TRPM8: A Therapeutic Target for Neuroinflammatory Symptoms Induced by Severe Dry Eye Disease.

Dry eye disease (DED) is commonly associated with ocular surface inflammation and pain. In this study, we evaluated the effectiveness of repeated instillations of transient receptor potential melastatin 8 (TRPM8) ion channel antagonist M8-B on a mouse model of severe DED induced by the excision of extra-orbital lacrimal and Harderian glands. M8-B was topically administered twice a day from day 7 until day 21 after surgery. Cold and mechanical corneal sensitivities and spontaneous ocular pain were monitored at day 21. Ongoing and cold-evoked ciliary nerve activities were next evaluated by electrophysiological multi-unit extracellular recording. Corneal inflammation and expression of genes related to neuropathic pain and inflammation were assessed in the trigeminal ganglion. We found that DED mice developed a cold allodynia consistent with higher TRPM8 mRNA expression in the trigeminal ganglion (TG). Chronic M8-B instillations markedly reversed both the corneal mechanical allodynia and spontaneous ocular pain commonly associated with persistent DED. M8-B instillations also diminished the sustained spontaneous and cold-evoked ciliary nerve activities observed in DED mice as well as inflammation in the cornea and TG. Overall, our study provides new insight into the effectiveness of TRPM8 blockade for alleviating corneal pain syndrome associated with severe DED, opening a new avenue for ocular pain management.

Effect of Harderian adenectomy on the statistical analyses of mouse brain imaging using positron emission tomography.

Positron emission tomography (PET) using 2-deoxy-2-[(18)F] fluoro-D-glucose (FDG) as a radioactive tracer is a useful technique for in vivo brain imaging. However, the anatomical and physiological features of the Harderian gland limit the use of FDG-PET imaging in the mouse brain. The gland shows strong FDG uptake, which in turn results in distorted PET images of the frontal brain region. The purpose of this study was to determine if a simple surgical procedure to remove the Harderian gland prior to PET imaging of mouse brains could reduce or eliminate FDG uptake. Measurement of FDG uptake in unilaterally adenectomized mice showed that the radioactive signal emitted from the intact Harderian gland distorts frontal brain region images. Spatial parametric measurement analysis demonstrated that the presence of the Harderian gland could prevent accurate assessment of brain PET imaging. Bilateral Harderian adenectomy efficiently eliminated unwanted radioactive signal spillover into the frontal brain region beginning on postoperative Day 10. Harderian adenectomy did not cause any post-operative complications during the experimental period. These findings demonstrate the benefits of performing a Harderian adenectomy prior to PET imaging of mouse brains.

Establishment of the mild, moderate and severe dry eye models using three methods in rabbits.

BACKGROUND: Dry eye (DE) is a common eye disease, and appropriate animal models are essential to explore the pathogenesis and therapy of DE. In this study, we aimed to establish rabbit models by three methods. METHODS: In group A, the lacrimal gland, Harderian gland, and nictitating membrane of the left eyes were surgically removed. In group B, the bulbar conjunctiva of the left eyes was burned with 50% trichloroacetic acid. In group C, both methods above were used. The right eyes served as normal controls. The Schirmer I test (SIt), fluorescein staining, and impression cytology were evaluated at baseline and on days 28, 42, and 56. RESULTS: Both the SIt and goblet cell density were significantly lower in operated eyes compared to the control eyes, while the corneal fluorescein staining scores in the operated eyes were significantly higher than in the control eyes on days 28, 42, and 56 (p < 0.05, p < 0.01 or p < 0.001). The SIt and goblet cell densities in groups B and C were significantly lower than group A on days 28, 42, and 56 (p < 0.05, p < 0.01 or p < 0.001). In addition, the corneal fluorescein staining scores in group C were significantly higher than either group A or group B on days 28, 42, and 56, while fluorescein staining scores were higher in group B than group A on days 42 and 56 days (p < 0.05, p < 0.01 or p < 0.001), with mean score 3.8 ± 1.30 (group A), 7.4 ± 1.14 (group B) and 10.8 ± 1.30 (group C) on day 56. CONCLUSIONS: Results suggest that three separate DE models, with mild, moderate, and severe manifestations of DE, could be stably established, in which conjunctival goblet cells took an important role.

