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PURPOSE. The purpose of this study was to test the role of substance P (SP) and its receptor neurokinin 1 (NK1R) on ocular surface pain. METHODS. Eight-week-old C57BL6/N (wild type [WT]) and B6.Cg-Tac1tm1Bbm/J (TAC1-KO) male mice were used. 5 M NaCl was topically applied on the cornea, followed by topical fosaprepitant 2, 10, and 50 mg/mL; 4 mg/mL oxybuprocaine chloride, or 0.1% diclofenac. Th eye wiping test was used to quantify ocular surface pain. SP content was quantified in the tear fluid and trigeminal ganglia (TG), and TAC1 mRNA was assessed in the cornea. Corneas were immunostained for β3-tubulin and NK1R, or CD45, to quantify leukocyte infiltration. RESULTS. TAC1-KO mice displayed a significant reduction of ocular pain (P < 0.001). Similarly, a single dose of 10 or 50 mg/mL fosaprepitant applied topically to WT mice reduced ocular pain as compared to vehicle (P < 0.001). Fosaprepitant 2 mg/mL, instead, induced corneal analgesia only when it was administered for 10 days, 6 times/day (P < 0.05). Diclofenac or oxybuprocaine reduced corneal nociception when compared to vehicle or fosaprepitant (P < 0.05). Fosaprepitant or oxybuprocaine groups showed lower SP content in tear secretions and TG (P < 0.05), and reduction in TAC1 mRNA (P < 0.05), and leukocyte infiltration (P < 0.05) in the cornea. Colocalization of NK1R and β3-tubulin was detected in mouse corneas. CONCLUSIONS. Topical administration of the NK1R antagonist fosaprepitant effectively reduces ocular surface nociception by decreasing SP release in the tear fluid and TG, and corneal leukocyte infiltration. Fosaprepitant repurposing shows promise for the treatment of ocular pain.

Purpose Aging impairs corneal nerve density and sensitivity. Substance P (SP), a neuropeptide secreted by sensory nerves, regulates nerve morphology and nociception. Here, we investigate the relationship between aging, nerve morphology, and SP expression in mouse and human corneas. Methods SP levels in mouse corneas (wild type and substance P-knockout) and human corneas and tears were quantified with an ELISA assay. Corneal total nerve length (TNL) was measured with whole-mount β3-tubulin immunofluorescence in mouse and in vivo laser corneal confocal microscopy in humans. SP and β3-tubulin stained cross-sections were used to assess the colocalization of SP and nerves in human and mouse corneas. Ocular surface nociception was assessed with a wiping test in mice. Results SP colocalizes with sub-basal neurons in mice and humans. In WT mice, SP levels decrease with age (P = 0.0045, 8 vs. 52 weeks; P = 0.004, 26 vs. 52 weeks) as well as TNL (P = 0.018, 8 vs. 26 weeks; P = 0.0001, 8 vs. 52 weeks). Knockout mice show a greater TNL reduction (8 vs. 26 weeks, P = 0.0016) than WT mice. In the oldest WT and age-matched KO mice, nociception is impaired (P = 0.007 and P < 0.0001, respectively), and KO mice sensitivity is restored by topical SP treatment. In humans, SP levels are reduced in old subject corneas and correlate, in tears, with age (P = 0.0368); TNL also decreases in older patients (P = 0.0002). Conclusions Age-associated corneal nerve loss is paralleled by reduction of SP expression in mice and humans. SP promotes the maintenance of normal nerve morphology in the long term and modulates nociception in the cornea.

Background & Objectives: The aim of this study was to investigate the time-course of the molecular and histological changes induced by a double intratracheal (IT) administration of bleomycin (BLM) on lung tissue. Methods: Male SD rats were injected with BLM (2 U/kg, IT) or saline on day 0 and day 4 and sacrificed at day 7, 14, 21, 28 and 56 after BLM first dose. Several inflammatory and fibrotic biomarkers were assessed in lung, BALF and plasma by colorimetric or immunometric assays. Histomorphometric analysis of lung fibrosis was performed on Masson’s trichrome stained sections. Results & Conclusions: In lungs of BLM-treated animals, several ECM proteins were significantly increased at day 7 (30-, 40- and 80-fold increase for TIMP1, PAI-1 and SPP1, respectively), and progressively declined over time, reaching control values at day 56. A significant increase in lung hydroxyproline levels was evident starting from day 14 (2.4-fold increase) and was maintained over the duration of the study. Histomorphometric evaluation revealed a sustained fibrosis during the first three weeks (fibrotic area: 50%), followed by a progressive reduction at day 28 and 56 (30%). Overall, these results indicate that the most suitable time point for assessing lung fibrosis and its targeted interventions is between 21 and 28 days post-BLM, where the fibrotic response appears to be more stable and consistent. Additional investigations, including transcriptomic and proteomic approaches will be essential to obtain a more comprehensive characterisation of the present model.

