A Novel Ex Vivo Blinking Method for Comparing the Corneal Residence Time of New Shampoo Formulations.

In the cosmetics sector, many products such as shampoos have a probability of accidental ocular exposure during their routine use. One very specific safety parameter is the residence time of the substance on the corneal surface, as prolonged exposure may cause injury. In this study, we developed a system that simulates corneal exposure to blinking and tear flow, for comparing the corneal clearance times of viscous detergent formulations. The Ex Vivo Eye Irritation Test (EVEIT), which uses corneal explants from discarded rabbit eyes from an abattoir, was used as the basis for the new system. To simulate blinking, we developed a silicone wiping membrane to regularly move across the corneal surface, under conditions of constant addition and aspiration of fluid, to mimic tear flow. Six shampoo formulations were tested and were shown to differ widely in their corneal clearance time. Three groups could be identified according to the observed clearance times (fast, intermediate and slow); the reference shampoo had the shortest clearance time of all tested formulations. With this new system, it is now possible to investigate an important physicochemical parameter, i.e. corneal clearance time, for the consideration of ocular safety during the development of novel cosmetic formulations.

P13-A113†The EVEIT bioreactor system as platform for artificial cornea prototypes.

PURPOSE: Corneal donor tissue is in short supply. Only a fraction of the demand is satisfied. The tissues can vary in quality and sometimes have limited use. To address the issue, the generation of artificial corneal grafts is intensively researched.Various aspects of these prototypes need to be tested, ranging from structural integrity to cellular morphology. Our Ex Vivo Eye Irritation Test (EVEIT) is based on an air-lift organ culture system, where we currently are using rabbit corneas from food industry. We constantly expanded our capabilities in quantifying various parameters concerning metabolism, structural integrity and optical properties. This also opens up the possibility of using the system as a testing platform for prototypical artificial corneal constructs. METHODS: Various ophthalmological aspects can be investigated using the EVEIT system:Self-healing of superficial injuries and morphological characteristics can be observed over several days by live-tissue staining macroscopy.Metabolic parameters are recordable via the endothelial nutrient supply mechanism.Acute changes in internal pressure can be measured in the artificial anterior chamber with high resolution.Corneal barrier functions and pharmacokinetic properties can be quantified using photometric analysis methods.Dry-Eye model and established corneal edema models can be employed to test the efficacy of potential therapeuticsAdvanced 3D design and printing methods allow us to quickly adapt the bioreactor, for example, to incorporate human corneas or to improve the mobility of the system.In order to comply with the 3Rs principle, testing of several different chemicals on one cornea is now also possible with the aid of automated multi-applicationRecent developments of the EVEIT system include the engineering of an artificial eyelid model. RESULTS: Our long experience in using and optimizing the EVEIT system led to a unique adaptability to accommodate different testing conditions and requirements. Established disease models such as corneal edema and in dry eye syndrome (in process) are involved in testing new drugs. CONCLUSION: Our established EVEIT system, in addition to its experimental capabilities, could contribute to the development of artificial corneal grafts in the future, as we have shown in previous work. The flexibility of the system allows us to adjust and improve an enormous range of test conditions and parameters.

Impact of Latanoprost Antiglaucoma Eyedrops and Their Excipients on Toxicity and Healing Characteristics in the Ex Vivo Eye Irritation Test System.

INTRODUCTION: Corneal epithelial toxicity and delayed healing process have already been attributed to preservatives or some excipients. We study the effects of galenic components in antiglaucoma drugs such as benzalkonium chloride (BAC) or surfactants like macrogolglycerol hydroxystearate 40 (MGHS 40) on corneal toxicity in an ex vivo system mimicking chronic use. METHODS: Latanoprost-containing eyedrops are available with and without preservatives on the market. Unpreserved, they are available in different formulations with various excipients like MGHS at different concentrations (0%, 2.5%, and 5%). We studied these in the ex vivo bioreactor (EVEIT) on initially injured rabbit corneas. The drugs were applied six times daily for observation periods of 3 or 5 days. BAC, 5% MGHS 40 solution, and 0.18% hyaluronic acid served as controls. Macroscopic photographic, biochemical methods and corneal integrity quantification were used for evaluation. Toxicity was assessed by measuring wound healing and corneal fluorescein sodium permeability and was confirmed by histology studies. RESULTS: The BAC-preserved formulation resulted in high corneal toxicity, which was expected. Interestingly, the preservative-free (PF) formulation containing 5% MGHS 40, carbomer, macrogol 4000, and sorbitol showed the highest corneal toxicity, followed by the control formulation with equal MGHS 40 concentration, which presented significantly less damage. No toxicity was shown by eyedrops containing 2.5% MGHS 40 or salts only. CONCLUSION: Our study demonstrates a significant corneal toxicity of certain formulations of PF antiglaucoma ophthalmic drugs containing 5% MGHS 40 with other excipients compared to other formulations with lower MGHS 40 concentrations (2.5% or 0%), or even compared to the solution containing 5% MGHS alone. This suggests a concentration-dependent toxicity of MGHS 40, especially in interaction with other excipients, which may increase its epithelial toxicity, and that has to be considered in clinical glaucoma therapy. Further single-component formulation trials are needed to support this interpretation.

