Instructive role of a peripheral pattern for the central patterning of the trigeminal projection at the brainstem and thalamus revealed by an artificially altered whisker pattern.

The central patterning mechanism of neuronal circuits is an important issue in developmental neuroscience. We report here the role of a peripheral whisker pattern for the patterning of the trigeminal projection at the brainstem and thalamus in the mouse somatosensory system. The whisker pattern was manipulated by infecting the embryonic epidermis with adenovirus harboring Shh. The ectopic expression of Shh led to the induction of extra whiskers and displacement of whiskers, where these whiskers were histologically normal. The altered whisker pattern was isomorphically represented in the brainstem (barrelette: subnuclei principalis and subnuclei interpolaris), thalamus (barreloid) and cortex (barrel) as revealed by cytochrome oxidase staining. The barrelette-like pattern of the parvalbumin became discernible by immunostaining at P7 in subnuclei principalis and at P4 in subnuclei interpolaris in normal mice. These are the barrelette neurons projecting to the thalamus and the local circuit within the barrelette. The barrelette-like parvalbumin pattern also exhibits the altered whisker pattern induced by the adenovirus harboring Shh. These results highlight the role the peripheral whisker pattern for the central patterning of the brainstem, thalamus, and cortex in the mouse somatosensory system.

A proposal on the hard tissue remineralization in osteoarthritis of the knee joint investigated by FT-IR spectrometry.

An investigation was performed on hard tissues of the affected area in the cases of osteoarthritis of the knee joint by using Fourier transform infrared spectroscopy (FT-IR) to obtain the information on components and characteristics of the compounds in the hard tissues at the molecular level. Biological specimens used were: normal cortical bone, normal cancellous bone, sclerotic area of the affected site (subchondral bone), osteophyte and cartilage of the affected joint. FT-IR spectra of these specimens were measured and spectroscopic characteristics of various types of hard tissues were quantified. As the result of the analysis, signal ratio of PO4(3-) to amide I (PO4(3-)/amide I) was 0 in cartilage, and the ratio was at the highest in normal cortical bone of the ulna, being as high as 1.82. The signal ratio of the sclerotic area of the affected site was 1.69, and this was higher than the values in osteophyte (1.44) or cancellous bone of the tibia (1.61). It is inferred from these data that the degree of remineralization was considerably higher in osteoarthritis of the knee joint. On the other hand, in the osteophyte, which is frequently seen in the radiography of osteoarthritis, it was 1.44 for the attached osteophyte and 1.37 for the detached osteophyte, and these values were lower compared with the values in the sclerotic area of the affected site (1.69). From these results, it appears that remineralization of mineral components is very likely to occur under the acidic conditions in the articular region in this disease. We propose that the mechanism of hard tissue formation in the knee joint of osteoarthritis is a form of remineralization.

Component analysis and growth process of nasopharyngeal calculus as revealed by Fourier transform infrared (FT-IR) spectroscopy.

A quite rare case of nasopharyngeal calculus in a woman in her twenties associated with the nasal discharge of pseudomonas infection was reported. As the substance was irregularly large in size, we extracted it partially by piecemeal resection using forceps and also by cracking technique using the holmium yttrium-aluminum-garnet (YAG) laser, under saline irrigation and stereotactic microscopic navigator (SMN) system under endoscopic observation. The substance was firmly fixed to the pharyngeal tonsil bed. The final extract was a small piece of singly folded bandage, which is probably the focal background for calculus formation. In a cross section of calculus specimen removed during surgery, Fourier transform infrared (FT-IR) analysis revealed that a) signal ratio of methylene group (organic substance) to amide I (protein) was 21.6% at the nasal cavity side, gradually decreased toward nasal mucous membrane showing approximate 50%, b) signal ratio of amide I to P04(3-) (inorganic substance) ranged between 17.7% and 26.7% at the different sites and inside the calculus, the protein content was approximate 1/5 of the inorganic substance, and c) signal ratio of the methylene group to amide I at the nasal cavity site showed that their contents were almost equal. The quantity of the organic substance was estimated at approximate 1/2 quantity of the protein at both the central part and the part contacted with the mucous membrane. From these results, it seems that throughout the course of calculus growth, both inorganic substance and protein remain almost constant inside the calculus, while organic substance is released from the internal part of the calculus being probably formed at an early stage.

