Guided growth with aligned neurites in adult spiral ganglion neurons cultured in vitro on silicon micro-pillar substrates.

OBJECTIVE: Assessment of the relationship between the topographical organization of silicon micro-pillar surfaces (MPS) on guidance and neural alignment of adult spiral ganglion neurons (SGN) and use of the otosurgical approach as an alternative for the extraction and isolation of SGNs from adult guinea pigs. APPROACH: SGNs from adult guinea pigs were isolated using conventional and otosurgical approach for in vitro cell culturing on MPS of various micro-pillar widths (1-5.6 µm) and spacing (0.6-15 µm). Cell cultures were compared morphologically with neuronal cultures on control glass coverslips. MAIN RESULTS: We found enhanced SGN in vitro cultures in MPS areas with small and intermediate inter-pillar spacing (from 0.6 µm to 3.2 µm) as well as in MPS areas with wider pillars (from 1.8 µm to 4 µm) compared to MPS flat zones and control glass coverslips. Scanning electron microscopy (SEM) images highlighted how neurites of SGNs follow straight lines by growing on top and between micro-pillars. Only micro-pillars with small and intermediate pillar spacings favor neurite alignment along preferred angles (30°, 90°, and 150°), while pillars with wider spacing produced less aligned neurites. We found propensity of adult SGNs grown on MPSs to attain more bipolar and multipolar morphologies. Additionally, we observed reduced interaction between neuronal and glial cells compared to control glass coverslips. Finally, we found that the otosurgical approach was more beneficial for SGN survival on glass coverslips and MPS flat surfaces than the conventional method. SIGNIFICANCE: MPS with specific architecture supports the guided growth of adult SGNs in vitro and controls adult SGN development and behavior.

Advantageous environment of micro-patterned, high-density complementary metal-oxide-semiconductor electrode array for spiral ganglion neurons cultured in vitro.

This study investigated micro-patterned, high-density complementary metal-oxide-semiconductor (CMOS) electrode array to be used as biologically permissive environment for organization, guidance and electrical stimulation of spiral ganglion neurons (SGN). SGNs extracted and isolated from cochleae of P5-P7 rat pups and adult guinea pigs were cultured 1, 4 and 7 days in vitro on glass coverslips (control) and CMOS electrode array. The cultures were analyzed visually and immunohistochemically for SGN presence, outgrowth, neurite alignment, neurite length, neurite asymmetry as well as the contact of a neuronal soma and neurites with the micro-electrodes. Our findings indicate that topographical environment of CMOS chip with micro-patterned pillars enhanced growth, survival, morphology, neural orientation and alignment of SGNs in vitro compared to control. Smaller spacing (0.8-1.6 µm) between protruding pillars on CMOS led SGNs to develop structured and guided neurites oriented along three topographical axes separated by 60°. We found morphological basis for positioning of the micro-electrodes on the chip that was appropriate for direct contact of SGNs with them. This configuration allowed CMOS electrode array to electrically stimulate the SGN whose responses were observed with live Fluo 4 calcium imaging.

CORRELATION BETWEEN MAMMOGRAPHY DETECTED BREAST ARTERIAL CALCIFICATIONS AND LIFESTYLE RISK FACTORS.

The aim of the study was to investigate the association between some lifestyle-attributable risk factors of atherosclerosis, such as body mass index (BMI), oral contraceptives, hormone replacement therapy, smoking and alcohol consumption with breast arterial calcification (BAC) and its intensity on mammograms, and to assess the impact of these lifestyle risk factors on mammography findings of BAC. This prospective study included 300 women aged 47-69, i.e. a group of 149 women with BAC on mammograms and control group of 151 women without BAC. Self-reported BMI, use of oral contraceptives, hormone replacement therapy, smoking and alcohol consumption were recorded by medical interview. The presence of BAC and its intensity on mammography was compared according to the presence of high BMI and use of hormone therapy, smoking and alcohol consumption. The results showed the highest proportion of smokers (28.9%) in the group with mild BAC as compared with the groups without calcification (14.6%) and with intense calcification (12.1%). Women taking oral contraceptives had a higher level of calcified breast arteries but no significant between-group difference was found for high BMI, hormone therapy and alcohol consumption. Thus, study results showed the mammographic finding of BAC to be inadequate to identify women with some lifestyle-attributable risk factors such as BMI, hormone replacement therapy, smoking and alcohol consumption.

[CROATIAN GUIDELINES FOR DIAGNOSIS AND MANAGEMENT OF BENIGN PAROXYSMAL POSITIONAL VERTIGO (BPPV)]. / HRVATSKE SMJERNICE ZA DIJAGNOSTIKU I LIJECENJE BENIGNOGA PAROKSIZMALNOG POZICIJSKOG VERTIGA (BPPV-A).

BPPV is generally the most common cause of vertigo, caused by a pinch-off of tiny calcium carbonate crystals (called the otoconia or the otoliths) from the macula utriculi, most frequently due to the degenerative processes or a trauma, whereby the crystals, under the action of gravity in certain head positions coinciding with its direction, arrive to some of the semicircular canals, usually the posterior one, due to the existent anatomical circumstances and relationships, thus creating an inadequate stimulus of the cupular senses while floating through the endolymph and provoking symptoms of a strong and short-term dizziness. Two main clinical forms can be distinguished: canalolythiasis, with an accommodation of otolithic debris in the semicircular canal, and cupulolythiasis, with their location immediately next to the cupular sense. The diagnosis is established by a positive positioning test, Dix-Hallpike for the posterior and the supine roll for the lateral canal. Although one can expect a spontaneous recovery subsequent to few weeks or months, various methods of otolith repositioning to a less sensitive place lead to a prompt improvement while reducing or withdrawing the symptoms completely. These guidelines are intended for all who treat the BPPV in their work, with an intention to assist in the diagnosis and application of an appropriate therapeutic method.

