The problem with traditional research

[S Foote.]

Thanks S Foote for your reply! It makes sense. I'm just pondering some of the research I have read regarding crosstalk between what goes on "in" the follicle and the surrounding tissue. Could your theory have an impact on that signaling? Does edema effect signaling?

Also ran across this commentary:

http://www.nature.com/ki/journal/v84/n5/full/ki2013287a.html

excerpt:

That's a very interesting study thanks.

I suggest the signalling pathway between hair follicles and the surrounding tissue, involves the recognised growth control of normal contact inhibition. Any change in the resistance of the surrounding tissue to anagen follicle enlargement, must change the point at which contact inhibition turns off follicle enlargement. Increased fluid levels and pressures will tighten the tissue, increasing its resistance and you get smaller follicles. We all know about the tight scalp in male pattern baldness.

Originally, I suggest this relationship evolved to link hair production with the mammalian temperature control of variable dermal fluid levels/pressures. But of course changes in dermal fluid levels/pressures for any other reason will therefore also affect hair production.

 

[IDW2BB]

In this research, the lymphatic system first develops in the jugular region of the mouse. Is that also true of humans? I found this study to be very informative in understanding your theory S Foote. I'm sorry if you have mentioned it before but I have not noticed a reference to it.

Interested in your thoughts on the findings.




http://www.nature.com/emboj/journal/v31/n4/full/emboj2011456a.html

Combining our findings with published data, we now propose a model for how the size of the lymphatic vasculature is regulated in the developing mouse embryo (Figure 8J): an increasing amount of interstitial fluid changes the ECM scaffolds adjacent to LECs, so that these become stretched. This stretching activates β1 integrins on the LECs, which subsequently activate SFKs that propagate within the cells and tyrosine phosphorylate VEGFR3. The combined action of VEGF-C and mechanical stretching synergistically activates VEGFR3 signalling and LEC proliferation. Since the expanded lymphatic vasculature has an improved ability to drain fluid, the fluid pressure in the surrounding tissue decreases. This negative feedback reduces any further lymph vessel growth and might ensure that the lymphatic vasculature is appropriately sized to meet the tissue demand for fluid drainage. Since fluid injection into the embryonic mouse skin and the adult mouse ear also increased VEGFR3 tyrosine phosphorylation plus LEC proliferation (Figure 9), the mechanism proposed might broadly apply to lymph vessels in general.

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While I don't have access to the full paper, I thought this abstract with the above linked research was interesting as it relates to the benefit of 5ar inhibitors.

http://www.ncbi.nlm.nih.gov/pubmed/15897888

Upregulation of VEGF-C by androgen depletion: the involvement of IGF-IR-FOXO pathway.

Li J, Wang E, Rinaldo F, Datta K.


Source

Department of Biochemistry and Molecular Biology and Mayo Clinic Cancer Center, Mayo Clinic Foundation, Rochester, MN 55905, USA.

Abstract

Androgen ablation therapy is eventually followed by a more metastatic and androgen-refractory stage of prostate cancer. The detailed molecular mechanism of this gradual transition is not clearly understood. Recent reports correlate the high abundance of vascular endothelial growth factor-C (VEGF-C) to the lymph node metastasis seen in human prostate cancer (Tsurusaki et al., 1999). In this study, we report that androgen ablation in LNCaP cells augment the transcriptional upregulation of VEGF-C and the downregulation of the IGF-IR pathway, due to androgen withdrawal, is a potential mechanism for this observed VEGF-C transcription. Forkhead transcription factor FOXO-1, activated by SIRT-1, was identified as the downstream molecule within this pathway. Furthermore, the VEGF-C-induced increase of Bag-IL expression in LNCaP cells suggests that VEGF-C plays a role in the androgen-independent reactivation of the androgen receptor, resulting in androgen-refractory prostate cancer growth.