The effect of tranexamic acid on cochlear blood flow in guinea pigs measured by laser Doppler flowmetry.

OBJECTIVE: This study investigated the vasoactive effect of tranexamic acid on the cochlear blood flow in guinea pigs. METHOD: 3 ml solution (infusion speed, 0.5 ml/min) containing different concentrations of tranexamic acid was intravenously infused into 15 guinea pigs under general anesthesia. The guinea pigs were grouped according to four levels of dosage of the medicine (470 mg/kg, n=6; 220 mg/kg, n=3; 4 mg/kg, n=3; 1 mg/kg, n=3). Before administering medicine, saline solution was administered in similar volume and speed as a control. The cochleas were surgically exposed and laser Doppler flowmetry monitored cochlear blood flow volume (CBF). The left femoral artery was cannulated to permit a transducer to monitor systemic blood pressure (BP). RESULTS: (1) Stimulatory effect of tranexamic acid on CBF was dose-dependent at concentrations of 1-470 mg/kg and, (2) the time course of changes in CBF was almost identical to that in BP following tranexamic acid administration. CONCLUSION: Preliminary findings suggest that intravenous administration of tranexamic acid increases CBF due to vasomotorial mechanism effect on BP.

Histological reaction to hydroxyapatite in the middle ear of rats.

OBJECTIVE: Present study was performed to evaluate the histological response of rat middle ear mucosa following implantation of Apaceram granules, a synthetic dense hydroxyapatite [Ca10(PO4)6(OH)2], prepared from commercially available synthetic auditory ossicle, and to assess the precise histological response of the rat middle ear to implantation of Apaceram granules, by microscopic examination of mucosal tissue at various time points after implantation. METHODS: Apaceram granules were implanted in the temporal bulla of 32 rats. As control, sham surgery was performed in a group of ten rats. Bulla specimens were removed at 1, 3, 7, 14, and 30 days after surgery in the implant and control groups, and at 90, 180 and 300 days in the implant group. Specimens were decalcified, sectioned at a thickness of 6 microm, and stained with haematoxylin and eosin, and Mallory's azan for histological examination of mucosal tissue. RESULTS: Evidence of inflammatory reaction was slightly greater in the implant group than in controls. Lymphocyte and macrophage counts were higher in the implant group 1 day after surgery, but decreased to similar levels by day 3, and continued to decrease thereafter, and few were observed in the implant group at 300 days. Neutrophils observed at 1 day after surgery were not evident in either group at 3 days. Gradual fibrosis development continued in both groups over all time points studied. Foreign body giant cells were never observed in either group. No bony reaction was observed in any specimen. CONCLUSION: The results of this study suggest that Apaceram is biocompatible and suitable for reconstructive ear surgery.

Histological reaction of auditory bulla bone to synthetic auditory ossicle (Apaceram) in rats.

To investigate the biocompatibility of a synthetic auditory ossicle to host bone, small thin Apaceram disks composed of dense hydroxyapatite were implanted under the periosteum of the left auditory bulla in 32 rats for periods ranging from 1 day to 270 days. A sham operation performed on 10 rats served as a control. Decalcified histological sections stained with hematoxylin and eosin were observed using light microscopy. The experiment showed: 1) a time-dependent mature fibrous connective tissue surrounding the Apaceram disk, 2) no evidence of inflammatory reaction caused by the implant from 90 days after implantation until the end of the experiment, 3) no evidence of osteolysis by osteoclasts caused by the implant, and 4) direct contact of bone to the implant on the bone-disk interface at 180 and 270 days after implantation. The findings suggest that Apaceram has a high degree of implant biocompatibility, making it a satisfactory substitute biomaterial for otological reconstructive surgeries.