Treatment of severe keratoconjunctivitis sicca by parotid duct transposition after tympanic neurectomy in rabbits.

PURPOSE: To investigate the feasibility of parotid duct transposition after tympanic neurectomy to treat severe keratoconjunctivitis sicca (KCS) in rabbits. METHODS: Thirty rabbits were divided into three groups in experiment 1. One eye was operated on, and the contralateral eye served as the control. In the KCS group, the lacrimal gland, harderian gland, and nictitating membrane were removed. In the group with parotid duct transposition (DT), the parotid duct was transposed into the lower conjunctival fornix. In the group with parotid duct transposition after tympanic neurectomy (DTTN), the tympanic nerve was resected in addition to parotid duct transposition. Schirmer test was performed and density of corneal staining was determined monthly after surgery, and goblet cell density was measured at postoperative month 3. In experiment 2, the tympanic nerve was resected on one side in 12 rabbits. Both sides of the parotid gland were resected for histopathology at intervals of 2 months to 1 year after surgery. RESULTS: Tear secretion from operated eyes at rest increased significantly after surgery in the treatment groups compared with the KCS group. Tear secretion from operated eyes after chewing was significantly lower in the DTTN than in the DT group. The corneal staining scores were higher in the operated than in the control eyes of the three groups, without significant difference among the operated eyes. Parotid gland atrophy on the operated side occurred at postoperative month 4 and recovered to normal 1 year after surgery. CONCLUSIONS: Parotid duct transposition after tympanic neurectomy could effectively reduce gustatory epiphora but may be insufficient to promote ocular surface health.

Establishment of a rabbit model for keratoconjunctivitis sicca.

PURPOSE: To establish a rabbit model for keratoconjunctivitis sicca (KCS) to study autologous submandibular gland transfer for treating severe KCS. METHODS: In 2 groups of 10 rabbits, left eyes were operated and right eyes were controls. In the trichloroacetic acid-treated group, the lacrimal and harderian glands and nictitating membrane were removed surgically; the palpebral and bulbar conjunctiva were swabbed with 50% trichloroacetic acid. In the non-trichloroacetic acid-treated group, the lacrimal and harderian glands and nictitating membrane were surgically removed. The Schirmer test was performed preoperatively and 1, 2, 3, and 4 months postoperatively. Corneal densities of rose bengal and fluorescein staining were scored every month postoperatively. At 4 months, the cornea and bulbar conjunctiva were removed from operated and control eyes for histopathology. The upper bulbar conjunctiva was used to determine goblet cell density. RESULTS: Compared with preoperative conditions, tear secretion of operated eyes significantly decreased in both groups postoperatively, then gradually increased. Scores for corneal rose bengal and fluorescein staining were higher and conjunctival goblet cell density was lower in the operated eyes than in control right eyes in both groups, but no significant difference was found between the operated eyes of the two groups. Inflammatory histopathologic changes of the cornea and conjunctiva were not found in either of the eyes in the two groups. CONCLUSIONS: A new rabbit model for KCS could be created by either of these methods. Experimental KCS with reduction of tear production was possible with surgical ablation of the lacrimal and harderian glands and nictitating membrane. It is unnecessary to apply trichloroacetic acid to burn the conjunctiva. Our modified incision better exposed the surgical field.

Harderian gland adenectomy: a method to eliminate confounding radio-opacity in the assessment of rat brain metabolism by 18F-fluoro-2-deoxy-D-glucose positron emission tomography.

