Ear Of Guinea Pig Research Articles

免疫感作モルモットの内耳の電気生理学的検討

免疫感作モルモットの内耳の電気生理学的検討

Histamine involvement in the local and systemic microvascular effects produced by intradermal substance P

The cutaneous microvascular changes produced by intradermal substance P were quantitatively evaluated in both substance P-injected and contralateral, saline-injected guinea pig ears. Substance P evoked a dose-dependent increase in cutaneous microvascular permeability in both treated and untreated ears which was reduced, but not abolished, by a mepyramine-cimetidine combination. This indicates that the local effect of substance P on microvascular permeability and the effect on the contralateral ear (presumably the result of systemic substance P absorption) are both partially mediated by histamine. A cutaneous vasodilator response was also observed in substance P treated and contralateral ears, but a bell-shaped dose-response relationship was apparent. Unlike microvascular permeability, pretreatment with mepyramine and cimetidine failed to consistently attenuate the vasodilator response to substance P. Thus, a direct cutaneous vasodilator effect appears to predominate in both substance P-injected and saline-injected ears.

Long refractory period after one application of nonimmunologic contact urticaria agents to the guinea pig ear

The decrease in the swelling capacity and the length of the refractory period after nonimmunologic contact urticaria produced by one application of six human nonimmunologic contact urticaria agents was studied with the use of the guinea pig ear test. On retesting 1 day later, all substances (benzoic acid, cinnamic acid, cinnamic aldehyde, diethyl fumarate, methyl nicotinate, and dimethyl sulfoxide) showed reactions decreased by at least 50%. This decrease was most marked with cinnamic aldehyde (91% decrease), cinnamic acid (88%), and benzoic acid (86%). The tachyphylaxis was not specific to the substance producing it; reactivity to other contact urticaria agents decreased as well. The refractory period was 4 days after methyl nicotinate, 8 days after diethyl fumarate and cinnamic aldehyde, and 16 days after the other agents. These results suggest the following practical application: there is a need for (1) appropriate scheduling in the reuse of animals for testing for nonimmunologic contact urticaria and (2) an awareness of possible false-negative results in human tests for this form of urticaria because of tachyphylaxis.

Leukotriene B4 and 12-Hydroxyeicosatetraenoic Acid Stimulate Epidermal Proliferation In Vivo in the Guinea Pig

Epidermal proliferation was studied in the guinea-pig ear in vivo using the incorporation of [3H]thymidine into DNA as a measure of cell proliferation. Twenty-four-hour pretreatment of the skin topically with nanomolar amounts of either leukotriene B4 (LTB4), 12-hydroxyeicosatetraenoic acid, ionophore A23187, or the epidermal mitogen 12-O-tetradecanoylphorbol-13-acetate induced dose-dependent increases in epidermal proliferation. The stimulatory effect of LTB4 was stereospecific since a number of biologically inactive LTB4 stereoisomers had no effect.

An Evaluation of the Effectiveness of Azelaic Acid As a Depigmenting and Chemotherapeutic Agent

In the past five years, it has been reported that certain dicarboxylic acids (C8-C13) and azelaic acid (C9) (AZA), in particular, have a remarkable effect in the management of lentigo maligna, human malignant melanoma, and certain disorders of hyperpigmentation. Preclinical trials, therefore, were undertaken in order to evaluate the effectiveness of AZA as a depigmenting agent and as a chemotherapeutic agent. Twenty-seven uniformly black pigmented guinea pigs were given topical applications of various concentrations (3, 5, 10, 15, and 20%) of AZA preparations for 8 weeks, and their effects on the melanocytes of epilated skin of the backs and the nonepilated ears of guinea pigs were compared to the effects of well-known depigmenting agents. Whereas 4-isopropylcatechol, monobenzylether of hydroquinone, monoethylether of hydroquinone, hydroquinone, and 4-hydroxyanisole were found to be selectively cytotoxic to melanocytes in black-skinned guinea pigs, AZA has little or no visually recognizable effect on melanocytes in these animals. The therapeutic effect of local s.c. injections of various concentrations of AZA preparations on the development of s.c. implanted B-16 melanoma tumor was evaluated in 96 C57BL/6J mice. In addition, 31 BDF1 mice, implanted i.p. with B-16 melanoma tumor, were used to assess the effect of 100-500 mg/kg concentrations of AZA administered i.p. In both studies, AZA revealed no significant tumoristatic or tumoricidal effect on the size, color, and growth of melanoma. The effect of AZA was also evaluated on S-91A (melanotic or pigmented) and S-91B (amelanotic) human melanoma cells in culture. Low concentrations (10(-5) and 10(-3) M) of AZA had no inhibitory effect on the growth of these cells. Only at higher concentrations (greater than 10(-3) M) was a cytotoxic effect on cell viability observed. These observations indicate AZA is not selectively cytotoxic to normal and proliferative melanocytes and has no apparent inhibitory effect on the formative process of melanin pigmentation.

