A second purpose of this study was to reconcile these two reports. Methods: Intracellular hydrostatic pressures were measured using a microelectrode/manometer system. Lenses from TRPV1 or Cx50 null mice were studied. Specific inhibitors of Cx50 gap junction channels, NKCC, and Akt were used. Results: Either knockout of Cx50 or blockade of Cx50 channels completely eliminated the response to positive surface pressures. Knockout of Cx50 also caused a positive drift in surface pressure. The short-term (∼20-minute) response to negative surface pressures was eliminated by blockade of NKCC, but a long-term (∼4-hour) response restored pressure to zero. Both short- and long-term responses were eliminated by knockout of TRPV1 or inhibition of Akt. Conclusions: Hemichannels made from Cx50 are required for the response to positive surface pressures. Negative surface pressures first activate NKCC, but a backup system is inhibition of Na/K ATPase activity. Both responses are initiated by TRPV1 and go through PI3K/Akt before branching.