Long-term microstructural analyses of hydroxyapatite implanted in rats using laser-Raman spectrometry and scanning electron microscopy.

To investigate the long-term surface microstructure of a synthetic auditory ossicle (Apaceram) composed of dense hydroxyapatite (HA), thin HA disks were implanted subcutaneously into the interscapular regions of 12 rats. After 6, 14 and 20 months, implanted HA surfaces were observed using stereoscopic microscopy, scanning electron microscopy (SEM) and laser-Raman spectrometry. Visual observation by SEM at 6 months and by stereoscopic microscopy at 14 months indicated a progressive degradation of the HA disk surfaces implanted in the subcutaneous tissue. Visual observation by SEM at 14 and 20 months and by stereoscopic microscopy at 20 months indicated a progressive redeposition on the surfaces of the implants. Raman spectra compared half-peak breadths of v1 signal (PO4(3-), 960 cm(-1)) on the gray and white surface areas of implanted HA disks observed by stereoscopic microscopy. Analysis demonstrates that demineralization at 14 months and remineralization at 20 months occur on the gray areas; demineralization at 6 months and remineralization at 14 months occur on the white areas.

Expression of the Vax family homeobox genes suggests multiple roles in eye development.

BACKGROUND: Homeobox gene products are thought to regulate target genes involved in the regional specification of tissues and organs. Several genes play roles in eye development. RESULTS: We isolated two new mouse homeobox genes, Vax1 and Vax2, expressed in retina primordium. The two genes share the same gene organization, consisting of three exons with predicted amino acid sequences identical in the homeodomain but diversified in other regions. At 9. 5 days post coitum, both Vax genes were expressed in optic vesicles. At 11.5-14.5 dpc, Vax1 became restricted to the optic stalk, while Vax2 was expressed in the ventral half of the neural retina. Mapping of Vax2 between D6Mit3 and D6Mit8 on chromosome 6 indicated a possible linkage with Emx1. CONCLUSIONS: The expression patterns of Vax genes suggest separate and specific involvement in eye development. Vax1 may contribute to differentiation of the neuroretina, pigmented epithelium and optic stalk, while Vax2 could function in the establishment of the dorso-ventral axis of the retina and the visual system. The chromosomal location of Vax2 suggests that Vax2-Emx1 and Vax1-Emx2 arose by chromosome duplication of the same ancestral gene.

Long-term observation of subcutaneous tissue reaction to synthetic auditory ossicle (Bioceram) in rats.

To evaluate biocompatibility to tissue in long-term implantation, Bioceram discs made of aluminum oxide (Al2O3) were implanted subcutaneously within the interscapular region of 64 rats for six to 20 months. Histological sections stained with haematoxylin and eosin (H&E) and the surface of the implant material were observed using light microscopy. Different cell types and the thickness of fibrous capsules surrounding the implants were examined quantitatively by light microscopy. Small numbers of macrophages (2.8 +/- 0.7%) and lymphocytes (2.7 +/- 0.9%) were observed at six months after implantation, gradually decreasing to zero at 16, 18 and 20 months. Neither neutrophils nor foreign body giant cells were seen in any specimens. The thickness of fibrous capsules surrounding the implants was closely related to the shape of the implant, but there was no significant change between six and 20 months after implantation. No change in Bioceram surfaces were observed under stereoscopic microscopy from six to 20 months after implantation. The study results indicate that Bioceram is a satisfactory biocompatible material for reconstructive surgery from the viewpoint of long-term tissue response. Present results of experiments with Bioceram are also compared to previous results with Apaceram and different tissue responses of the two materials are discussed.

Thickness of fibrous capsule after implantation of hydroxyapatite in subcutaneous tissue in rats.

