Epithelial Nerve Research Articles

Altered corneal nerve morphology and epithelial wound healing in experimental lacrimodeficient dry eye

AbstractPurpose To analyze the morphological changes of corneal nerves and its influence on corneal epithelial wound healing in a lacrimodeficient dry eye model in the guinea‐pig.Methods Corneal nerve architecture and the rate of corneal epithelial wound healing were studied in guinea‐pigs to whom the main lacrimal gland was removed 4 weeks before (dry eye) and in control animals. Eyes were fixed, cryoprotected and incubated with neuronal class III Beta‐tubulin antibody. Epithelial migration rate (EMR) and estimated time of healing (ETH) were calculated for 2mm‐diameter epithelial corneal debridations stained with fluorescein, photographed regularly until complete closure and analyzed with image processing software.Results Density (16±6 vs 27±11 nerves/mm2) and length (115±45 vs 186±74 µm) of subbasal nerves decreased significantly in dry eye. Subbasal nerves were less branched and tortuous. Epithelial nerve terminals were also reduced. EMR decreased significantly (63±4 vs 110±1 µm/h) and ETH increased significantly (38.6±1.8 vs 20.1±0.1 h) in dry eye.Conclusion The morphological appearance and the decreased density, length and branches of corneal subbasal nerves are suggestive of corneal nerve degeneration at 4 weeks after lacrimal gland removal. Lacrimodeficient eyes presented also a slowing‐down in corneal epithelial wound healing suggesting an early trophic defect consecutive to nerve damage. (Supported by: SAF2008‐00529, CSD2007‐00023, BFU2008‐04425, BFU2009‐07793 and RETICS RD07/0062/0012 from Ministerio de Ciencia e Innovación, Spain, and the Leonardo da Vinci Lifelong Learning Program.)

Nanotechnology and the treatment of inner ear diseases.

Inner ear diseases such as sensorineural hearing loss and vertigo that cause degeneration of sensory-neural transducer epithelial cells and nerve cells are extremely common and significantly disabling conditions for which there are almost no effective remedies to date. Efforts are being made to produce agents that could prevent injury, limit its extent, and repair injury or regenerate new end organ and neural cells. These promising drugs or gene therapies may require local administration to the inner ear and even cell-specific targeting to provide adequate effective doses without systemic toxicity or side effects. Delivery of agents may require controlled or sustained release into the inner ear fluids, cellular cytoplasm, or nucleus of targeted cells. A number of classes of nanoparticles have been investigated that demonstrate the potential for accomplishing these challenging tasks. The strategies and experience to date for functionalizing nanoparticles for inner ear delivery and intracellular release of therapeutic agent into targeted cells will be reviewed in this article.

Innervation of the Mouse Cornea during Development

Dense innervation of the cornea is important for maintaining its homeostasis and transparency. Although corneal nerves have been well studied in adults, little is known about mammalian corneal innervation during development. This study provides a detailed profile of nerves at various stages of mouse cornea development. Mouse heads and corneas were collected at various stages of development including embryonic days (E)12.5 to E16.5, postnatal days (P)0, P10, three weeks after birth, and the adult. Corneas were immunostained with an anti-neuron-specific β-tubulin antibody (TUJ1). Fluorescently labeled nerves in whole-mount tissues and sections were imaged and analyzed for their axonal projections during eye development. The first nerve bundles appear at the periphery of the anterior portion of the eye by E12.5. Initial projection into the stroma occurs at E13.5 without formation of a pericorneal nerve ring. Between E13.5 and E16.5, nerve bundles project directly into the periphery of the presumptive cornea stroma. They branch repeatedly as they extend toward the cornea center and epithelium. Concomitantly, nerve bundles originating from four quadrants of the eye bifurcate into smaller branches that innervate the entire stroma. The first epithelial innervation occurs at E16.5. Epithelial nerves arrange into patterns that project toward the center subsequently forming a swirl at three weeks after birth, which becomes more pronounced in adults. Nerve bundles that arise from four quadrants of the eye innervate the mouse cornea. The nerve bundles directly innervate the stroma without forming a pericorneal nerve ring. Radial arrangement of epithelial nerves gradually becomes centrally oriented, subsequently forming a swirl pattern.