I wonder if this lymphatic signaling were to occur in close proximity to the dermal papilla if perhaps some signals may be crossed? Seems to me that the DP signaling and lymphatic signaling are happening on the same dance floor. I wonder if their dance partners cut-in every once in a while?

 

[S Foote.]

In this research, the lymphatic system first develops in the jugular region of the mouse. Is that also true of humans? I found this study to be very informative in understanding your theory S Foote. I'm sorry if you have mentioned it before but I have not noticed a reference to it.

Interested in your thoughts on the findings.




http://www.nature.com/emboj/journal/v31/n4/full/emboj2011456a.html



- - - Updated - - -

While I don't have access to the full paper, I thought this abstract with the above linked research was interesting as it relates to the benefit of 5ar inhibitors.

http://www.ncbi.nlm.nih.gov/pubmed/15897888






I wonder if this lymphatic signaling were to occur in close proximity to the dermal papilla if perhaps some signals may be crossed? Seems to me that the DP signaling and lymphatic signaling are happening on the same dance floor. I wonder if their dance partners cut-in every once in a while?

I think the first study is interesting, because it shows a 'Hydraulic' feedback of pressure/ lymph vessel development. This 'may' be something that happens in the male pattern baldness scalp, to try to balance the fluid build up. However if the restriction is a back pressure effect as suggested, the fluid has nowhere to go anyway while this backpressure still exists. Here again we don't have much research into the state of lymph vessels in the male pattern baldness area. I would be willing to bet they would be in a similar state to those in the lipedematous alopecia study I referenced in this thread.

Regarding the second study, I don't think there is any significant molecular signalling going on here. I think any molecular changes in follicles and lymph vessels, are downstream of the basic pressure mechanism. We have to remember that it is basic pressure changes for whatever reason, that link hair growth/loss changes according to the whole body of evidence.

 

[IDW2BB]

http://www.thefreelibrary.com/Anato...ystem--key+to+lymphatic+drainage.-a0161013430

Deep Lymphostasis

Lymphostasis can occur anywhere in the deep system, leading to serious problems (1). When the superficial vessels are overloaded, normal drainage pathways become congested, causing backflow to occur. The deep channels are affected (1). This system is so extensive, with deep lymphatic nodes and vessels situated so deeply in the body, that it is evident that exercises and outside pressure alone cannot clear all the congestion that often occurs at particular points or areas (4).

Deep breathing helps, inhalation decreases the pressure in the chest sucking lymph flow along. It is squeezed forward on exhalation (5). Unfortunately it is not enough to clear all deep lymphostasis. There are instances when stimulation of the superficial system has to be relied upon, when the deep lymphatic system is blocked (1).

Most techniques currently used predominantly access the superficial system. The skin and subcutaneous tissue are the areas that directly respond to complex physical therapy (CPT) (1).

 

[S Foote.]

http://www.thefreelibrary.com/Anato...ystem--key+to+lymphatic+drainage.-a0161013430

Thanks for that, interesting read. Demonstrates that others recognise the principle of lymph back flow.

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I think these two diagrams tell a story of androgen related hair growth, and the lymphatic system. These are two of the pics I could not use directly in my paper for copyright reasons.

 

[IDW2BB]

S Foote, I would like your opinion on the following studies once you get the time:



http://www.pnas.org/content/110/51/E4950.long

Mechanistic Insights Underlying the Age-Related Decline in HFSC Activity.
Tracing mechanism, we were led to BMP-regulated Nfatc1 and NFATc1 activity as a key contributor to the enhanced internal threshold that aging HFSCs display when challenged with anagen cues. By selectively forcing Nfatc1 down-regulation (by blocking BMP activity) and/or NFATc1 (by blocking NFATc1 transcriptional activity), we provided compelling evidence that failure to properly down-regulate Nfatc1/NFATc1 during HFSC activation delays entry of aging HFs into the hair cycle. Collectively, these changes render aging HFSCs less competent to respond to anagen cues and proliferate. Although not sufficient on its own, local environment does contribute to this response, as BMP levels are elevated in the surrounding adipose tissue of aged mice. This imposes a higher environmental threshold for the activation of aged HFSCs, as revealed by our hair-plucking experiments. Overall, the outcome is a less coordinated, asynchronous anagen among neighboring HFs, a lethargic response of HFSCs to anagen activation cues and a thinning of hair density with age.