Defining corneal chemical burns: A novel exact and adjustable ocular model.

INTRODUCTION: Live-animal-free ocular toxicity models and tests are a necessity in multiple branches of medicine, industry and science. Corneal models with adjustable ranges of injury severities do not exist. In this work, a novel and precise and dose - response method to induce and observe ex vivo corneal chemical burns has been established. METHODS: The EVEIT (Ex Vivo Eye Irritation Test) is based on an ex vivo corneal organ model for rabbit corneas from food industry. Further, a highly precise three - axis workstation has been employed to apply liquid corrosive, sodium hydroxide (NaOH), droplets in a nanolitre (nL) range onto the corneal surface. Optical Coherence Tomography (OCT) has been used to observe and quantify the elicited changes in the corneal layers. RESULTS: The speed and intervals of single nanodroplet application played a crucial role in the extent of the corneal changes. Similar total volumes applied at low frequencies elicited deep and extensive changes in the corneal layers whereas high application frequencies elicited comparatively superficial changes. Increasing NaOH concentrations effected measurably increasing corneal changes. Increasing the volume of applied NaOH also showed an increase in corneal changes. CONCLUSIONS: OCT imaging proved to be effective in observing, documenting and quantifying the changes in the corneal layers. The ex vivo model, in conjunction with the novel application method was able to induce and display distinctive and consistent correlations between NaOH volume, concentration and elicited corneal changes. This ex vivo ocular chemical burn model provides a consistent in vitro basis for pharmaceutical and toxicological experiments and investigations into corneal chemical burn mechanisms and treatment.

High-Frequency Application of Cationic Agents Containing Lubricant Eye Drops Causes Cumulative Corneal Toxicity in an Ex Vivo Eye Irritation Test Model.

Purpose: High-frequency applied cetalkonium chloride (CAC) and benzalkonium chloride (BAC) 0.02% did not hamper corneal healing in a living rabbit model of induced corneal erosion. In contrast, the ex vivo eye irritation test (EVEIT) shows inhibition of healing for these substances. In a systematic ex vivo reproduction of the in vivo experiments, we discuss the background of these differences. Methods: Excised rabbit corneas (n = 5 per group) were cultured in artificial anterior chambers (EVEIT). Four erosions were induced for each cornea before starting regular 21 installations/day over 3 days of (1) CAC containing eye drops (Cationorm®), (2) 0.02% BAC. Corneal fluorescein staining, quantification of glucose-/lactate consumption, and histology were performed. Results: BAC 0.02% treated corneas showed increased epithelial lesions from 10.13 ± 0.65 mm2 to 10 ± 0.8 mm2 on day 0, to 86.82 ± 5.18 mm2 (P < 0.0001) by day 3. After a trend toward smaller lesions for CAC on day 1, erosion sizes increased significantly by day 3 from 9.82 ± 0.30 mm2 to 29.51 ± 16.87 mm2 (P < 0.05). For 1 cornea, corneal erosions nearly disappeared on day 3 (0.89 mm2). Corneal lactate increased significantly for BAC and CAC, whereas glucose concentrations were unchanged. Histology revealed disintegration of the corneal structures for both compounds. Conclusions: The data underline the EVEIT as a predictive toxicity test to show side effects in a time-compressed manner. The consistency of these predictions was previously demonstrated by the EVEIT for BAC, phosphate buffer, and others. The EVEIT is suited for a chronic application prediction of tolerability and toxic side effects of eye drops in particular, and other chemicals in general.

Corneal bioprinting utilizing collagen-based bioinks and primary human keratocytes.