Localization of NK1 receptors and roles of substance-P in subepithelial fibroblasts of rat intestinal villi

Subepithelial fibroblasts of the intestinal villi, which form a contractile cellular network beneath the epithelium, are in close contact with epithelial cells, nerve varicosities, capillaries, smooth muscles and immune cells, and secrete extracellular matrix molecules, growth factors and cytokines, etc. Cultured subepithelial fibroblasts of the rat duodenal villi display various receptors such as endothelins, ATP, substance-P and bradykinin, and release ATP in response to mechanical stimulation. In this study, the presence of functional NK1 receptors (NK1R) was pharmacologically confirmed in primary culture by Ca(2+) measurement, and the effects of substance-P were measured in an acute preparation of epithelium-free duodenal villi from 2- to 3-week-old rats using a two-photon laser microscope. Substance-P elicited an increase in the intracellular Ca(2+) concentration and contraction of the subepithelial fibroblasts in culture and the isolated villi. The localization of NK1R and substance-P in the villi was examined by light and electron microscopic immunohistochemistry. NK1R-like immunoreactivity was intensely localized on the plasma membrane of villous subepithelial fibroblasts in 10-day- to 4-week-old rats and mice and was decreased or absent in adulthood. The pericryptal fibroblasts of the small and large intestine were NK1R immuno-negative. These villous subepithelial fibroblasts form synapse-like structures with both substance-P-immunopositive and -immunonegative nerve varicosities. Here, we propose that the mutual interaction between villous subepithelial fibroblasts and afferent neurons via substance-P and ATP plays important roles in the maturation of the structure and function of the small intestine.

Mapping the entire human corneal nerve architecture

We developed an approach to generate a three-dimensional map that facilitates the assessment of epithelial nerve density in different corneal areas to define aging and gender influence on human corneal nerve architecture. Twenty-eight fresh human eyes from 14 donors of different ages were studied. Corneal nerves were stained and consecutive images acquired with a fluorescence microscope, recorded at the same plane, and merged for viewing the complete epithelial and stromal nerve architecture. After whole mount examination, the same cornea was also used for transection. Stromal nerves entered the cornea in a radial pattern, subsequently dividing into smaller branches. Some branches connected at the center of the stroma, but most penetrated upward into the epithelium. No differences were observed between nerve densities in the four corneal quadrants. Epithelial innervation in the limbal and most of the peripheral area was supplied by a superficial network surrounding the limbal area. Central epithelial nerves were supplied by branches of the stromal nerve network. Epithelial nerve density and terminal numbers were higher in the center of the cornea, rather than the periphery. There were no differences in epithelial nerve density between genders, but there was a progressive nerve density reduction concomitant with aging, mainly in eye samples of donors 70-years of age and older. The modified technique of tissue preparation used for this study allowed for observation of new nerve structure features and, for the first time, provided a complete view of the human corneal nerve architecture. Our study reveals that aging decreases the number of central epithelial nerve terminals, and increases the presence of irregular anomalies beneath the basal layer.

“Burning Tongue” and “Burning Tip”: The Diagnostic Challenge of the Burning Mouth Syndrome

To investigate the clinical features of burning mouth syndrome (BMS) in a large cohort of patients and to correlate them with the results of tongue biopsy. We screened 98 patients complaining of oral burning pain for at least 6 months. Forty-two patients were excluded after screening for contact sensitivity to dental materials, food allergies, tongue injuries, malignancies, connective tissue and metabolic disorders, oral infectious diseases, vitamin deficiencies, and other systemic diseases known to cause neuropathy. Fifty-six patients underwent neurologic examination and assessment of pain intensity, depression, anxiety, quality of sleep, and quality of life. Tongue biopsy with the quantification of epithelial nerve fibers (ENF) was performed in 51 patients. Compared with 9 healthy participants (4.13+/-1.85 SD), epithelial innervation density was significantly reduced in 38 patients (1.35+/-1.46 SD; P<0.0001) and normal in 13 patients (6.1+/-2.19 SD). The clinical features differed in the two groups: patients with reduced ENF density complained of pain in the whole tongue, lips, hard palate, and alveolar ridges, reported dysgeusia and xerostomia in 29% of cases (P<0.001), and 24% of them were depressed. Patients with normal innervation complained of pain on the tip of the tongue, reported dysgeusia and xerostomia in 7.7% of cases, and 54% of them were depressed (P<0.017). The diagnostic criteria for BMS are not defined yet and the relationship with depression and anxiety is debated. We proposed a biopsy-supported approach for the diagnosis. Our study shows that BMS can present with two distinct clinical pictures and that tongue biopsy can contribute to the assessment of the diagnosis. Mood disorders occur frequently and should be considered when approaching patients and treatment options. These observations could help physicians in identifying patients with BMS and addressing them with the appropriate diagnostic work-up and treatment.