Species specificity of nonimmunologic contact urticaria: Guinea pig, rat, and mouse

In order to find the most suitable animal species for studies on nonimmunologic contact urticaria (NICU), human NICU agents: 20% benzoic acid (BA), 10% sorbic acid (SA), 15% cinnamic acid (CA), 20% cinnamic aldehyde (CAL), 1.0% diethyl fumarate (DEF), 0.2% methyl nicotinate (MN), all in absolute ethyl alcohol, and 100% dimethyl sulfoxide (DMSO) were each applied to the earlobes of guinea pigs, rats, and mice. Guinea pig ear reacted with swelling to all agents tested. The mean maximal increase in thickness of the guinea pig ear, measured with a micrometer, was 114 +/- 46%. No response to BA, SA, CA, DEF, or MN was noted in the rat or mouse ear. However, DMSO and CAL caused significant ear swelling both in the mouse and the rat. Thus, the guinea pig ear is more sensitive to a variety of human NICU agents than is rat or mouse ear. The striking differences in species reactivity to NICU agents suggest the possibility that there are several mechanisms (mediators) involved in reactions to different substances. The guinea pig ear swelling test remains the best quantitative animal method available for screening human NICU agents.

The effect of BW 540C, a novel anti-inflammatory agent, on ultraviolet-induced physical and histological changes in guinea-pig skin.

We have examined the effects of various topical formulations of BW 540C, a novel dual inhibitor of the cyclo-oxygenase and lipoxygenase pathways of arachidonic acid metabolism, utilizing the model of ultraviolet irradiation of guinea-pig ears. During a series of studies, BW 540C preparations were compared with a range of corticosteroids, a cyclo-oxygenase inhibitor and a placebo using both subjective and objective measurements. Results showed BW 540C base to be the best overall agent for treating the pathological changes seen in this model.

The effect of hydrocortisone and cyclosporin A on bacillus Calmette-Guérin epithelioid cell granulomas

The injection of bacillus Calmette-Guérin (BCG) intradermally into the ear of guinea pigs leads to the formation in the draining lymph node of granulomas containing epithelioid cells with rough endoplasmic reticulum (RER) and an absence of phagocytosed material. BCG granulomas in hydrocortisone- or cyclosporin A (CsA)-treated animals contain mononuclear phagocytes with no RER. In CsA-treated animals, these cells contain fragments of phagocytosed organisms. CsA was given at two doses, 25 mg/kg orally and 50 mg/kg ip. The higher dose completely suppressed the delayed hypersensitivity (DH) response to purified protein derivative (PPD) but the lower dose did not affect the level of the DH response, but had a profound effect on epithelioid cell formation. The role of lymphokines in the maturation of the monocyte/ macrophage to epithelioid cells is discussed.