Corneal transplantation is the treatment of choice for patients with advanced corneal diseases. However, the outcome may be affected by graft rejection, high associated costs, surgical expertise, and most importantly the worldwide donor shortage. In recent years, bioprinting has emerged as an alternative method for fabricating tissue equivalents using autologous cells with architecture resembling the native tissue. In this study, we propose a freeform and cell-friendly drop-on-demand bioprinting strategy for creating corneal stromal 3D models as suitable implants. Corneal stromal keratocytes (CSK) were bioprinted in collagen-based bioinks as 3D biomimetic models and the geometrical outcome as well as the functionality of the bioprinted specimens were evaluated after in vitro culture. We showed that our bioprinting method is feasible to fabricate translucent corneal stromal equivalents with optical properties similar to native corneal stromal tissue, as proved by optical coherence tomography. Moreover, the bioprinted CSK were viable after the bioprinting process and maintained their native keratocyte phenotypes after 7 days in in vitro culture, as shown by immunocytochemistry. The proposed bioprinted human 3D corneal models can potentially be used clinically for patients with corneal stromal diseases.

The Ex vivo Eye Irritation Test (EVEIT) model as a mean of improving venom ophthalmia understanding.

Snakes belonging to the genus Naja (Elapid family), also known as "spitting cobras", can spit venom towards the eyes of the predator as a defensive strategy, causing painful and potentially blinding ocular envenoming. Venom ophthalmia is characterized by pain, hyperemia, blepharitis, blepharospasm and corneal erosions. Elapid venom ophthalmia is not well documented and no specific treatment exists. Furthermore, accidental ejection of venom by non-spitting vipers, as Bothrops, also occurs. The Ex vivo Eye Irritation Test model (EVEIT) has enabled important progress in the knowledge of chemical ocular burns. Considering the lack of experimental animal model, we adapted the EVEIT to study venom ophthalmia mechanisms. Ex vivo rabbit corneas were exposed to venoms from spitting (Naja mossambica, Naja nigricollis) and non-spitting (Naja naja, Bothrops jararaca and Bothrops lanceolatus) snakes, and rinsed or not with water. The corneal thickness and the depth of damage were assessed using high-resolution optical coherence tomography (HR-OCT) imaging and histological analysis. All Naja venoms induced significant corneal edema, collagen structure disorganization and epithelial necrosis. Corneas envenomed by African N. mossambica and N. nigricollis venoms were completely opaque. Opacification was not observed in corneas treated with venoms from non-spitting snakes, such as the Asian cobra, N. naja, and the vipers, B. jararaca and B. lanceolatus. Moreover, Bothrops venoms were able to damage the epithelium and cause collagen structure disorganization, but not edema. Immediate water rinsing improved corneal status, though damage and edema could still be observed. In conclusion, the present study shows that the EVEIT model was successfully adapted to set a new experimental ex vivo animal model of ophthalmia, caused by snake venoms, which will enable to explore new therapies for venom ophthalmia.

The ex vivo eye irritation test (EVEIT) model as a mean of improving venom ophthalmia understanding

Snakes belonging to the genus Naja (Elapid family), also known as "spitting cobras", can spit venom towards the eyes of the predator as a defensive strategy, causing painful and potentially blinding ocular envenoming. Venom ophthalmia is characterized by pain, hyperemia, blepharitis, blepharospasm and corneal erosions. Elapid venom ophthalmia is not well documented and no specific treatment exists. Furthermore, accidental ejection of venom by non-spitting vipers, as Bothrops, also occurs. The Ex vivo Eye Irritation Test model (EVEIT) has enabled important progress in the knowledge of chemical ocular burns. Considering the lack of experimental animal model, we adapted the EVEIT to study venom ophthalmia mechanisms. Ex vivo rabbit corneas were exposed to venoms from spitting (Naja mossambica, Naja nigricollis) and non-spitting (Naja naja, Bothrops jararaca and Bothrops lanceolatus) snakes, and rinsed or not with water. The corneal thickness and the depth of damage were assessed using high-resolution optical coherence tomography (HR-OCT) imaging and histological analysis. All Naja venoms induced significant corneal edema, collagen structure disorganization and epithelial necrosis. Corneas envenomed by African N. mossambica and N. nigricollis venoms were completely opaque. Opacification was not observed in corneas treated with venoms from non-spitting snakes, such as the Asian cobra, N. naja, and the vipers, B. jararaca and B. lanceolatus. Moreover, Bothrops venoms were able to damage the epithelium and cause collagen structure disorganization, but not edema. Immediate water rinsing improved corneal status, though damage and edema could still be observed. In conclusion, the present study shows that the EVEIT model was successfully adapted to set a new experimental ex vivo animal model of ophthalmia, caused by snake venoms, which will enable to explore new therapies for venom ophthalmia.