http://www.ncbi.nlm.nih.gov/pubmed/19233265

excerpt:

Abstract

NFATc1 transcription factor is critical for lineage selection in T-cell differentiation, cardiac valve morphogenesis and osteoclastogenesis. We identified a role for calcineurin-NFAT signaling in lymphatic development and patterning. NFATc1 was colocalized with lymphatic markers Prox-1, VEGFR-3 and podoplanin on cardinal vein as lymphatic endothelial cells (LEC) are specified and as they segregate into lymph sacs and mature lymphatics. In NFATc1 null mice, Prox-1, VEGFR-3 and podoplanin positive endothelial cells sprouted from the cardinal vein at E11.5, but poorly coalesced into lymph sacs. NFAT activation requires the phosphatase calcineurin. Embryos treated in utero with the calcineurin inhibitor cyclosporine-A showed cytoplasmic NFATc1, diminished podoplanin and FGFR-3 expression by the lymphatics and irregular patterning of the LEC sprouts coming off the jugular lymph sac, which suggests a role for calcineurin-NFAT signaling in lymphatic patterning. In a murine model of injury-induced lymphangiogenesis, NFATc1 was expressed on the neolymphatics induced by lung-specific overexpression of VEGF-A. Mice lacking the calcineurin Abeta regulatory subunit, with diminished nuclear NFAT, failed to respond to VEGF-A with increased lymphangiogenesis. In vitro, endogenous and VEGF-A-induced VEGFR-3 and podoplanin expression by human microvascular endothelial cells was reduced by siRNA to NFATc1, to levels comparable to reductions seen with siRNA to Prox-1. In reporter assays, NFATc1 activated lymphatic specific gene promoters. These results demonstrate the role of calcineurin-NFAT pathway in lymphangiogenesis and suggest that NFATc1 is the principle NFAT involved

both are free access.

 

[IDW2BB]

http://www.ncbi.nlm.nih.gov/pubmed/24517869

Abstract

BACKGROUND:

Cilostazol, an inhibitor of phosphodiesterase type III, is an antiplatelet agent and vasodilator. Some clinical reports have suggested that this drug can improve progressive and refractory lymphedema.

OBJECTIVE:

In this study, we investigated whether cilostazol has the potential to proliferate lymphatic vessels and to improve lymphatic function using human lymphatic endothelial cells (LECs) and mouse lymphedema models.

METHODS:

Human LECs were counted at several time points while they were cultured in the presence of cilostazol and/or protein kinase A inhibitor. After receiving a diet including 0.1% cilostazol or control diet, skin tissue and lymphatic function of k-cyclin transgenic (kCYC+/-) mice, which have pernicious lymphatic dysfunction, was analyzed. A different lymphedema model was generated in wild type mice by excising circumferential tail skin to remove the superficial lymphatics. After oral administration of cilostazol, tail lymphedema was examined in this mouse model.

RESULTS:

Proliferation of LECs was promoted in a dose-dependent manner, which was partially inhibited by a protein kinase A inhibitor. Lymphatic vessel count increased in the cilostazol-treated kCYC+/- mice over that in the non-treated mice. Lymph flow improved in cilostazol-fed kCYC+/- mice as assessed by subcutaneous injection of Evans blue dye into the footpad. Oral administration of cilostazol also decreased lymphedema in a tail of wild type mice.

CONCLUSION:

Cilostazol promoted growth of human LECs and improved lymph flow and lymphedema in two different mouse lymphedema models. These results suggest that cilostazol would be a promising agent for the treatment of lymphedema.