The 18F isotope of fluoro-2-deoxy-D-glucose (FDG) is a radiotracer commonly used in positron emission tomography (PET) for determining regional metabolic activity in the brain. However, in rats and many other species with nictitating membranes, harderian glands located just behind the eyes aggressively incorporate 18F-FDG to the extent that PET images of the brain become obscured. This radioactive spillover, or 'partial volume error,' combined with the limited spatial resolution of microPET scanners (1.5 to 2 mm) may markedly reduce the ability to quantify neuronal activity in frontal brain structures. Theoretically, surgical removal of the harderian glands before 18F-FDG injection would eliminate the confounding uptake of the radioactive tracer and thereby permit visualization of glucose metabolism in the frontal brain. We conducted a pilot study of unilateral harderian gland adenectomy, leaving the contralateral gland intact for comparison. At 1 wk after surgery, each rat was injected intravenously with 18F-FDG, and 40 min later underwent brain microPET for 20 min. Review of the resulting images showed that the frontal cortex on the surgical side was defined more clearly, with only background 18F-FDG accumulation in the surgical bed. Activity in the frontal cortex on the intact side was obscured by intense accumulation of 18F-FDG in the harderian gland. By reducing partial volume error, this simple surgical procedure may become a valuable tool for visualization of the frontal cortex of rat brain by 18F-FDG microPET imaging.

Construction and evaluation of a non-laser optical system for photodynamic process excitation

Objetivo: Descrever a construção de uma fonte de luz não-laser, a partir de uma lâmpada de Tungstênio e filtros óticos adequados e demonstrar que sua eficiência em estudos fotodinâmicos, mediados por protoporfirina IX, é semelhante a do laser de Hélio Neônio. Métodos: As regiões Infravermelha e Azul do espectro de emissão óptica de uma lâmpada de Tungstênio foram convenientemente descartadas por processos de absorção, enquanto que a fração centrada na região do vermelho foi removida com o uso de um filtro de interferência. O efeito fotodinâmico foi estudado, em glândula Harderiana de ratos Wistar em razão da produção endógena de protoporfirina IX (PpIX) por estas glândulas. Foram utilizados 20 ratos. Cada animal teve as duas glândulas expostas cirurgicamente, sendo uma delas tratada com a fonte não-laser e a outra mantida como controle. Após tratamento por 30 minutos os animais foram sacrificados e suas glândulas removidas para estudo histológico. Os resultados foram comparados a estudos realizados com laser de Hélio Neônio, já publicados. Resultados: A luz produzida pelo equipamento está centrada em torno de (636 ± 6,5) nm, fornecendo uma densidade de potência de 11,3 mW/cm2. Os efeitos fotodinâmicos produzidos na glândula Harderiana, podem ser observados tanto por espectroscopia de fluorescência como por microscopia ótica. Conclusão: Não foram observadas diferenças significativas nos resultados do efeito fotodinâmico obtidos com a fonte de luz não-laser proposta, em comparação aos resultados conhecidos com o uso do laser de Hélio Neônio.

[The possible role of the harderian gland in the chemical communication of the hamster (Mesocricetus auratus Waterhouse, 1839)]. / Vozmozhnaia rol' garderovoi zhelezy v khimicheskoi kommunikatsii khomiaka (Mesocricetus auratus Waterhouse, 1839).

The hypothesis was tested on involvement of the Harderian gland in chemical communication of the Syrian hamster (Mesocricetus auratus), a species in which smelling plays a leading role in initiation of many forms of social behavior. Experiments have been carried out, in which homo- and heterospecific olfactory stimuli were presented to recipients (adult males, n = 45). When the Harderian gland homogenates from males and females of the same species were presented in microtubes, the recipient males examine the female Harderian gland homogenates reliably longer. In an "open field" chamber the males spent more time near the box with the sawdust bedding from intact females than with the sawdust bedding from males or Harderectomized females. The control box was of least interest to them. When the immobile model was presented the frequency of behavioral elements of the male recipients characteristic of the reaction to a female decreased while that of elements typical for the reaction to a male increased in a sequence: female--male with applied vaginal secretory substance--male with the applied female Harderian gland homogenate--male. When the Harderian gland homogenates from the Syrian hamster and Campbell hamster female were presented, the homospecific stimulus was examined longer. Thus, the Harderian glands of the Syrian hamster produce olfactory stimuli with an attractive effect and containing information about species and sex. Besides, the Harderian gland homogenate masks the smell of an immobile male and stimulates, to some extent, elements of sexual behavior.