The Ex vivo Eye Irritation Test (EVEIT) model as a mean of improving venom ophthalmia understanding

Snakes belonging to the genus Naja (Elapid family), also known as "spitting cobras", can spit venom towards the eyes of the predator as a defensive strategy, causing painful and potentially blinding ocular envenoming. Venom ophthalmia is characterized by pain, hyperemia, blepharitis, blepharospasm and corneal erosions. Elapid venom ophthalmia is not well documented and no specific treatment exists. Furthermore, accidental ejection of venom by non-spitting vipers, as Bothrops, also occurs. The Ex vivo Eye Irritation Test model (EVEIT) has enabled important progress in the knowledge of chemical ocular burns. Considering the lack of experimental animal model, we adapted the EVEIT to study venom ophthalmia mechanisms. Ex vivo rabbit corneas were exposed to venoms from spitting (Naja mossambica, Naja nigricollis) and non-spitting (Naja naja, Bothrops jararaca and Bothrops lanceolatus) snakes, and rinsed or not with water. The corneal thickness and the depth of damage were assessed using high-resolution optical coherence tomography (HR-OCT) imaging and histological analysis. All Naja venoms induced significant corneal edema, collagen structure disorganization and epithelial necrosis. Corneas envenomed by African N. mossambica and N. nigricollis venoms were completely opaque. Opacification was not observed in corneas treated with venoms from non-spitting snakes, such as the Asian cobra, N. naja, and the vipers, B. jararaca and B. lanceolatus. Moreover, Bothrops venoms were able to damage the epithelium and cause collagen structure disorganization, but not edema. Immediate water rinsing improved corneal status, though damage and edema could still be observed. In conclusion, the present study shows that the EVEIT model was successfully adapted to set a new experimental ex vivo animal model of ophthalmia, caused by snake venoms, which will enable to explore new therapies for venom ophthalmia.

Comparison of the effects of various lubricant eye drops on the in vitro rabbit corneal healing and toxicity.

Ingredients of lubricant eye drops are potentially harmful to the ocular surface. The products Optive, Optive Fusion, Neopt were tested regarding corneal irritability versus Vismed Multi and 0.01% benzalkonium chloride as negative and positive control, respectively. Formulas (30-40µl per hour) were applied hourly in-vitro for six days on rabbit corneas (n=5, per product) cultured in artificial anterior chambers (EVEIT system). Initially, four corneal abrasions (2.4-4.6mm2) were induced. All defects were monitored during drop application by fluorescein stains and photographs. To ensure corneal vitality, glucose and lactate concentrations in artificial anterior chamber fluids were determined photometrically. All products showed a complete corneal healing on day 2. Thereafter, all five Optive-treated corneas developed progressive fluorescein-positive epithelial lesions until day six (24.96µm, ±21.45µm, p<0.01). For Optive Fusion three corneas showed corneal erosions on day six (23.11µm, ±37.02µm, p>0.5) while Vismed Multi did not adversely affect the corneal integrity. Glucose/lactate concentrations remained unchanged while lubricants were applied. Histology revealed epithelial loss and severe alterations of the superficial stroma for Optive. Optive Fusion displayed a comparable pathology. Neopt did not significantly affect the corneal healing and integrity. This study suggested a cumulative corneal toxicity of Optive and, to a lesser extent, Optive Fusion most likely caused by its oxidative preservative, SOC. Clinical data are needed to clarify the application frequency at which corneal toxicity might occur. Neopt and Vismed Multi did not affect the corneal integrity.

The Impact of Glaucoma Medications on Corneal Wound Healing.