This comes in a topical I believe?

 

[IDW2BB]

A few free articles regarding this discussion. Looks like Lymph is BAD for immune privilege.


http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3959614/

Like other scientists, immunologists use two types of approaches to research: one reduces the problem to its parts; the other studies the emergent phenomenon produced by the parts. Scientists that reduce the problem to its parts are sometimes called reductionists. The conclusions of reductionist experiments are often applied to the greater whole, when in actuality they may only apply to that particular experimental set. We, reductionists, are the ones who think our immune behavior exists solely because of genes, the presence of TGFβ, the presence of inflammatory cytokines, and appearance of a receptor. We tend to interpret the outcome in the colors of our interests (ergo “to a hammer, everything is a nailâ€). We measure parts and from the parts, we draw parallels to far-removed outcomes in terms of health and disease. More often than not, however, the results from the study of the parts do not predict the whole, and often the whole becomes greater than the sum of its parts. For example, a toll-like receptor does one thing when there is a bacterial invasion but the same toll-like receptor may lead to a different outcome when activated by danger signals during injury.

The approach that is perhaps more applicable to biology, and more specifically, to immunology, is the chaos theory. The chaos theory deals with the multiple layers of conditions and unexpected turns and restarts that can effect the outcome. It is applicable to studies of dynamic systems like the weather, and in our case, dynamic biological/immunological systems. The chaos theory points out that small differences in initial conditions make it impossible to predict the outcome. Thus, the behavior of the parts does not make the outcome predictable.

Our point? Immune privilege is broadly understood as the ability of the tissue to actively regulate and direct immune responses that take place in its “territory.†The articles in this Research Topic in Frontiers in Immunology, “Good news–bad newsâ€, support the idea that to understand how immune privilege works, one has to understand the dynamics of the different tissues in terms of initiation, expression, regulation, and behavior, before one can begin to predict an outcome.

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3954811/

Corneal immune privilege

The concept of corneal immune privilege is entertained by observations of high survival rates of allogeneic corneal grafts despite HLA mismatching between donor and host tissues. It is the uniquely configured corneal anatomy and physiology of the anterior chamber that evade a host immune response through low immunogenicity and generation of alloantigen tolerance [18,19,43]. The cornea is a uniquely avascular tissue and free of lymphatics, preventing direct access of the immune system to the cornea through lack of vasculature, and barring free transport of antigens and APCs to T cell-rich secondary lymphoid organs through absence of lymph vessels. Further, all layers of the cornea have low constitutive expression of MHC-I and –II antigens, limiting immunogenicity to foreign antigens. Even though DCs are present both in the central and peripheral cornea, they exist in an immature, inactivated state, maintaining immune quiescence in a healthy cornea. The cornea expresses many cell membrane-bound molecules that guard the cornea from immune-mediated inflammation and induce apoptosis of immune effector cells. These molecules include complement regulatory proteins (CRP), Fas ligand (FasL), MHC-Ib and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL). FasL (CD95L), a pro-apoptotic molecule, is expressed by the corneal epithelium and endothelium. FasL serves to destroy polymorphonuclear neutrophils (PMNs) and effector T cells that express its receptor Fas/CD95, promoting immune quiescence while protecting against immune-mediated graft rejection [44,45]. The corneal epithelium, stroma and cells of the ciliary body also express programmed death ligand-1 (PD-L1), which upon interaction with its cognate receptor (PD-1) on T cells leads to inhibition of T cell proliferative capacity, induction of apoptosis and suppression of IFN-γ secretion [46], promoting graft survival [47,48]. Expression of PD-1 by T cells is regulated by Notch signaling [49]. PD-1 inhibits T cell proliferation though suppression of Ras and Akt signaling pathways which inhibit transcription of SKP2 leading to upregulation of transforming growth factor-beta (TGF-β)-specific transcription factor Smad3, resulting in cell cycle arrest of T cells [50].