A comparison of the gland of Harder response and head-associated lymphoid tissue (HALT) morphology in chickens and turkeys.

The immunofunctional response of the gland of Harder (GH) was compared in chickens and turkeys using an in vivo assay previously developed for use in chickens. The GH were surgically removed (GHx) from leghorn chicks at 1 day of age and from poults at 2 days of age. Intact birds of each species served as controls. During the fourth week of age, both GH-intact and GHx chicks were exposed to killed Brucella abortus antigen by the ocular or intraperitoneal route. One week later, serum and tears were collected and assayed for antibodies to B. abortus. In addition, all birds were killed at the end of the trial period, and the heads were fixed and processed for histologic examination. Various components of the head-associated lymphoid tissue (HALT) including the GH, nasal glands, lacrimal glands, lacrimal ducts, eyelid conjunctiva, and nasal cavity mucosa/submucosa, were evaluated microscopically using a scoring system to estimate quantity and degree of development of immune tissue in those sites. Results of all analyses indicate that functional response and morphology of the HALT are comparable in turkeys and chickens.

Role of harderian glands in resistance against Mycoplasma gallisepticum challenge.

Harderian glands of one-day-old chickens were surgically removed. At one week old, these chickens and controls from which these tissues were not removed, were vaccinated intranasally with a temperature-sensitive mutant of Mycoplasma gallisepticum. Humoral and local immunity were measured by means of antibody in sera and tracheal washings, respectively. Protection was measured by resistance to intra-air-sac challenge with the S6 strain of M gallisepticum. There was no discernible difference in either humoral or local antibody response between vaccinated chickens from which the glands had been removed and control birds. In addition, both groups were significantly protected against air-sac challenge compared with unvaccinated controls. These results indicate that removal of the Harderian glands neither affects the production of antibody to M gallisepticum, nor alters the effectiveness of temperature-sensitive M gallisepticum vaccination. The role that the Harderian glands play in resistance to M gallisepticum is therefore questioned.

The effect of Harderian adenectomy on the antibody response in chickens.

Intact chicks and those that had their glands of Harder (GH) removed (GHx) at 1 day of age were studied for their response to optically or intraperitoneally (IP) applied antigens. Following exposure of the chicks to sheep red blood cells (SRBCs), killed Brucella abortus, or bovine serum albumin (BSA), serum and tear samples were collected and assayed for antibodies. Of the two sources of antibodies, the serum generally had higher levels than did the tears. The only exception to this occurred in the intact chicks inoculated by the eye, in which serum and tear levels were equivalent. With SRBCs, no difference could be detected between the two routes of inoculation. However, IP inoculation produced higher levels of antibody in the serum of intact and GHx chicks inoculated with B. abortus or BSA and in the tears of the GHx chicks exposed to B. abortus. Removal of the GH resulted in a consistent decrease in antibody levels in the tears, regardless of the route of exposure. Although this effect was noted with all three antigens, it was more pronounced in the trials using B. abortus and BSA. This finding is discussed in terms of describing the importance of the GH as a source of antibodies to optically applied antigens, and its importance as a route of circulating antibody egress. Furthermore, the feasibility of using the antibody response in tears to a test antigen is discussed as a means of measuring the immune status of a functioning GH.

Neuropeptides and the innervation of the avian lacrimal gland.