The JAK/STAT signalling plays a key role in both innate and adaptive immunity and it has been reported that JAK inhibition can reduce allergen-induced airway inflammation in different animal models of asthma. Here we studied the effects of the JAK inhibitor tofacitinib, orally administered at 15 and 30 mg/kg b.i.d., in two HDM-induced mouse models of asthma, one sensitive (HDM/Alum) and the other insensitive to inhaled steroids (HDM/CFA): modulation of inflammatory cell recruitment and cytokines in BALF along with the lung STAT3 phosphorylation, as a target engagement marker of JAK inhibition, were evaluated. In the HDM/Alum model characterised by eosinophilic inflammation and Th2 cytokine profile, tofacitinib, administered only during the HDM challenge, significantly reduced eosinophil recruitment at 30 mg/kg (41 ± 8%, p Conversely, in the HDM/CFA model, characterised by a predominant neutrophilic component and mixed Th1 and Th2 cytokine profile, tofacitinib failed to modulate inflammatory cell recruitment in BALF, even when administered during both the sensitisation and the challenge phase. Overall, these data suggest that the JAK/STAT pathway may represent a relevant target for eosinophilic allergic forms of asthma.

The cornea is normally devoid of blood and lymphatic vessels; however, a number of infectious/inflammatory diseases can induce corneal neovascularization (CNV). Tumor necrosis factor (TNF)-α, a well known pro-inflammatory cytokine, acts on the vascular endothelium by promoting vasodilatation, edema, and leukocyte recruitment, which are all commonly associated with the development of CNV. Corneal neovascularization is the second cause of blindness worldwide; hence, pharmacological TNF-α inhibition might represent an attractive therapeutic option. Although none of the existing TNF-α antagonists has been registered as a CNV inhibitor, three of them (etanercept, adalimumab, and infliximab) have been proposed to control ocular inflammation. More specifically, it has been demonstrated that infliximab is also effective in reducing hemangiogenesis and lymphangiogenesis in different animal models of CNV. In this article, we review the role of TNF-α on the ocular surface and, in particular, its specific role in the process of CNV. Moreover, we review existing literature and speculate on the potential role of TNF-α inhibitors in the treatment of CNV.

HTLV Tax proteins (Tax-1 and Tax-2) are known to be able to transactivate several host cellular genes involved in complex molecular pathways. Here, we describe a stable and regulated high-level expression model based on Tet-On system, to study the capacity of Tax-2 to transactivate host genes. In particular, the Jurkat Tet-On cell line suitable for evaluating the ability of Tax-2 to stimulate transactivation of a specific host gene, CCL3L1 (C-C motif chemokine ligand 3 like 1 gene), was selected. Then, a plasmid expressing tax-2 gene under control of a tetracycline-response element was constructed. To avoid the production of a fusion protein between the report gene and the inserted gene, a bidirectional plasmid was designed. Maximum expression and fast response time were achieved by using nucleofection technology as transfection method. After developing an optimized protocol for efficiently transferring tax-2 gene in Jurkat Tet-On cellular model and exposing transfected cells to Dox (doxycycline, a tetracycline derivate), a kinetics of tax-2 expression through TaqMan Real-time PCR assay was determined.

Purpose The aim of this study was to test the efficacy of Neurokinin-1 Receptor (NK-1R) antagonist −Fosaprepitant− in inducing regression of established corneal neovascularization (CNV). Methods Twenty C57BL/6 mice underwent alkali burn. Seven days later, when corneal neovessels had developed, they received Fosaprepitant 10 mg/ml, administered topically six times a day in the right eye for 10 days. In parallel, a group of 20 causticated mice was treated with normal saline, as control. A second independent experiment was also performed (n = 10/group). Finally, ten healthy mice received the same topical treatment for 10 days to evaluate Fosaprepitant safety. Haemangiogenesis and lymphangiogenesis were measured by means of vesselj plugin (imagej). Secondary endpoints, such as leucocyte infiltration, corneal opacity and corneal fluorescein staining were also evaluated. Inflammatory cell composition was assessed by flow cytometry. Differences between groups were assessed using unpaired t-test, Mann–Whitney U-test or two-way anova, as appropriate. Results Topical Fosaprepitant administration induced a significant reduction of (i) CD31+ blood corneal neovessels (−27%, p = 0.0132), (ii) LYVE1+ lymphatic corneal neovessels (−31%, p = 0.0118) and (iii) CD45+ leucocyte infiltration (−36%; p = 0.0237). The second independent experiment confirmed these data. Moreover, Fosaprepitant-treated corneas showed a reduction in opacity, no impairment in corneal fluorescein staining and decreased infiltration of neutrophils (−72%, p