Neuroprotectin D1 Synthesis and Corneal Nerve Regeneration after Experimental Surgery and Treatment with PEDF plus DHA

This study was conducted to define whether pigment epithelial-derived growth factor (PEDF), together with docosahexaenoic acid (DHA), enhances the synthesis of neuroprotectin D1 (NPD1) and the regeneration of corneal nerves damaged after surgery. Corneal stromal dissection was performed in the left eyes of adult New Zealand rabbits treated with DHA+PEDF, PEDF, or DHA for 6 weeks. In vivo confocal images of the corneas were obtained at 2, 4, and 8 weeks, and nerve areas were quantified. At 8 weeks after treatment, corneas were stained with tubulin betaIII antibody, and the epithelial nerve area and the sub-basal and stromal nerve plexus were quantified. At 1 week and 2 weeks after treatment, lipids were extracted from corneas, and the synthesis of NPD1 was analyzed by mass spectrometry. Epithelial cell density was quantified by confocal microscopy 8 weeks after surgery. In vivo confocal images at 2 and 4 weeks after surgery showed a 2.5-fold increase in corneal nerve area in PEDF+DHA-treated animals compared with control animals. Increased nerve surface areas in epithelia, subepithelia, and stroma were observed in rabbits treated for 8 weeks with PEDF+DHA. PEDF or DHA alone did not produce a significant increase. NPD1 synthesis peaked at 1 week and was four times higher in the PEDF+DHA-treated group than in the controls. PEDF+DHA promotes the regeneration of corneal nerves. Neurotrophin-mediated NPD1 synthesis is suggested to precede nerve regeneration by demonstration of its accumulation upon addition of DHA and PEDF at earlier time points. Therefore, this signaling mechanism upregulates corneal nerve regeneration and may be targeted in neurotrophic keratitis, dry eye after refractive surgery, and other corneal diseases.

Herpes Zoster Radiculopathy Treated with Fluoroscopically-Guided Selective Nerve Root Injection

Background: Varicella-zoster virus, a member of the herpes virus family, is a neurotrophic virus that primarily affects afferent sensory neurons. Reactivation of latent virus within the dorsal root ganglion and axoplasmic transport to epithelial nerve terminals causes the segmental cutaneous rash and neuralgic pain characteristic of herpes zoster. Setting: Outpatient orthopedic practice. Case Description: A 75-year-old male developed a herpetic rash followed by burning pain in the right L5 distribution. The pain was exacerbated by standing or walking. Six weeks later, the rash had improved, but the patient developed a right foot drop requiring use of a molded ankle-foot orthosis. MRI of the lumbar spine revealed mild degenerative changes without evidence of significant spinal stenosis or disc disease. Electrodiagnostic studies confirmed the diagnosis of right L5 radiculopathy. Results: The patient had dramatic improvement of pain and weakness after undergoing a fluoroscopically guided right L5 selective nerve root block with Depo-Medrol and Lidocaine. He then began a course of physical therapy and, 6 weeks later, had only trace weakness of the ankle dorsiflexor group on the right side. The patient has continued without significant weakness or pain since the procedure and has returned to normal functioning. Discussion: This case demonstrates apparent treatment of a relatively uncommon phenomenon, herpes zoster radiculopathy, using selective nerve root block. Limitations: There is a limited amount of data regarding this disorder presently available regarding Herpes Zoster Radiculopathy. A second limitation would be an inability to exclude spinal pathology as an alternative etiology of this patient’s condition. Conclusion: Cases of herpes zoster-induced radiculopathy may become more frequent, as evidenced by the increasing number of cases of herpes zoster in the United States noted epidemiologically. Key words: Herpes zoster, shingles, lumbar radiculopathy, lumbar stenosis, dermatome, electrodiagnosis, selective nerve, root injection

Expression of transforming growth factor-beta (TGF-beta) in chronic idiopathic cough.