The present study evaluates in rats the histomorphometrical thickness of fibrous capsules that surround hydroxyapatite (HA) disks after implantation. HA disks were implanted into the subcutaneous tissue of 79 rats for 1 day to 20 months. Decalcified histological sections stained with hematoxylin and eosin were examined. Fibrous capsule thickness (FCT) was measured using an objective micrometer. On the fourteenth day, primary fibrous capsules formed around implants. From that time point FCT increased with time of implantation. Within a given sample, FCT differed from one portion of the fibrous capsule to another, depending on which site faced the disks. FCT was thickest at the upper and lower portions of the disks, thinner at the lateral portions, and thinnest at the upper and lower ring-shaped portions. Two possible explanations for the above findings are discussed in this paper: (1) The area of contact between disk and tissue differs. (2) Chemical stimulation of implanted material caused by demineralization and remineralization may result from the varying thicknesses of fibrous capsules. FCT from upper and lower portions of HA disks increased by over 200% in the first 10 months and steadily increased about 20% over the next 10 months. Many studies have concluded that HA is useful for reconstructive surgery, so the long-term effects of FCT need further study.

Subcutaneous inflammatory reaction to a synthetic auditory ossicle (Bioceram) in rats.

Cellular response and inflammatory reaction to synthetic auditory ossicle (Bioceram) made from aluminium oxide are investigated. Local inflammatory effects are important in wound healing and in determining biocompatibility of an implant, necessitating the study of biologic effects of implants, especially inflammation and fibrous capsule formation. Bioceram discs were implanted subcutaneously in the interscapular region of rats for various periods of time, ranging from 1 day to 300 days. Histological sections 6 microns thick were stained with haematoxylin and eosin. Cell types around the implants were examined quantitatively by light microscopy. Inflammatory cell reaction to Bioceram decreased rapidly within 14 days, similar to the reaction in control groups. From 30 days to 300 days after implantation, there was continuous reduction to very low levels for macrophages and lymphocytes, but fibrous connective tissue capsule around implants matured. Preliminary results suggest that Bioceram is a satisfactory biocompatible material for reconstructive surgery from the viewpoint of cellular response. We also briefly discuss the different tissue responses in light of our previous study on hydroxyapatite (Apaceram).

Evaluation of tinnitus patients by peroral multi-drug treatment.

To evaluate patients complaining of subjective tinnitus, this study examined their response to peroral betahistine mesilate, vitamin B complex and diazepam in combination. Because three drugs were used together, it remains to be seen whether a single drug or a combination of drugs was effective. We issued questionnaires to 67 patients with tinnitus associated with sensorineural hearing loss of unknown etiology or tinnitus, despite normal hearing in pure tone audiometry and lack of distinct systemic disorders. Our original questionnaire contained seven items and allotted points for each item to facilitate evaluation. After prescribing the above drugs and observing patients' progress for 5 weeks, 50 of the 67 subjects were evaluated again by the same questionnaire. The present study evaluates tinnitus of patients as an example of clinical applications; this was not a controlled double blind study. It was found that, after patients took the prescribed medication, the total number of points were significantly reduced (paired t-test, P < 0.001). After medication, cases of bilateral tinnitus were significantly reduced from 27 to 14, and cases of two types of tinnitus sound, were significantly decreased from 22 to 11 (chi 2-test, P < 0.05). After 5 weeks of administration, 54% of patients felt treatment had been effective. Preliminary results suggest this peroral multi-drug treatment may provide relief for some patients with subjective tinnitus. However, long-term efficacy of the treatment was not investigated.

A subcutaneous tissue reaction in the early stage to a synthetic auditory ossicle (Bioceram) in rats.

The discs of synthetic auditory ossicle (Bioceram), which are composed of aluminium oxide (Al2O3), were implanted subcutaneously in the interscapular region of 16 rats. The implanted specimens were removed at 1, 3, 7 and 14 days after implantation. The decalcified 6 microns thick sections were stained with H.E. and cell types around the implants were counted microscopically. We found that an acute inflammatory reaction occurred at one day, in which macrophages and neutrophiles predominated, and almost disappeared at about 7 days after implantation. Fibrosis began to be observed at 3 days. During this early stage, foreign body giant cells were found in only one specimen at 3 days. These findings, in comparison with those in the controls, showed that the chemical irritation of Bioceram to the subcutaneous tissue is slight, although the physical and/or chemical irritation of Bioceram lasts continuously and induces fibrosis around the bioimplant. The results so far suggest that Bioceram seems to be a satisfactorily biocompatible material, at least within the extent of 2 weeks.