The anterior chamber is rich in soluble immunosuppressive factors such as TGF-β, alpha-melanocyte stimulating hormone (α-MSH), calcitonin gene-related peptide (CGRP), CRP, somatostatin (SOM), indoleamine dioxygenase (IDO), vasointestinal peptide (VIP) and macrophage migration inhibitory factor (MIF), which inhibit T cell and complement activation [18,51]. The most notable contribution is that of anterior chamber-associated immune deviation (ACAID), an alloantigen-specific peripheral immune tolerance to antigens in the anterior chamber, capable of deviating the systemic cytotoxic immune response [52,53]. ACAID suppresses delayed-type hypersensitivity (DTH) response and maintains humoral immunity, promoting graft survival [54,55]. Antigens within the anterior chamber are recognized and processed by F4/80+ APCs that orchestrate allotolerance by upregulating the expression of TGF-β with downregulation of the co-stimulatory molecule CD40/CD40L and interleukin-12 (IL-12) [53,56,57]. Suppression of DTH is brought about by migration of these APCs to the spleen through vascular elements in the trabecular meshwork, and together with splenic accessory immune cells, alloantigen-specific tolerance is achieved [58–60]. While the effect of ACAID on DTH is unequivocal, its impact on the regulation of cytotoxic T lymphocytes (CTL) is more complex, being dictated by the nature of the antigens present in the anterior chamber. Some groups have demonstrated that CTL function remains intact through expression of CTL precursors and effectors in the spleen and lymph nodes of animals inoculated with tumor cells in vitro [60,61], making the hypothesis of a suppressed CTL response an unlikely explanation for tumor growth in ACAID. In contrast, other groups that used a soluble antigen for intracameral inoculation observed inhibition of antigen-specific CD8+ T cell responses, confirming the antigen-dependent effect of ACAID on CTL [62–66]. Since CTL responses contribute to allogeneic corneal graft rejection even though they are not known to be directed against MHC alloantigens [67–70], the effect of ACAID on CTL function against MHC antigens and the involvement of FoxP3+ regulatory T cells (Treg) in modulating CD8+ T cell function during ACAID have been explored [71]. Both CD4+ T and CD8+ T cell populations in the spleen proliferate upon MHC alloantigen-specific ACAID induction, however, once ACAID is expressed, the percentages of these T cells decrease substantially, suggesting ACAID-mediated inhibition of both CD4+ T and CD8+ T cell function. Therefore, we now know that solubility of the antigen is not a necessary determinant of ACAID-mediated CTL immune suppression. Therefore measures to promote ACAID-mediated inhibition of DTH and CTL could prove beneficial in prolonging graft survival. Interestingly, while FoxP3+ regulatory T cells (Treg) increase upon ACAID induction, they have not been shown to be directly involved in the modulation of ACAID-mediated MHC alloantigen-specific T cell function and response [71].