Purpose To determine whether the inhibition of Substance P (SP) activity can reduce corneal neovascularization (CNV) by means of local administration of high-affinity, competitive, tachykinin 1 receptor (NK1R) antagonists Lanepitant and Befetupitant. Methods We performed a safety and efficacy study by using (1) two different C57BL/6 mouse models of CNV: alkali burn and sutures; (2) different concentrations; and (3) different routes of administration: topical or subconjunctival. Clinical examination endpoints, SP levels, CNV index, and leukocyte infiltration were measured. Results Substance P increased after injury in the corneal epithelium of both CNV models, and later in the suture model. Topical Lanepitant was nontoxic to the ocular surface and effective in reducing hemangiogenesis and lymphangiogenesis, corneal SP levels, and leukocyte infiltration, as soon as 4 days later in the alkali burn model. Topical Lanepitant, up to 7 days, was ineffective in the suture model. However, subconjunctival Lanepitant was effective in reducing lymphatic CNV, leukocyte infiltration, and SP levels in the suture model, after 10 days. Additionally, in the alkali burn model, subconjunctival Lanepitant significantly reduced blood CNV, corneal perforation rate, opacity, and leukocyte infiltration, and improved tear secretion. Finally, topical application of Befetupitant reduced CNV in the alkali burn model but was toxic owing to the vehicle (dimethyl sulfoxide [DMSO]); hence, Befetupitant was not tested in the suture model. Conclusions The NK1R antagonist Lanepitant is safe for the ocular surface and effective in reducing both corneal hemangiogenesis and lymphangiogenesis, and leukocyte infiltration. We suggest that inhibition of NK1R may represent an adjunctive tool in the treatment of CNV.

PURPOSE To quantify blood and lymph angiogenesis in mouse corneal flat mounts by means of a novel plug-in for ImageJ, called VesselJ, based on a dynamic threshold algorithm. METHODS Corneal neovascularization (CNV) was induced in the right corneas of 20 C57BL6/N mice by means of alkali burn (n = 10) or intrastromal sutures (n = 10). All corneal flat mounts were stained for blood vessels with CD31 and for lymphatics with LYVE1. Three independent operators measured blood and lymphatic CNV with both a published manual method (mCNV) and VesselJ (automatic method; aCNV). RESULTS Both methods showed a strong reliability, defined as intraclass correlation coefficient (ICC) > 0.90, in quantifying hemangiogenesis for sutures and alkali burn. However, reliability of lymphatic mCNV varied from moderate in alkali burn (ICC: 0.700) to poor in sutures (ICC: 0.415), whereas it remained high in aCNV (alkali ICC: 0.996; sutures ICC: 0.959). Among sutures, a significant correlation between mCNV and aCNV was found among all the three operators for blood vessels and just for one operator for lymphatic vessels (P < 0.001). In the alkali burn model, correlation between blood mCNV and aCNV was significant for all operators after excluding three noisy flat mounts (P < 0.001), whereas no significant correlation was seen for lymphatic vessels. CONCLUSIONS VesselJ is a semiautomatic, reliable, and fast method to quantify corneal hem- and lymphangiogenesis in corneal flat mounts. VesselJ can be easily used in the sutures model; it should be applied to other models (e.g., alkali burn) only after checking for background hyperfluorescence. Italian Abstract.

Purpose The purpose of this study was to investigate the role of substance P (SP) in patients affected with corneal neovascularization (CNV) and in three different Tac1-knockout (KO) murine models of CNV. Methods SP levels in tears were measured with a multiplex bead assay. The extent of human CNV was quantified as number of affected corneal quadrants. Murine CNV was induced in both strains by means of total disepithelization, alkali burn, and intrastromal sutures. After death, CNV (blood and lymphatic) and leukocyte infiltration were quantified by CD31, LYVE1, and CD45 immunofluorescence, respectively. Trigeminal ganglions were collected for quantitatitive PCR IL1β quantification. Hematoxylin-eosin corneal cross sections and whole-mounted β-3-tubulin nerve staining were used to compare anatomy and nerve density of wild-type (WT) versus Tac1-KO normal mice. Results SP tear levels correlate positively with CNV extension in patients (r = 0.49, P = 0.03). After disepithelization, Tac1-KO corneas showed reduced blood and lymphatic vascularization (-34% and -51% respectively) compared with the WT counterpart. CD45+ leukocytes infiltrating the cornea were reduced in Tac1-KO mice as opposed to WT in the disepithelization (P = 0.0001), alkali burn (P = 0.0258), and suture (P = 0.0149) models. Tac1-KO mice showed reduced IL1β expression in the trigeminal ganglion. Normal WT and Tac1-KO corneas did not show significant differences in transparency, thickness, and nerve density. Conclusions Our results suggest (1) the involvement of SP in human CNV; (2) the key role of SP in promoting inflammatory CNV in three different mouse models; and (3) that absence of SP is not associated with obvious ocular surface pathology in a KO model.