Long-term observation of subcutaneous tissue reaction to synthetic auditory ossicle (Apaceram) in rats.

The present study evaluates histological characteristics of the soft tissue response to long-term implantation of Apaceram discs composed of dense hydroxyapatite in rats. Discs were implanted into the subcutaneous tissue of 76 rats for six to 20 months. Decalcified histological sections stained with haematoxylin and eosin (H & E) and Mallory's azan were examined. Different cell types surrounding implants were counted. The greatest proportion of macrophages was found at six months (13.5 per cent). This proportion gradually decreased to four per cent at 20 months. Small numbers of lymphocytes and foreign body giant cells were observed in every group, but neither neutrophils nor osteogenesis were observed in any specimens. Results of the present study and previous related studies indicate that despite reappearance of a small number of macrophages six months after implantation, Apaceram is useful for reconstructive surgery.

Regionalized expression of the Dbx family homeobox genes in the embryonic CNS of the mouse.

Here we report the identification of a novel homeobox gene family Dbx in mouse, which consists of Dbx and Dbx2. The two genes share similar structural organization and are encoded by different chromosomes. The predicted Dbx and Dbx2 proteins share 85% identity in their homeodomain amino acid sequences, but otherwise showed no significant similarity. Characterization of the expression of these two genes in the embryos suggested their role in the development of the CNS. In the forebrain, Dbx is expressed in various regions, while Dbx2 showed a more restricted pattern of expression. In the midbrain, the expression domains of Dbx and Dbx2 overlap along the dorso-lateral wall of the ventricle. In the hindbrain and spinal cord, both genes are expressed in the boundary separating the basal and alar plates, which seems to correspond to the sulcus limitans. Expression of the Dbx/Dbx2 genes is restricted to the ventricular region of the embryonic CNS except for that of Dbx in the septum of the telencephalon. Together these observations indicate possible participation of the members of the Dbx family in regionalization of the CNS. While the expression of Dbx was restricted to the CNS, Dbx2 was also expressed in some of the mesenchymal cells, such as limb buds and tooth germs.

Chronic Prototheca meningitis.

A case of chronic meningitis caused by the achloric alga Prototheca wickerhamii is described, which has persisted for more than 6 years despite treatment with various antifungal agents. For the last year no treatment has been given, but the patient has no complaints.

Demonstrations of de-and remineralization mechanism as revealed in synthetic auditory ossicle (Apaceram) of rats by laser-Raman spectrometry.

In investigations of the process of bone destruction caused by chronic otitis media complicated with cholesteatoma, we proposed previously a hypothesis to explain its mechanism. In the present study, we substitute a synthetic auditory ossicle (Apaceram) for the bone to simplify the model system of experiments for our hypothesis. Its process was studied on a model simulation in vitro, in vivo and clinically at molecular level, using laser-Raman spectrometry. An increase of conductivity in both the saline solution and double-distilled water immersed Apaceram indicated demineralization depending on time lapse. The process of demineralization was revealed by the Raman spectral profiles as shown by narrowing a half-peak breadth (p < 0.01) of v1 signal (PO4(3-), 960 cm-1) and the disappearance of the shoulder at circa 950 cm-1 on the Apaceram surface implanted for 6 months. On the other hand, the process of remineralization was revealed in vivo by the spectral profiles: 1) a broader half-peak breadth (p < 0.01) of v1 signal on the Apaceram surface implanted for 10 months than that implanted for 6 months; 2) a weak reappearance of the shoulder at ca. 950 cm-1 on the Apaceram surface after 10 months; 3) four signals (v1 through v4) of PO4(3-) on the HOAP observed for inside the Apaceram shaft and 4) a weak v1 signal on both the granular fluorescent substance in the clinical case and the high density area of subcutaneous tissue after contact with the Apaceram in rats for 3 months. Judging from these investigations, it is proposed that the mechanism of bone destruction associated with cholesteatoma is a form of de- and remineralization.