In patients with chronic idiopathic cough, there is a chronic inflammatory response together with evidence of airway wall remodelling and an increase in airway epithelial nerves expressing TRPV-1. We hypothesised that these changes could result from an increase in growth factors such as TGFβ and neurotrophins.We recruited 13 patients with persistent non-asthmatic cough despite specific treatment of associated primary cause(s), or without associated primary cause, and 19 normal non-coughing volunteers without cough as controls, who underwent fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) and bronchial biopsies.There was a significant increase in the levels of TGFβ in BAL fluid, but not of nerve growth factor(NGF) and brain-derived nerve growth factor(BDNF) compared to normal volunteers. Levels of TFGβ gene and protein expression were assessed in bronchial biopsies. mRNA expression for TGFβ was observed in laser-captured airway smooth muscle and epithelial cells, and protein expression by immunohistochemistry was increased in ASM cells in chronic cough patients, associated with an increase in nuclear expression of the transcription factor, smad 2/3. Subbasement membrane thickness was significantly higher in cough patients compared to normal subjects and there was a positive correlation between TGF-β levels in BAL and basement membrane thickening.TGFβ in the airways may be important in the airway remodelling changes observed in chronic idiopathic cough patients, that could in turn lead to activation of the cough reflex.

Structure and function of the arolium of Mantophasmatodea (Insecta)

All species of the insect order Mantophasmatodea characteristically keep the 5th tarsomere and pretarsus (arolium plus two claws) turned upwards and off the substrate. The unusually large arolium was studied in two species of Mantophasmatodea using bright field light microscopy, reflection microscopy, fluorescence microscopy, TEM, SEM, and Cryo-SEM. It contains an epithelial gland, numerous tracheoles, and nerves. The gland consists of enlarged epithelial cells with large nuclei, mitochondria, RER, golgi complexes, microtubules, and numerous secretion vesicles. Evidence for exocytosis of the vesicles into the gland reservoir between the epithelial gland and the thick cuticle could be observed. Cryo-SEM revealed that the ventral side of the arolium and distal part of its dorsal side are covered with a liquid film. Fluid footprints of arolia of individuals walking on a glass plate also indicate the presence of secretory fluid on the arolium surface. Behavioral experiments using animals with ablated arolia showed that representatives of Mantophasmatodea do not need their arolia to detect and respond to vibratory communication signals nor to catch small to medium-sized prey. Individuals with ablated arolia were not able to move upside down on a smooth glass plate. We conclude that Mantophasmatodea use their arolia for attachment when additional adhesion force is required (e.g. windy conditions, handling large prey, mating). They can bring their arolia in contact with the surface in a very fast reflex (18.0 +/- 9.9 ms). The secretory fluid found on the surface is produced by the gland and transported to the outside, presumably through small pore channels, to enhance adhesion to the substrate.

A TR(I)P to Pruritus Research: Role of TRPV3 in Inflammation and Itch

Pruritus in eczema and other inflammatory skin diseases is an unsolved problem. Recent findings strongly indicate that histamine is not the only mediator of pruritus. Subtypes of the transient receptor potential (TRP) ion channel superfamily are expressed by sensory nerves, keratinocytes, and certain leukocytes. Temperature, pH changes, and certain toxins activate TRPs. New evidence indicates that the vanilloid type 3 (TRPV3) channel is crucially involved in pruritic dermatitis, making it a good candidate for future therapy in skin inflammation and pruritus.

Control of Patterns of Corneal Innervation by Pax6

Corneal nerves play essential roles in maintaining the ocular surface through provision of neurotrophic support, but genetic control of corneal innervation is poorly understood. The possibility of a neurotrophic failure in ocular surface disease associated with heterozygosity at the Pax6 locus (aniridia-related keratopathy [ARK]) was investigated. Patterns of corneal innervation were studied during development and aging in mice with different Pax6 dosages and in chimeras. Immunohistochemistry and ELISA-based assays were used to determine the molecular basis of defects seen in Pax6 mutants, and wound healing assays were performed. In adults, the Pax6(+/-) epithelium was less densely innervated than the wild-type epithelium, and radial projection of epithelial nerves was disrupted. Neurotrophic support of the corneal epithelium appeared normal. Directed nerve projection correlated with patterns of epithelial cell migration in adult wild-types, but innervation defects observed in Pax6(+/-) mice were not fully corrected in wound healing or chimeric models where directed epithelial migration was restored. Pax6 dosage nonautonomously controls robust directed radial projection of corneal neurons, and the guidance cues for growth cone guidance are not solely dependent on directed epithelial migration. There is little evidence that ARK represents neurotrophic keratitis.