Morphological and electrophysiological study of experimental endolymphatic hydrops in guinea pig

Endolymphatic hydrops was experimentally induced in guinea pig ears by obliteration of the endolymphatic sac. One, two, four, and eight months after obliteration the animals were sacrificed for histological examination. Immediately before sacrificing the animal, compound action potentials (AP) and summating potentials (SP) were measured from both, the hydropic and the control ear. After one month, a distention of Reissner's membrane (hydrops) was found which reached the bony wall of scala vestibuli in 50% of the ears. After eight months the majority of the guinea pig ears showed extensive hydrops. In addition, we found thin connections between Reissner's membrane (RM) and the bony wall, ruptures of RM, thickening of RM and defects in the mesothelial cells, edema followed by atrophy of the stria vascularis, and loss of spiral ganglion cells and nerve fibers. The outer hair cells, followed by the inner hair cells, showed progressive degeneration starting at the apical end of the cochlea. One month after obliteration the AP threshold for the hydropic ear did not differ from the threshold for the control ear. Two months after obliteration small AP‐threshold shifts were found which increased up to 40 dB after eight months. For 2‐ and 4‐kHz stimuli an enhanced negative SP was found after one month when the extracochlear electrode was placed near the apex. After four and eight months the enhanced negative SP turned into a reduced SP. The enhanced SP is ascribed to the hydrops provoking a deviant resting position of the basilar membrane. The decrease in AP and SP amplitude two and more months after obliteration is ascribed to the loss of hair cells and spiral ganglion cells.

Contact urticaria from diethyl fumarate.

The contact urticariagenic properties of diethyl fumarate were studied using the guinea pig ear swelling assay and by open application on the human upper back skin. Diethyl fumarate caused non-immunologic contact urticaria in both human and guinea pig skin, and the reactions exhibited similar dose dependency and timing of maximal response.

Function of the Stria Vascularis in Endolymphatic Hydrops

We examined electronmicroscopically the inner ear of guinea pigs sensitized with the stria vascularis of rabbits. 25, 000 units of HRP were injected through a catheter in the right jugular vein. The tracer was observed in the stria vascularis, around the capillary vessels, in the marginal cells and in the intercellular spaces of these animals. The region of the intermediate cells in the stria vascularis was stained with HRP, and vacuolar degeneration was present in the marginal cells. This implies that the parenchymatous cells of the stria vascularis are damaged. We examined the effect of anaphylatoxins on the stria vascularis.Ten guinea pigs were injected with Zymosan+guinea pig serum+e-aminocaproic acid and 10 guinea pigs with guinea pig serum+e-aminocaproic acid into the carotid artery. Under anesthesia with nembutal, these animals were perfused with 10% neutral formalin and immobilized. Tissues were examined after HE staining. The stria vascularis of various cochlear turns showed marked atrophy except in the basal turn, and also some animals indicated the mild endolymphatic hydrops. These findings suggest that endolymphatic hydrops and other inner ear pathological conditions are related to anaphylotoxins.Furthermore, electronmicroscopic findings included hyperpermeability of the stria vascularis. The increase of permeability may be due to the leakage in intercellular spaces or in the destroyed region of the endothelial cells in addition to the usual vesicular transport.We conclude that the stria vascularis plays an important role in the development of endolymphatic hydrops.

Effect of neonatal conductive hearing loss on neuronal size in the ventral cochlear nucleus of guinea pigs

The left ear of newborn guinea pigs was closed by removing the skin and cartilage of the external auditory meatus and pulling the surrounding tissue over the opening with silk sutures. At 60 days of age the animals were anesthetized with chloral hydrate and auditory brainstem responses (ABR) were measured before and after destroying the intact right ear. After ABR testing the animals were killed and their cochleas were examined histologically. Right and left brainstems were embedded in paraffin, serially sectioned and stained with cresyl violet. The cross‐sectional area of four different cell types was measured, using a total of 210 cells of each type from each side. For all four groups; large spherical cells, small spherical cells, pale globular cells, and octopus cells, soma size was significantly smaller (P < 0.01) on the left side. This result for guinea pigs, a precocial animal, agrees with earlier findings for an altrical animal the mouse [D. B. Webster, Exp. Neurol. 79, 130–140 (1983)]. [Supported by NIH training grant NSD‐07058 and the Louisiana Lions Eye Foundation.] Present address: Dept. of Biological Sciences, Loyola University, New Orleans, LA 70118.

Distribution of HRP in the inner ear after injection into the middle ear cavity.