The enhancement of cellular infiltration and vascularisation of a collagenous dermal implant in the rat by platelet-derived growth factor BB.

The implantation of collagen-based dermal substitutes offers one means of management of full-thickness skin lesions. We have examined the effect of the recombinant BB homodimer of platelet-derived growth factor (rPDGF-BB) on the extent of cellular infiltration and vascularisation of collagen sponges implanted into full-thickness excision wounds in rats. Histological examination of sponges excised 14 and 21 days post-implantation in dose-response studies in which 0-4 micrograms rPDGF-BB were applied to the undersurface of each sponge, immediately prior to its implantation, demonstrated a progressively increased infiltration of host cells, especially fibroblasts, and enhanced capillary formation. With 4 micrograms rPDGF-BB, an enhanced infiltration of fibroblasts into sponges was already apparent 3 days post-implantation, and enhanced capillary formation was noticeable after 7 days. This neovascularisation was noted to be associated with improved survival of autologous split-thickness skin grafts applied to the sponges immediately following their implantation.

Subcutaneous tissue reaction to synthetic auditory ossicle (Apaceram) in rats.

A study was carried out in order to obtain further information about the soft tissue response to thin Apaceram discs of dense hydroxyapatite (HA) implanted in rats for various periods of time between one day and 10 months. The Apaceram discs were implanted subcutaneously into the interscapular region of 33 rats. A sham operation was performed on eight rats used as controls. Decalcified histological sections stained with haematoxylin and eosin and Mallory's azan were examined and the different cell types found around the implants were counted. It was found that an acute inflammatory reaction occurred after one day and disappeared at about two weeks after implantation. In the test groups, macrophages and lymphocytes disappeared about one week later, and no inflammatory reaction was observed from one to three months. However, a tissue reaction occurred at six months with the appearance of macrophages and lymphocytes, and decreased gradually at 10 months. Meanwhile, a few foreign body giant cells at the Apaceram-tissue interface and a thick layer of fibrous connective tissue around the Apaceram disc were observed at 10 months. No osteogenesis was observed in any specimen. The results obtained so far suggest that Apaceram is still a useful material for reconstructive surgery, despite the possible appearance of a slight macrophage reaction at six months.

Characteristics of neo red cells, their function and safety: in vivo studies.

A new type of artificial oxygen carriers, the Neo Red Cells (NRCs) have been developed and investigated for oxygen transporting efficiency and safety in experimental animals. Stroma free hemoglobin from outdated human red blood cells together with inositol hexaphosphate as an allosteric effector under sterile, pyrogen free condition were encapsulated in liposomes and then were coated with polyethylene glycol bond to hydrogenated soy phosphatidylethanolamine as a surface modifier to prevent aggregation of NRCs in plasma. The efficiency of the NRCs in tissue oxygenation was studied in rabbits which were made severely anemic by drawing 85% of their blood and immediately replacing it with NRC solution. The animals, all recovered to pre-anemic conditions within 6-8 hr and lived normally until being sacrificed, 6 months after the exchange transfusion. The circulation half-life and tissue distribution of NRCs were studied using radiolabeled NRCs. Within the circulation, the half-life of NRCs was 21 hr and extravascularly, they were distributed mainly in and metabolized by the reticuloendothelial system within 7 days. Our observations suggest that the NRCs prepared and investigated in this study are efficient oxygen carriers without causing serious adverse reactions and can be prepared free from pathogenic micro-organisms by special filtration technique before encapsulation of Hb. Currently, experiments are ongoing to control auto-oxidation of oxyHb to metHb which is higher in NRCs than in native red cells at physiological conditions.