PURPOSE: To evaluate the impact of antiglaucoma drugs on the corneal healing process and corneal toxicity. MATERIALS AND METHODS: Four eye drops to treat glaucoma--Xalatan (latanoprost 50 µg/mL; Pfizer), Monoprost (latanoprost 50 µg/mL; Théa Pharma), Taflotan Sine (tafluprost 15 µg/mL; Santen Pharmaceutical Co.), Travatan (travoprost 40 µg/mL; Alcon), and 0.02% benzalkonium chloride (BAC) solution and HyloComod (1 mg/mL sodium hyaluronate; Ursapharm) as positive and negative control were tested regarding corneal irritability and effect on corneal healing. Formulas were tested over 3 days and administered 6 times daily on rabbit corneas cultured on an artificial anterior chamber (the Ex Vivo Eye Irritation Test system). Initially, 4 corneal abrasions (2.5 to 5.7 mm2) were applied. All defects were monitored during drug application by fluorescein stains and photographs. Glucose/lactate concentrations were monitored for corneal metabolic activity evaluation. RESULTS: For Xalatan and BAC, the corneal erosion size increased from 14.65 to 66.57 mm2 and 14.80 to 87.26 mm2. Travatan and Taflotan Sine did not interfere with corneal healing. Monoprost delayed corneal healing. For Xalatan and BAC, histology showed severe alteration of the superficial cornea. An increase in anterior chamber lactate concentration indicates corneal toxicity for Xalatan, BAC, and Monoprost. CONCLUSIONS: Corneal toxicity of Xalatan is most probably caused by BAC. Monoprost delays corneal healing, which is not well understood. The Monoprost effects could be caused by its additive, macrogolglycerolhydroxystearate 40. This excipient is a known skin irritant, and its concentration is relatively elevated in Monoprost, 50 mg/mL, compared with its active ingredient, latanoprost (0.05 mg/mL).

The Ex Vivo Eye Irritation Test as an alternative test method for serious eye damage/eye irritation.

Ocular irritation testing is a common requirement for the classification, labelling and packaging of chemicals (substances and mixtures). The in vivo Draize rabbit eye test (OECD Test Guideline 405) is considered to be the regulatory reference method for the classification of chemicals according to their potential to induce eye injury. In the Draize test, chemicals are applied to rabbit eyes in vivo, and changes are monitored over time. If no damage is observed, the chemical is not categorised. Otherwise, the classification depends on the severity and reversibility of the damage. Alternative test methods have to be designed to match the classifications from the in vivo reference method. However, observation of damage reversibility is usually not possible in vitro. Within the present study, a new organotypic method based on rabbit corneas obtained from food production is demonstrated to close this gap. The Ex Vivo Eye Irritation Test (EVEIT) retains the full biochemical activity of the corneal epithelium, epithelial stem cells and endothelium. This permits the in-depth analysis of ocular chemical trauma beyond that achievable by using established in vitro methods. In particular, the EVEIT is the first test to permit the direct monitoring of recovery of all corneal layers after damage. To develop a prediction model for the EVEIT that is comparable to the GHS system, 37 reference chemicals were analysed. The experimental data were used to derive a three-level potency ranking of eye irritation and corrosion that best fits the GHS categorisation. In vivo data available in the literature were used for comparison. When compared with GHS classification predictions, the overall accuracy of the three-level potency ranking was 78%. The classification of chemicals as irritating versus non-irritating resulted in 96% sensitivity, 91% specificity and 95% accuracy.

Osmolarity of prevalent eye drops, side effects, and therapeutic approaches.

PURPOSE: Little is known about how the osmolarity of ophthalmic formulations affects the ocular surface. Because hyperosmolar eye drops could be therapeutic for treating corneal edema, this article presents an ex vivo model of corneal edema for testing ophthalmic drugs based on their osmolarity. The respective osmolarity of common eye drops found in the German market is also analyzed here. METHODS: For modeling corneal edema, an Ex Vivo Eye Irritation Test was used to simulate an ocular anterior chamber with a physiological corneal barrier. To induce corneal edema, the anterior chamber was supplied with a hypoosmolar medium (148 mOsm/L) for 24 hours. Preserved and preservative-free 5% sodium chloride (hyperosmolar Omnisorb and Ocusalin 5% UD) were used for 1 hour, on 5 corneas each, to test their efficiency to reduce corneal edema in this model. Corneal thickness was determined by optical coherence tomography. Osmolarity of 87 common eye drops was measured by freezing point osmometry. RESULTS: Ex vivo, the tested hypoosmolar condition induced corneal edema from 450 µm (±50 µm) at baseline to 851 µm (±94 µm, P < 0.0001). Omnisorb and Ocusalin 5% UD significantly reduced the corneal thickness by 279 µm (±28 µm, P < 0.001) for Omnisorb and 258 µm (±29 µm, P < 0.001) for Ocusalin 5% UD. Forty-three (49%) of the tested products had an osmolarity below and 44 (51%) above the physiological tear osmolarity of 289 mOsm/L. Osmolarity values of less than 200 mOsm/L were found in lubricant drops. The highest osmolarity was detected in Omnisorb (1955 mOsm/L). CONCLUSIONS: The Ex Vivo Eye Irritation Test has proven to be a reliable novel model of corneal edema for evaluating osmotic eye drops. Osmolarity measurements revealed a wide range from hypotonic to hypertonic formulations for commonly marketed ophthalmic drugs.