Immunology of Corneal Graft Rejection

Corneal graft rejection occurs when the host immune response is directed toward antigens in the donor corneal button, leading to tissue destruction brought about by cells and mediators of the innate and adaptive immune responses. An immune response may target any of the main layers of the cornea selectively, or, in combination. Compromise of the corneal epithelium and stroma may be reversible, but, rejection of the endothelium invariably results in irreversible endothelial cells loss and may result in permanent graft failure, if not treated judiciously [72]. Sensitization of the host to donor antigens forms the “afferent†arm, also known as the induction phase of corneal allograft rejection. This allorecognition process is orchestrated by APCs presenting donor antigens to naïve T cells in draining lymph nodes in either a direct or indirect fashion [18]. The direct pathway constitutes presentation of donor antigens to naïve T cells directly by donor APCs through non-self MHC-II recognition on their surface, resulting in proliferation of direct alloreactive T effector cells [19]. In contrast, the indirect pathway yields donor antigens to host APCs that travel the cornea, capture donor antigens, and transport them to draining lymph nodes where antigen presentation occurs through recognition of self MHC-II by naïve T cells [19]. While initially believed to be a phenomenon brought about exclusively by the indirect pathway [67], accumulated evidence indicates that both the direct and indirect pathways are implicated in the immune-mediated rejection of orthotopic corneal allografts, especially in high-risk corneal beds with higher immunogenicity and compromised immune privilege [73–78]. The cornea harbors resident populations of the most potent bone marrow-derived epithelial and stromal APCs [22,79], namely, DCs, which are pivotal to the modulation of corneal immunogenicity [80]. These resident DCs are uniformly immature and MHC-II low/negative in the corneal center, but with a change in the microenvironment of the cornea from a quiescent to an inflammatory state, as in corneal transplantation, they express MHC-II and other co-stimulatory molecules, as well as increase in density [22,79,81,82]. More recently additional subpopulations of corneal DCs have been identified, adding to the complexity of the corneal immune system [83–86]. Once DCs undergo maturation, they express co-stimulatory molecules such as CD80, CD86 and CD40 [81], as well as differential adhesion molecules, that activate T cell receptors and induce T cell proliferation through concurrent release of cytokines. Among such cytokines are IL-1, -6 and -12 released by the APCs [80].

Lymph nodes serve as the priming hub for T cell allosensitization and activation, which then drives the subsequent “efferent†arm, or the expression phase, of immune-mediated graft rejection. It is this phase that results in the actual destruction of the graft, making lymph nodes profoundly critical to the process of rejection [87]. In support of the importance of draining lymph nodes in the rejection process, several murine studies have demonstrated that cervical lymphadenectomy prior to orthotopic corneal transplantation yields near complete graft acceptance along with suppressed allospecific DTH response, regardless of the pre-operative risk [77,88]. Following sensitization and activation of naïve T cells, cytokines and chemokines released induce proliferation and trafficking of these alloreactive T cells to the cornea through expression of specific combinations of adhesion molecules [89]. Chemokines (chemotactic cytokines), are small-molecule-weight cytokines that modulate recruitment of leukocytes and immune cells to the inflamed cornea [80,89]. Immune-mediated damage to the graft begins with the release of cytokines, such as tumor necrosis factor-alpha (TNF-α) and IL-1, secondary to the mechanical trauma of surgery. In the setting of high-risk corneal transplantation, cytokines further induce the production of various early chemokines. Overexpression of chemokines monocyte chemotactic protein-1 (MCP-1), chemokine C-C motif ligand 2 (CCL2), regulated on activation normal T cell expressed and secreted (RANTES; CCL5), macrophage inflammatory protein (MIP), MIP-1α (CCL3) and MIP-1β (CCL4) in acute graft rejection leads to additional recruitment of APCs and T cells into the cornea [18,90–92].