The distribution patterns of horseradish peroxidase (HRP) reaction products in the inner ears of guinea pigs were studied after injections into the middle ear cavities and perilymphatic and subarachnoid spaces. The normal round window membrane resisted HRP penetration from the middle ear side, but when it became pathological after repeated applications, its permeability increased. HRP deposits were found in the cochlear and vestibular sensory cells and in the lumen of the endolymphatic sac. HRP reaction products were minimal at the cochlear apex even after long survival times, suggesting that perilymph flow, if it exists, is rather weak toward this direction. Whereas the stria vascularis is impermeable to HRP, the vestibular dark cells were accessible; thus, the metabolic activity of the dark cells can be more readily controlled by drug applications through the middle ear cavity. The finding of HRP deposits on the scala vestibuli surface of Reissner's membrane and the absence of HRP in the upper portion of the spiral ligament at the basal turn suggests that the oval window is a secondary route of passage for these particles from the middle ear cavity to the inner ear. In order to determine the route of HRP into the endolymphatic sac from the middle ear cavity or scala tympani, the cochlear and/or vestibular aqueducts were obliterated singly or together. The route of HRP was determined to be the vestibular aqueduct. HRP is believed to enter the sac lumen through Reissner's and saccular membranes and the sac epithelium. Drugs and other large molecular substances instilled in or gaining access to the middle ear cavity may reach the endolymphatic sac causing its functional alteration.

CARBONIC ANHYDRASE ACTIVITY IN THE INNER EAR

The histochemical and cytochemical localization of carbonic anhydrase activity in the inner ear of guinea pig was studied with Hansson's method and modified Yokota's method. All cells possessing the cell membrane infoldings are proved to have the enzymatic activity. The cochlear hair cells show strong enzymatic activity while the vestibular hair cells show no appreciable activity. The supporting cells of both cochlear and vestibular hair cells also show the enzymatic activity. The reaction products showing the enzymatic activity are observed in the cytoplasm, nucleus and cell membrane of the stained cells with both light microscope and electron microscope. The stria vascularis, vestibular dark cells and endolymphatic sac are proved to have the enzymatic activity. The cells, which are already proved to have Na+-K+-ATPase activity, show the enzymatic activity. The regulation of the fluid and ion in the inner ear is probably a complex mechanism involving not only various cells but also several enzymes such as carbonic anhydrase, Na+-K+-ATPase and adenylate cyclase.

Experimental otitis media in guinea pigs. Serum antibody response to Bacteroides fragilis, Propionibacterium acnes and Peptostreptococcus anaerobius upon challenge.

The production of antibodies in serum was studied after inoculation of Bacteroides fragilis, Propionibacterium acnes or Peptostreptococcus anaerobius into the middle ear of guinea pigs. Inoculation of B. fragilis resulted in IgG and IgM serum antibodies at an early stage of the infection, while IgA antibodies were seldom detected and at a later stage. In contrast, inoculation of P. acnes or P. anaerobius induced no serum antibody response. Of these three species B. fragilis induced the most intense otitis, whereas P. anaerobius failed to induce otitis.

A Comparison of the Conjugation of DNTB and Other Dinitrobenzenes with Free Protein Radicals and Their Ability to Sensitize or Tolerize

Of several dinitrobenzenes tested, 2,4-dinitrothiocyanatebenzene (DNTB) was found to be the only one that did not induce contact sensitivity when applied to the guinea pig ear epicutaneously, but when applied epicutaneously it induced tolerance to 2,4-dinitrofluorobenzene (DNFB). The manner in which DNFB, DNTB, and other dinitrobenzene compounds conjugated in vitro to soluble proteins, at physiologic pH, was examined. By measuring the free amino and sulfydryl radicals in the protein before and after conjugation, it was possible to determine to which groups the hapten was bound. It was found that although all the haptens bound to the free sulfydryl groups, DNTB was the only one that did not bind to amino groups. It is suggested that to be an epicutaenous tolerizer, as opposed to sensitizer, a hapten should bind to sulfydryl groups exclusively. It is hoped that a search for agents binding in a similar manner will reveal epicutaneous tolerizers for important industrial sensitizers.