http://hdl.handle.net/20.500.11768/96868 Purpose To define proangiogenic angiopoietin 2 (ANG2) expression and role(s) in human and mouse vascularised corneas. Further, to evaluate the effect of ANG2 inhibition on corneal neovascularisation (CNV). Methods CNV was induced in FVB mice by means of intrastromal suture placement. One group of animals was sacrificed 10 days later; corneas were immunostained for ANG2 and compared with (i) mouse non-vascularised corneas and (ii) human vascularised and non-vascularised corneas. A second group of CNV animals was treated systemically with an anti-ANG2 antibody. After 10 days, the corneas were whole-mounted, stained for CD31 and LYVE1 and lymphatic/blood vessels quantified. In another set of experiments, the corneal basal Bowman membrane was either (i) removed or (ii) left in place. After 2 or 10 days the corneas were removed and immunostained for collagen IV, ANG2, CD31, LYVE1, CD11b and MRC1 markers. Results In human beings and mice, ANG2 is expressed only in the epithelium, and, mildly, in the endothelium, of the avascular cornea. Instead, it is expressed in the epithelium, endothelium and stroma of vascularised corneas. Disruption of the Bowman membrane is associated with a significant increase of (i) ANG2 stromal expression and (ii) proangiogenic macrophage infiltration in the corneal stroma. Finally, blocking ANG2 significantly reduced hemangiogenesis, lymphangiogenesis and macrophage infiltration. Conclusions Balancing proper healing and good vision is crucial in the cornea, constantly exposed to potential injuries. In this paper, we suggest the existence of a mechanism regulating the onset of inflammation (and associated CNV) depending on injury severity. ZB 0 Z8 0 ZR 0 ZS 0

Neuropeptides, and specifically Substance P (SP), can crucially contribute to the ocular inflammatory response. SP is an undecapeptide that is secreted from sensory nerve endings and from various immune cells during inflammation. SP modulates ocular inflammation through its binding with the high-affinity neurokinin-1 receptor (NK-1R). This receptor is expressed on nerves, immune cells, and epithelial cells. SP is a key mediator of neurogenic inflammation as it induces increased microvascular permeability, vasodilatation, plasma extravasation, and subsequent tissue edema. In addition, macrophages can release inflammatory mediators such as interleukins, chemokines, and growth factors in response to SP stimulation. Inhibition of SP activity, either through blockade of the neuropeptide release or the use of SP receptor antagonists, ameliorates ocular inflammation, it restores immune privilege and improves a number of clinical endpoints associated with inflammation, such as corneal opacity, ocular perforation, and angiogenesis. This review of the literature will summarize the role of SP in the ocular inflammatory response (with an emphasis on the ocular surface). In addition, it will review the therapeutic effects of SP blockade to control ocular inflammation (i) in animal models and (ii) in highly prevalent human diseases.

PURPOSE To test whether a corneal injury can stimulate inflammation in the trigeminal ganglion (TG), a structure located in the brain. METHODS At 4 and 8 days after alkali burn induced in the right eyes of mice, in vivo magnetic resonance imaging (MRI) of the brain was done before and after ultrasmall superparamagnetic iron oxide nanoparticle (USPIO) contrast to track macrophages. Trigeminal ganglia were stained for Prussian Blue and inflammatory cell markers. Interleukin-1β, TNF-α, and VEGF-A transcripts were quantified on days 1, 4, and 8, and 4 days after corneal topical anti-inflammatory treatment with 0.2% dexamethasone. The expression of Substance P and its receptor NK-1R was also measured in the TG on day 4. RESULTS Corneal alkali burn induced leukocyte infiltration, including T cells, in the right TG at 4 and 8 days. In vivo MRI showed an increased contrast uptake in the right TG, which peaked at day 8. Prussian Blue(+) USPIO(+) macrophages were observed in the right TG and exhibited an M2 phenotype. The M2-macrophage infiltration was preponderant in the TG after damage. The proinflammatory cytokines Substance P and NK-1R were significantly increased in both the TGs. The expression of IL-1β and VEGF-A was significantly reduced in the right TG with dexamethasone treatment. CONCLUSIONS We suggest, for the first time, inflammatory involvement of brain structures following ocular surface damage. Our findings support the hypothesis that the neuropeptide Substance P may be involved in the propagation of inflammation from the cornea to the TG through corneal nerves.