Once the graft and infiltrating leukocytes release late chemokines, guidance of alloreactive T cells towards the graft begins [19,93]. Alloreactive T cells then migrate to the cornea where they recognize donor MHC antigens, and also induce the development of memory T cells so that an immune response may be mounted against the same antigens upon re-exposure as in the case of a re-graft [19]. The primary cellular mediators of graft rejection are CD8+ CTL, and CD4+ T-helper (Th) lymphocytes, otherwise known as DTH cells. Even though the role of CTL in corneal graft rejection remains somewhat controversial, they are believed to be sufficient but not necessary for corneal graft rejection [69,94]. Based on the types of cytokines secreted by Th lymphocytes, T cells can be further classified into Th1, Th2 and the more recently discovered Th17 cells [95,96]. Th1 cells are largely considered to be the primary effector cells in corneal graft rejection [19,97]. CD4+ Th1 cells secrete IL-2, IFN-gamma and lymphotoxin, which lead to inflammation as an attack on the inciting antigen. IL-2 is critical to a sustained immune response by its positive feedback on T and B cell activation and proliferation. IFN-γ ensures that macrophages are activated at the site of inflammation, and facilitates further expression of MHC-II antigens in the donor button. Th17 cells on the other hand secrete IL-17, IL-21 and IL-22 [98]. TGF-β is the key differentiation factor for Th17 cells, which acts in concert with IL-6 or IL-21 and IL-23 serves as a stabilizing factor for maintaining the Th17 lineage [99–103]. IL-1 signaling also comes into play via IL-1β-mediated regulation of the dendritic cell-mediated Th17 cell differentiation pathways and maintenance of cytokine expression in Th17 cells [104]. Interestingly, TGF-β is also an inducer of CD4+CD25+Foxp3+ Tregs [105]. Therefore, generation of Th17 cells or CD4+CD25+Foxp3+ Tregs is largely dictated by the cytokine milieu of the tissue microenvironment [99,106,107]. Murine studies demonstrate increased expression of Th17 cells in the early stages of corneal allograft rejection followed by predominance of a Th1 response in the late stage [108]. However, Th17 do not orchestrate the corneal immune response in graft rejection independently. The role of IL-17 in graft rejection is somewhat limited. While some studies using monoclonal antibodies against IL-17 successfully demonstrate a moderate increase in murine corneal allograft survival [109], IL-17 knockout studies in mice failed to show improved graft survival [108]. This has been postulated to be as a result of an emerging Th2 response, which then mediates graft rejection [109,110]. However, there still remains controversy regarding clearly defined pathways given the complex interplay of immune cells in corneal allograft rejection. Murine IFN-γ and IL-17 knockout studies have shown that even MHC-matched corneal allografts are rejected in an IFN-γ- and IL-17-independent manner, suggesting mediators other than the simplistic model of Th1, Th2 or Th17 pathways [111].

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3954555/

Discussion.

PG analogs are the most frequently used topical ocular hypotensive medications for their apparently good safety/efficacy profile [11]. However, the generally good hypotensive outcomes have tended to overshadow the significant number of patients who undergo ocular surface discomfort. Therefore, prophylaxis and treatment of ocular surface toxic reactions associated with commercial PG analogs have become important clinical issues. In the present study, we utilized a clinically relevant animal model to examine the corneal alterations following exposure to commercial solution of latanoprost, travoprost and bimatoprost.

We have now shown that optical application of commercial PG analogs can quickly break down the barrier integrity of corneal epithelium without causing significant changes in aqueous tear production, corneal fluorescein and Rose bengal scores, and BUT. The findings indicated that the barrier function of corneal epithelium is very sensitive to the toxicity of commercial PG analogs. Kusano et al [17] recently developed an in vivo corneal TER measurement system that can be used to measure the barrier function of the intact fresh cornea in live rabbits. Nakagawa et al [14] reported that exposure to commercial PG analogs induced the disruption of the barrier function of stratified cultivated human corneal epithelial cell sheets. Our study extends these findings and confirms that in vivo toxic effect of commercial PG analogs on corneal epithelial barrier function is dependent on BAK concentrations. We found that commercial latanoprost (containing 0.02% BAK) has less toxic effects on the barrier function than 0.02% BAK. In conjunctival cell culture, the active component of PG analogs produced a marked reduction of various inflammatory cytokines [18], [19], and were responsible for protective effects against BAK toxicity by their antioxidative properties [20]. We data suggest possible protective effect of the active component latanoprost on corneal epithelium from barrier function disruption induced by BAK.