Expression of S100A6 protein in a broad spectrum of cutaneous tumors using tissue microarrays

S100A6, a calcium-binding protein in the S100 family, has been observed in melanocytic nevi, neural tumors, fibrohistiocytic tumors and is overexpressed in melanoma. Previous studies reported S100A6 expression in atypical fibroxanthomas (AFX) but not in a small number of desmoplastic melanomas (DM). Limited data on S100A6 expression in cutaneous epithelial tumors exists in the literature. The goal of this study was to determine the specificity and sensitivity of S100A6 protein in a spectrum of cutaneous mesenchymal or epithelial tumors. Tissue microarrays of cutaneous epithelial neoplasms, mesenchymal neoplasms, DM and malignant peripheral nerve sheath tumors (MPNST) were stained with S100A6 antibody. Eleven basal cell carcinomas (BCC) failed to express S100A6, whereas all 10 squamous cell carcinomas (SCC) expressed S100A6. Four of seven microcystic adnexal carcinomas (MAC) stained for S100A6. Tumors with duct differentiation variously expressed S100A6 protein, with two hidradenomas showing the strongest staining. Malignant spindle cell tumors, with the exception of 13 of 30 MPNST, had a high incidence of S100A6 positivity. S100A6 expression may distinguish SCC from BCC, MAC from BCC and hidradenoma from other adnexal tumors. S100A6 expression favors DM over MPNST but overlap limits its diagnostic use.

Overexpression of the anaphylatoxin receptors, complement anaphylatoxin 3a receptor and complement anaphylatoxin 5a receptor, in the nasal mucosa of patients with mild and severe persistent allergic rhinitis

Although the complement anaphylatoxin peptides, complement anaphylatoxin 3a (C3a) and complement anaphylatoxin 5a (C5a), are implicated in the inflammatory process in allergic rhinitis, a direct interaction between allergic mucosa and complement receptors has not been demonstrated. We investigated the expressional levels and distributional patterns of the C3a receptor (C3aR) and C5a receptor (C5aR) in normal, mild, and severe persistent allergic nasal mucosa. Immunohistochemistry and Western blotting using C3aR and C5aR antibodies were applied to normal nasal, mild, and severe persistent allergic nasal mucosa. Complement anaphylatoxin 5a receptor was detected in the inflammatory cells of normal and allergic nasal mucosa, and its expression level was significantly higher in allergic nasal mucosa than normal nasal mucosa. C3aR in normal and allergic nasal mucosa was commonly expressed in nonmyeloid cells such as epithelial cells, submucosal glands, and nerve fibers. In addition, C3aR was expressed in the endothelium of cavernous sinuses and the surrounding perivascular muscle layer in severe persistent allergic nasal mucosa, but not in normal and mild allergic nasal mucosa. Western blotting demonstrated that the expression level of C3aR was significantly increased in severe persistent allergic nasal mucosa compared with normal and mild nasal mucosa. On the basis of the location of C3aR and C5aR, C5aR may play a role in activation of inflammatory cells, whereas C3aR may mediate mucus secretion and mucosal swelling in allergic nasal mucosa, especially severe persistent allergic nasal mucosa.

Age-Related Changes in Rat Corneal Epithelial Nerve Density

To determine the effect of aging on corneal epithelial nerve density in an animal model. Corneal whole mounts from rats aged 6, 12, 18, and 24 months were stained immunohistochemically with antisera against the pan-neuronal marker neurotubulin. Epithelial nerve terminals and subbasal nerves in standardized 1-mm(2) central and peripheral zones from each cornea were drawn using a drawing tube attached to a light microscope. Images were scanned, and nerve densities were calculated as the percentage of each 1-mm(2) area occupied by nerves. The diameters of subbasal nerves in 6- and 24-month old animals were measured. Subbasal nerve vortices were analyzed qualitatively with reference to location, morphologic appearance, and directionality. Epithelial nerve terminal density decreased by approximately 50% between 6 and 24 months. The rate of decline was roughly linear and similar in both central and peripheral cornea. In contrast, subbasal nerve density increased by more than 50% between 6 and 24 months in both central and peripheral cornea. The mean diameter of corneal subbasal nerves decreased approximately 30% (0.384 microm vs. 0.271 microm) between 6 and 24 months. The morphologic appearance and directionality of the subbasal nerve vortex demonstrated considerable interanimal variability and did not correlate with age. Rat corneal nerve terminal density decreases, but corneal subbasal nerve density increases, as a function of age. The age-related loss of nerve terminal density seen in the rat cornea is in keeping with the decreased corneal sensitivity reported in elderly humans and may contribute to the pathogenesis of dry eye disease in aged persons.