Growth Stimulation and Tumor Promotion in Skin

Stimulation of epidermal growth in adult mouse skin can be induced by chemical agents, such as phorbol esters and other skin mitogens, or by mechanical means, such as skin massage and skin wounding. It leads to different kinds of epidermal hyperproliferation, according to interference with mechanisms of endogenous growth control (G1 chalone) and to mediation by endogenous regulatory factors (prostaglandins). Certain phorbol esters and skin wounding induce epidermal hyperproliferation and, in addition, a metaplastic process. Another property of these metaplasiogenic mitogens is their tumor-promoting efficacy in mouse skin, which has been initiated by a carcinogen in a subthreshold dose. Tailor-made phorbol esters allow the subdivision of the process of tumor promotion into two stages. In the first--probably irreversible--stage, a single application of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) or wounding brings about the events critical and obligatory for promotion, whereas in the second--probably reversible--stage, repetitive applications of an "incomplete promoter" evoke epidermal hyperplasia necessary to make the tumors visible. Adult guinea pig epidermis in vivo, as well as primary cell cultures derived from adult guinea pig ear epidermis, responds to the proliferative effects of phorbol esters such as TPA along a similar sequence of biochemical events as mouse skin in vivo. The in vitro approach allows the study of the molecular events involved in the mechanism of action of phorbol esters in more detail.

Production of cytotoxic canthin-6-one alkaloids by Ailanthus altissima plant cell cultures.

Ailanthus altissima (Mill.) Swingle was established as callus- and cell-suspension cultures. Canthin-6-one and 1-methoxycanthin-6-one were isolated by a combination of preparative tlc and preparative hplc. The two alkaloids were identified by their uv, ms, and 1H-nmr spectra. The combined yield of the two alkaloids was 1.38% of dry weight from callus and 1.27% of dry weight from cell suspensions. The cytotoxicities of canthin-6-one, 1-methoxycanthin-6-one, 5-methoxycanthin-6-one, and canthin-6-one-3-N-oxide to guinea pig ear keratinocytes have been compared, and the IC50 values range from 1.11 to 5.76 micrograms/ml. There is no significant difference in activity among these four cytotoxic alkaloids.

Adenylate cyclase activity during growth and maturation of keratinocytes: comparison of two methods of study

The adenylate cyclase activity of primary cultures of guinea-pig ear keratinocytes was studied at different times by challenge with various agonists after labelling of ATP by exposure of the cells to [3H]-adenine. The [3H]-cAMP (cyclic adenosine-3',5-monophosphate) formed was assayed by scintillation counting. In other experiments, cells were exposed to the equivalent amount of unlabelled adenine, challenged and cAMP was then assayed by radioimmunoassay. These absolute amounts were compared to [3H]-cAMP levels expressed as percentage conversion of [3H]-ATP. Comparison of the two methods suggested that the use of the prelabelling method would result in underestimation of cAMP formed. It was found that the response of the enzyme to different agonists changed with the length of time of the cells in culture.

Studies on histamine-induced cutaneous flare in the guinea pig.

Evidence for the involvement of both H1- and H2-receptors in histamine-induced cutaneous flare reactions has been obtained. The guinea pig ear was used as the site of histamine injection since, unlike virtually all other skin areas in non-humans examined to date, it displays a similar response to that which occurs in human skin, namely the Lewis triple response. The flare reaction to histamine in the guinea pig ear was established as an indirectly mediated, neurovascular phenomenon by demonstrating that the vascular changes which occur in the region of the flare are incompatible with histamine directly stimulating the cutaneous vasculature and also by showing that histamine-induced cutaneous flare reactions are reduced by local anaesthesia. Having verified the authenticity of the flare reaction in the guinea pig ear, the role of H1- and H2-receptors in histamine-induced flare reactions was examined. Mepyramine and Cimetidine both significantly reduced the flare reaction produced by histamine. In addition, 2-(2-aminoethyl) pyridine, a selective H1-receptor agonist, was found to elicit a dose-dependent flare response.