Corneal epithelium serves as a functional barrier between the external and internal ocular environment. Both tight and adherens junctions contribute to the establishment and maintenance of this barrier. ZO-1 is expressed in the superficial cell layer of the corneal epithelium and has been considered a marker of the tight junction [16], [21]. Recently, we found that topical application of BAK results in the redistribution of ZO-1 and in the disruption of the barrier function of corneal epithelium [15], [16]. In this study, we found that control eyes exhibited a continuous linear pattern of tight junction proteins staining at cell-cell boundaries in the surface of the rabbit corneal epithelium in vivo. In the eyes treated with commercial PG analogs, ZO-1 and occludin immunoreactivity was discontinuous, suggestive of disruption of tight junction. We noted that commercial PG analogs induced dispersion of ZO-1 and occludin from the interfaces of neighboring corneal superficial epithelial cells without affecting the localization of E-cadherin and β-catenin on day 5. These findings indicate that topical application of commercial PG analogs can disrupt the barrier integrity of the cornea as a consequence of dispersion of TJ proteins from their normal locus at superficial epithelial layer. We also noted that commercial PG analogs induced dispersion of E-cadherin and β-catenin at the superficial layer of the corneal epithelium on day 30. These findings suggested that the long-term use of commercial PG analogs induces the disruption of AJs between superficial cells in the rabbit corneal epithelium in vivo.

Both tight and adherens junctions are associated with the actin cytoskeleton, which plays an important role in the development and maturation of intercellular adhesion. It has been demonstrated that BAK can induce contraction of cortical actin filaments at junctional structures, which results in disruption of corneal epithelial cell barrier function [22]. In this study, we found that in commercial PG analogs treated eyes, F-actin immunoreactivity was patchy in the superficial epithelium. Our study has demonstrated that commercial PG analogs break down the barrier integrity of the corneal epithelium, concomitant with the disruption of cell junction and actin cytoskeleton between superficial cells in the rabbit corneal epithelium in vivo.

Chinnery et al. reported that the corneal stroma of normal mouse is endowed with a small number of long (>300 μm) and fine (<0.8 μm) membrane nanotube-like structures with expression of histocompatibility complex class II molecules [23]. They showed that the frequency of these nanotubes was significantly increased in corneas subjected to trauma and LPS, suggesting that these structures play an important role in vivo in cell-cell communication between widely spaced dendritic cells during inflammation [23]. We have previously demonstrated existence of long and hypereflective membrane bridge-like structures in intact rabbit corneal anterior stroma [15]. In this study, we noted that 5 days after application of commercial bimatoprost, the number of membrane bridge-like structures in central anterior stroma increased from 17.2±1.5 per square millimeter to 35±6.6 per square millimeter. This finding suggests that these membrane bridge-like structures have an important role in vivo in commercial bimatoprost induced corneal stromal inflammation. We also noted that the density of these structures was less increased in the eyes treated with commercial travoprost, latanoprost and 0.02% BAK. Guenoun et al. investigated the effect of commercial PG analogs on conjunctiva-derived epithelial cells [18]. They found that although bimatoprost contains less BAK that do travoprost and latanoprost, the expression levels of various inflammatory cytokines were higher than those obtained with two other PG analogs [18]. Our results support this observation.

We investigated the effect of commercial PG analogs on cell proliferation and apoptosis of rabbit corneal epithelium. It has been shown that in 3D-reconstituted corneal epithelium, the number of apoptotic and proliferative cells increased following exposure to BAK containing commercial PG analogs [24]. Our study provides direct evidence that commercial PG analogs can induce corneal epithelial cell apoptosis in a BAK-concentration-dependent manner without affecting cell proliferation in vivo.

A significant difference between the commercial PG analogs is the concentration of the preservative BAK. BAK can affect the tear film in vivo due to its detergent effect [25]. In this study, we found that after commercial PG analogs treatment dry eye symptoms occurred only in the eyes treated with latanoprost, which contains the highest concentration of BAK. This finding is agreement with a recent study reporting significant decreases in Schirmer score and goblet cell density and increase in fluorescein score in rabbits after 30 days treatment of commercial latanoprost [26]. Our group has proven that the reduced aqueous tear production in eyes treated with BAK is a consequence of corneal nerve damage [27]. Further studies are needed to determine the effect of commercial PG analogs on corneal nerves.