In vivo Confocal Microscopy for the Evaluation of Corneal Microstructure in Keratoconus

Purpose: To evaluate the corneal microstructure in keratoconus with in vivo confocal microscopy (IVCM). Methods: Unscarred corneas of 68 patients with keratoconus and 22 controls were evaluated with slit lamp examination, corneal topography, and IVCM (Confoscan 3.0, Vigonza, Italy). One eye was randomly chosen for analysis. Keratocyte, endothelial cell and basal epithelial densities, sub-basal and stromal nerve structure, and severity of stromal haze were evaluated. Results: Compared with corneas of control subjects, patients with keratoconus had a significantly lower anterior stromal keratocyte density (1279 ± 197 cells/mm2 vs. 1132 ± 178 cells/mm2, p = 0.002), lower midstromal keratocyte density (904 ± 213 cells/mm2 vs. 770 ± 120 cells/mm2, p < 0.001), lower posterior stromal keratocyte density (935 ± 113 cells/mm2 vs. 725 ± 113 cells/mm2, p < 0.001), lower endothelial cell density (2924 ± 300 cells/mm2 vs. 2719 ± 279 cells/mm2, p = 0.004), lower basal epithelial cell density (5987 ± 699 cells/mm2 vs. 4365 ± 537 cells/mm2, p < 0.001), lower sub-basal long nerve density (32.0 ± 6.5 nerves/mm2 vs. 19.6 ± 6.5 nerves/mm2, p < 0.001), thicker sub-basal (3.2 ± 0.4 μm vs. 3.7 ± 1.1 μm, p = 0.01) and stromal nerves (5.0 ± 1.2 μm vs. 8.0 ± 2.9 μm, p < 0.001), and higher proportion of corneas with haze (40.9% vs. 92.6%, p < 0.001). A history of contact lens use (n = 12) was not associated with lower keratocyte, endothelial cell, or basal epithelial cell counts. Conclusions: Corneal microstructure is abnormal in patients with keratoconus. Keratocyte and endothelial cell loss appears to be present in keratoconic corneas.

Innervation of Tissue-Engineered Corneal Implants in a Porcine Model: A 1-Year In Vivo Confocal Microscopy Study

To examine the pattern of nerve regeneration within tissue-engineered corneal substitutes grafted into host porcine corneas over a 1-year postoperative period. Biodegradable corneal substitutes from cross-linked collagen were implanted into the left eyes of 12 pigs by deep lamellar keratoplasty. Regeneration of severed nerves into the central implant region was investigated with in vivo confocal microscopy. Both implant-recipient and control (right) eyes were examined before surgery and 2, 6, 10, and 12 months after surgery, to quantify the number, density, diameter, and branching of nerve fiber bundles at various corneal depths. Transmission electron microscopy was used to confirm the presence of nerve bundles. Two months after surgery, corneal nerve ingrowth was observed within the deep anterior stroma, with a number and density of regenerated nerves significantly higher than in nonsurgical control eyes (P < 0.01). Nerves within the superficial anterior stroma regenerated by 6 to 10 months after surgery, and the first subbasal epithelial nerves were seen 10 months after surgery. After 1 year, subbasal nerve density recovered to preoperative levels. Nerve fibers in the deep anterior stroma remained significantly thinner relative to control eyes after 1 year (P < 0.001), where both superficial anterior and subbasal nerve diameter did not change relative to control eyes. The pattern of reinnervation within tissue-engineered corneal substitutes has been quantified in vivo. Innervation proceeded rapidly in the deep anterior stroma, followed by repopulation of more superficial regions. One year after surgery, nerve density within the tissue-engineered cornea increased or remained unchanged relative to controls in all corneal regions examined.