orneal nerves are known to con-tribute significantly to ocularsurface integrity. A normal cornealinnervation is required for a normaltear secretion and flow. In fact, dryeye disease is defined as a disorder ofthe lacrimal functional unit, an inte-grated system comprising the sensoryand motor nerves (DEWS 2007). Cor-neal nerve depletion reduces tearsecretion by decreasing reflex-inducedlacrimal secretion and by reducing theblink rate. Moreover, corneal nervessecrete up to 17 different mediatorssuch as nerve growth factor and oth-ers, which have profound effects oncorneal surface homoeostasis (Mu¨lleret al. 2003).Damage to the sensory nerves in theocular surface, particularly the cornea,is common in a number of conditions,including refractive surgery, keratopl-asty and even normal ageing (He et al.2010). Corneal nerve damage oftenoccurs in concomitance with inflam-mation elicited by surgery or drugs;hence, it is difficult to precisely dissectthe role of corneal nerves in dry eye.We recently developed a murinemodel of corneal denervation (TSE,Trigeminal Stereotactic Electrolysis)where we selectively ablate the oph-thalmic branch of the trigeminal nerve(Ferrari et al. 2011). We used thismodel to test whether and to whichextent selective sensory nerve ablationcould induce dry eye in terms of (i)tear secretion and (ii) corneal fluores-cein staining.Eight eyes of eight C57BL⁄6 micewere used. Animals were examinedbefore and 7 days after TSE. Theeffectiveness of the procedure wasconfirmed by the absence of blinkreflex (Ferrari et al. 2011). At the endof the procedure, a complete tars-orrhaphy was performed to minimizethe effect of absent blink reflex. Theanimals were examined using a biomi-croscope, and tear production wasquantified with phenol red thread test.Finally, the corneas were stained withfluorescein and scored according tothe National Eye Institute GradingSystem.Before TSE was performed, biomi-croscopy revealed a normal cornea.Corneal fluorescein staining was nega-tive before TSE (score, 0) and phenoltest was 2.59 ± 0.44 mm (mean ±SD). Corneal reflex was tested with acotton thread and found to be presentin all the eyes. Following TSE, corneafluorescein staining score was10.39 ± 2.20 (mean ± SD), phenoltest decreased to 0.52 ± 0.36 mm(mean ± SD) and corneal reflex wasabsent in all the eyes (Fig. 1). AfterTSE, we found significant differencesin both the fluorescein score (p

PURPOSE To evaluate the safety/efficacy of topical infliximab, an anti-TNF-α monoclonal antibody, in a mouse model of ocular surface scarring. METHODS Twenty alkali burn mice were treated with infliximab (10 mg/mL) topically 6 times a day, while 20 alkali burn mice received saline for 7 days. Corneal opacity, epithelial wound healing, and ocular phimosis were examined at the slit-lamp. Tear production was quantified with phenol red thread test. Immunofluorescence for infliximab penetration, TNF-α localization, CD45+ cell infiltration, PAS, and Masson's trichrome staining were evaluated on ocular globes and eyelids. TNF-α and IL-1β expression levels were measured on treated murine corneas and eyelids. Finally, quantification of corneal CD31+ blood vessels and LYVE1+ lymphatic vessels were evaluated on 10 additional alkali burn mice receiving either infliximab or saline, after 14 days. RESULTS Topical infliximab penetrated the cornea and the conjunctiva and was not toxic (negative fluorescein stain). Its molecular target, TNF-α, was detected in the cornea after injury. Infliximab significantly reduced corneal perforation, opacity index, phimosis, leukocyte infiltration, and fibrosis in the eyelids. It also significantly prevented goblet cell infiltration in epithelial cornea and loss in the conjunctiva (P < 0.05), improved tear secretion and epithelial healing (P < 0.05). Finally, it significantly reduced both corneal hem- (P < 0.05) and lymphangiogenesis (P < 0.01). CONCLUSIONS Infliximab penetrates the cornea and is safe to the ocular surface in an animal model of ocular surface scarring. We suggest that topical application of infliximab may be a useful treatment in ocular caustications.