The chicken Harderian gland, the major lacrimal gland, has two major cell populations: a cortical secretory epithelium and a medullary interstitial cell population of lymphoid cells. There is an extensive acetylcholinesterase (AChE) network throughout the gland, as well as catecholamine positive fibers among the interstitial cells. There are substance P-like (SPLI) and vasoactive intestinal polypeptide-like (VIPLI) immunoreactive fibers throughout the gland. These fibers are particularly dense and varicose among the interstitial cells. The adjacent pterygopalatine ganglion complex has neuronal somata that exhibit VIPLI and were AChE-positive. This ganglion complex also contains SPLI and catecholamine-positive fibers. In regions of the ganglion, the somata appear surrounded by SPLI varicosities. Surgical ablation of the ganglion eliminated or reduced the VIPLI, AChE and catecholamine staining in the gland. The SPLI was reduced only in some regions. Ablation of the superior cervical ganglion or severance of the radix autonomica resulted in the loss of catecholamine staining in the pterygopalatine ganglion and the gland. Severance of the ophthalmic or infraorbital nerves had no effect on the VIPLI or the SPLI staining pattern in the gland.

Experimental nasal dermatitis in the Mongolian gerbil: effect of bilateral harderian gland adenectomy on development of facial lesions.

Two groups of adult Mongolian gerbils (Meriones unguiculatus) of mixed ages and sex were used to study the effect of bilateral Harderian gland adenectomy on development of nasal dermatitis. One group of gerbils underwent bilateral Harderian gland adenectomies, while the other group underwent sham surgeries, leaving the Harderian gland intact. All animals in both groups were fitted with Elizabethian collars to prevent self-grooming, allowing a buildup of nasolacrimal or Harderian gland secretions near the medial canthus of the eye and at the external nares. Twenty-six of 27 animals with intact Harderian glands developed nasal and facial lesions within 20 days. None of the 27 Harderian gland adenectomized animals developed nasal or facial lesions. Apparently, accumulation of Harderian gland secretions is involved in the pathogenesis of nasal dermatitis in the Mongolian gerbil.

A new rabbit model for keratoconjunctivitis sicca.

The authors created a new rabbit model for keratoconjunctivitis sicca by cauterizing the lacrimal gland excretory duct and surgically removing the nictitating membrane and harderian gland. Although the slit-lamp examination findings remained normal for the first 8 wk postoperatively, tear-film osmolarity was elevated by postoperative day 1. Corneal epithelial glycogen levels declined progressively, and conjunctival goblet cell density remained decreased. Multiple controls indicated that closure of the lacrimal gland excretory duct was required for elevation of tear film osmolarity, which, in turn, was required for persistent ocular surface disease.

Lacrimal gland duct cysts.

Lacrimal gland duct cysts develop insidiously in the superotemporal cul de sac. They may be preceded by trauma, infection, or inflammation of the conjunctiva. They are often asymptomatic but may induce discomfort, a sensation of fullness, a visible mass, lid distortion or ectropion. Fluctuation in size with weeping or environmental stimuli is seen. Histopathology is variable. Six cases are described to demonstrate the features. Complete excision of each cyst by meticulous dissection through a conjunctival approach was performed. Total resection is the optimal technique to prevent recurrence.

Further observations on functional deletion of paraocular glands in the fowl (Gallus domesticus).

Methods are described for inoculation of the fowl Harderian gland with sclerosing reagent and for its surgical excision. Though very destructive the former procedure proved the less reliable in achieving the gland's complete functional deletion. Three weeks after surgical removal of the Harderian glands the lachrymal glands of 10-week-old fowls were heavier and contained more immunocompetent cells than the glands of intact and sham operated birds. When adult birds deprived of both paraocular glands were given sheep erythrocytes or Newcastle disease virus by eye drop they developed slightly higher than normal titres of serum antibody but failed to produce lachrymal antibody.

Removal of the lachrymal gland and ligation of the Harderian gland duct in the fowl (Gallus domesticus): procedures and sequelae.

Surgical methods are described for removal of the lachrymal gland and ligation of the Harderian gland duct in the fowl. Total or partial cystic degeneration of the Harderian gland and loss of immunoglobulins from lachrymal fluid was evident in three of six adult birds 10 weeks after operation. Functional deletion of these paraocular glands is thus feasible and can be used for investigations of local immunity of the oculonasal region.