In vivo confocal microscopy in the normal corneas of cats, dogs and birds

To evaluate the applicability of in vivo confocal microscopy (IVCM) in veterinary ophthalmology and analyze the morphology of living, healthy cornea. ANIMALS EXAMINED: Thirty-seven dogs, 34 cats and five birds. Various corneal sublayers were visualized in the central region using an in vivo confocal corneal microscope (HRTII/RCM). An investigation method was developed and adapted for use on animals with varying skull forms and eye positions. Real-time images of the epithelial cells, the corneal stroma and the endothelial layer were obtained. The corneal stromal nerve trunks and the subepithelial and basal epithelial nerve plexus were visualized. In dogs, full corneal thickness (FCT) was 585 +/- 79 microm (mean +/- SD) and endothelial cell density (ECD) 3175 +/- 776 cells/mm(2) (mean +/- SD). In cats, FCT was 592 +/- 80 microm and ECD 2846 +/- 403 cells/mm(2). There were no significant differences between canine and feline FCT and ECD and no morphologic differences could be seen between dogs and cats. The bird images revealed a number of structural differences. Noninvasive IVCM allows accurate detection of corneal sublayers, corneal pachymetry, endothelial cell density and corneal innervation in various animal species. For clinical usage, patients must be under general anesthesia. The confocal images provided anatomic reference images of various healthy corneal structures in dogs, cats and birds.

Immunohistochemical localization of a retinoic acid-like receptor in nerve cells of two colonial anthozoans (Cnidaria)

Retinoic acid is known to induce vertebrate stem cells to differentiate into a variety of cell types, including neurons. Although retinoic acid was reported to affect morphogenetic pattern specification in the hydrozoan Hydractinia (Müller, W.A., 1984. Retinoids and pattern formation in a hydroid. J. Embryol. Exp. Morph. 81, 253–271) and a retinoid RXR receptor was cloned in the jellyfish Tripedalia (Kostrouch, Z., Kostrouchova, M., Love, W., Jannini, E., Piatigorsky, J., Rall, J.E., 1998. Retinoic acid X receptor in the diploblast, Tripedalia cystophora. Proc. Natl. Acad. Sci. U.S.A. 95, 13442–13447), the cellular targets of retinoids were not investigated. We used Western immunoblotting and immunohistochemistry to investigate the presence and cellular distribution of a RXR-like receptor in the sea pansy Renilla koellikeri and in the staghorn coral Acropora millepora (Cnidaria, Anthozoa). Western blots revealed a 64 kDa protein from a sea pansy extract in a band that co-migrated with a RXR protein from the rat brain. Using antibodies raised against an epitope of human alpha RXR, we visualized putative ectodermal sensory cells in the polyp column of the adult sea pansy. Immunoreactivity was absent in staghorn coral larvae but present in the polyp column of adult colonies in the form of clusters of neuron-like cells in the basiectoderm near the ectoderm-mesoglea interface. These observations suggest that a RXR-like receptor is involved in epithelial nerve cell specification in adult anthozoans and that this role is conserved throughout evolution.

Asthma/Allergic Airways Disease: Does Postnatal Exposure to Environmental Toxicants Promote Airway Pathobiology?

The recent, dramatic increase in the incidence of childhood asthma suggests a role for environmental contaminants in the promotion of interactions between allergens and the respiratory system of young children. To establish whether exposure to an environmental stressor, ozone (O3), and an allergen, house dust mite (HDMA), during early childhood promotes remodeling of the epithelial-mesenchymal trophic unit (EMTU) of the tracheobronchial airway wall by altering postnatal development, infant rhesus monkeys were exposed to cyclic episodes of filtered air (FA), HDMA, O3, or HDMA plus O3. The following alterations in the EMTU were found after exposure to HDMA, O3, or HDMA plus O3: (1) reduced airway number; (2) hyperplasia of bronchial epithelium; (3) increased mucous cells; (4) shifts in distal airway smooth muscle bundle orientation and abundance to favor hyperreactivity; (5) interrupted postnatal basement membrane zone differentiation; (6) modified epithelial nerve fiber distribution; and (7) reorganization of the airway vascular and immune system. cyclic challenge of infants to toxic stress during postnatal lung development modifies the EMTU. This exacerbates the allergen response to favor development of intermittent airway obstruction associated with wheeze. And, exposure of infants during early postnatal lung development initiates compromises in airway growth and development that persist or worsen as growth continues, even with cessation of exposure.