Comparison of the lubricant eyedrops Optive®, Vismed Multi®, and Cationorm® on the corneal healing process in an ex vivo model.

PURPOSE: To evaluate the impact of lubricant eyedrops on the corneal healing process and corneal toxicity. METHODS: Optive® and Cationorm® were tested regarding corneal irritability against Vismed Multi® and 0.01% benzalkonium chloride as negative and positive control, respectively. Formulas were applied on rabbit corneas (n = 5) cultured on artificial anterior chambers (EVEIT system) hourly over 3 days. Initially, 4 corneal abrasions (2-5.4 mm²) were induced. All defects were monitored during drug application by fluorescein stains and photographs. To ensure corneal vitality, glucose and lactate concentrations were determined photometrically in artificial anterior chamber fluids. Corneal fluorescein sodium permeability was tested as an indicator of the corneal barrier function. RESULTS: Optive® and Vismed Multi® showed a complete corneal healing on day 2. In one cornea (Optive®), erosion reoccurred on day 3. Erosion sizes of Cationorm®-treated corneas increased significantly from 12.20 mm² to a subtotal erosion of 51.89 mm² on day 3. Histology revealed epithelial loss and severe alterations of the superficial stroma for Cationorm®. Glucose and lactate concentrations did not change after application of Optive® and Vismed Multi®. In contrast, Cationorm®- and BAC-treated corneas showed a significant increase in lactate concentrations. CONCLUSIONS: Vismed Multi® application resulted in rapid corneal healing. Whether the toxicity seen for Optive® in one cornea is a valid result should be examined further. Cationorm® showed considerable corneal toxicity that could be caused by its additive, cetalkonium chloride. Otherwise, the electrostatic properties of Cationorm® led to a drug film on the area of epithelial loss that could hinder epithelial cell migration and adhesion in order to heal the lesion.

Adhesion of human mesenchymal stem cells can be controlled by electron beam-microstructured titanium alloy surfaces during osteogenic differentiation.

Several studies focusing on bone tissue engineering demonstrated that given microstructuring of an implant surface has a strong effect on its interaction with cells, and their adhesion and differentiation. In the present study, geometrically structured titanium alloy surfaces are shown to be able to guide cell adhesion during differentiation in vitro. For this reason, using an electron beam texturing technique, TiAl6V4 surfaces were selectively targeted in the micrometer range. The effect of such textured titanium alloy surfaces on cell adhesion during osteogenic differentiation was analyzed for human mesenchymal stem cells (MSC), the natural precursor cells of bone tissue. Cytotoxicity, cell viability and differentiation were analyzed. Immunofluorescence stainings demonstrated that in contrast to MSC in an expansion medium, MSC in an osteogenic induction medium produce adhesion proteins such as ß3-integrins and thereby connect in an oriented way to the generated microstructures on titanium alloy surfaces. These results are of relevance for developing tailored titanium alloy implant surfaces which exhibit an improved cell response.

Nerve growth factor injection into semispinal neck muscle evokes sustained facilitation of the jaw-opening reflex in anesthetized mice -- possible implications for tension-type headache.

Nociceptive input from neck muscles probably plays a role in the pathophysiology of tension-type headache. In order to elaborate an animal model, the impact of noxious input from neck muscles on orofacial sensorimotor processing was investigated by electrophysiological means in anesthetized mice. Group IV muscle afferents of the semispinal neck muscle were excited by local injection of nerve growth factor (NGF, 0.8 microM, 20 microl). Orofacial sensorimotor processing was monitored by the jaw-opening reflex (JOR) elicited by electric tongue stimulation. After unilateral NGF injection into the right neck muscle (n = 10), JOR integral (+89%) and duration (+9%) increased and latency decreased (-5%) for at least 1 h. Bilateral injection of NGF (n = 10) into neck muscles induced an increase of JOR integral (+111%) and duration (+20%) and a reduction of latency (-9%). This facilitation of the JOR lasted for at least 90 min without any downward drift (n = 5). Electric JOR threshold diminished after NGF injection. After intramuscular injection of isotonic saline into the right semispinal neck muscle (20 microl), the JOR remained unchanged (n = 10). Local NGF injection into neck muscles evoked noxious input to the brainstem that induced a sustained central facilitation of the JOR for more than 1 h. This long-term facilitation of orofacial sensorimotor processing by a singular NGF injection possibly reflects plastic changes of nociceptive synaptic processing that may be involved in the pathophysiology of headache.