Nasopharyngeal bacterial flora in healthy preschool children during winter-spring months.

The paper aimed to determine the incidence of colonization of Streptococcus pyogenes, Streptococcus pneumoniae, Haemophilus influenzae type b and Neisseria meningitidis in the nasopharynges of healthy children in two preschool institutions during winter and spring months, without using antimicrobial treatment or serotyping of these bacteria. In addition to colonization of the above bacteria, the research that continued for 3 months monitored the length of their persistence in and disappearance from children's nasopharynges, children's health statuses, and provision of adequate medical interventions in children demonstrating clinical signs of disease. The ultimate aim of the paper was based on contributing to clearer and more accurate determination of a medical procedure in case of a positive result for bacteria intended to be found in the nasopharynx of a healthy child who spends time in a preschool institution.

Long-term functional outcomes after 10 years of bilateral cochlear implantat use.

The aims were to determine the benefit of bilateral cochlear implantation in a 20 years old patient implanted in Croatia on hearing and speech development. The male patient, after 10 years of deafness, got cochlear implants Med-EL Combi 40+ on both sides in one-stage surgery. The etiology of his deafness was posttraumatic meningitis. Auditory capacity and speech recognition tests were performed for both ears separately and together Average hearing level on the right ear with right cochlear implant switched on started at 62 dB 1 month after the cochlear implantation and was on 55 dB after 10 years. Average hearing level on the left ear with left cochlear implant switched on started at 55 dB 1 month after the cochlear implantation and was on 32 dB after 10 years. Average hearing level on the both ears with 2 cochlear implants switched on started at 35 dB 1 month after the cochlear implantation and was on 27 dB after 10 years. Long-term functional outcomes with bilateral cochlear implantation provides advantages over unilateral implantation including improved hearing level, speech perception in noise and improved sound localization.

Thermography-measured effect of capsaicin, methylprednisolone and mitomycin on the survival of random skin flaps in rats.

AIMS: This study was designed to determine if steroids, capsaicin and mitomycin improved skin flap survival in rats, and it has confirmed that thermography is an effective method to measured skin flap vitality. METHODS: Forty female Wistar rats were randomised into four groups. A standardised full thickness inferiorly based dorsal random-pattern skin flap was raised, and a gelatine sponge was placed before suturing between the flap and its recipient bed, soaked with 0.9 % saline in the control group and with capsaicin, methylprednisolone and mitomycin in the experimental groups. Vitality of the flap and of the survival area was measured by infrared thermography. RESULTS: Methylprednisolone statistically significant decrease skin flap necrosis, approximately 56%, when compared with controls (P< 0.05). CONCLUSION: Corticosteroids applied subcutaneously in a single dose on a gelatine sponge produced a statistically significant improvement of the survival of random skin flaps in rats, whereas capsaicin and mitomycin showed no improvement.

Hidradenoma of the external auditory canal: clinical presentation and surgical treatment.

The aim of this article is to present clinical features, diagnostic procedures and surgical treatment of a rare ear tumor. We report a case of 78 year old female with hidradenoma of the external auditory canal. Patient had a sensation of pain and fullness with permanent ottorhea from the right ear for one year Temporal bone computed tomography showed a tumor of the external ear, 6 centimeters in diameter, without bone, temporomandibular joint or intracranial invasion; the tumor was limited medially by the tympanic membrane. Biopsy was performed and pathohistology finding was: hydradenoma nodulare atypicum. Surgical intervention and wide tumor removal in general endotracheal anesthesia was performed. One year after the surgery there was no sign of tumor recurrence. Hidradenoma is rare ear tumor arising from the epithelial cells of sweat glands of the external auditory canal. Radiological evaluation and pathohistology confirmation of hidradenoma is necessary and wide excision of the tumor is the treatment of choice.

Bacterial colonization and granulocyte activation in chronic maxillary sinusitis in asthmatics and non-asthmatics.

The impact of bacterial colonization on the severity and pattern of chronic inflammation in rhinosinusitis is not clear. In this study, it was hypothesized that bacterial colonization of the sinus mucosa would have a greater impact on inflammatory response modulation in asthmatic patients than in non-asthmatic patients with chronic rhinosinusitis. In order to test this hypothesis, granulocyte activation was measured and related to bacteria identified in the sinus lavage. Lavages from the maxillary sinuses of 21 asthmatic and 19 non-asthmatic patients with chronic rhinosinusitis (CRS) were microbiologically examined for aerobic and anaerobic growth. Eosinophil cationic protein (ECP), an eosinophil activation marker, and myeloperoxidase (MPO), a neutrophil activation marker, were measured in the sinus lavages. Bacteria were recovered in 20/32 samples from the asthmatics and in 21/33 samples from the non-asthmatics. Gram-positive aerobes and anaerobes were slightly more common than Gram-negative bacteria. A different bacterial profile was found when comparing Gram-negatives between the groups. Concentrations of MPO were significantly higher in samples with bacterial recovery from asthmatic patients, compared to sterile samples of both groups. Concentrations of ECP in the samples from asthmatic patients were significantly higher than in the controls, with no significant difference related to bacterial colonization. Bacterial colonization in chronically inflamed sinuses may have an impact on neutrophil granulocyte activation in patients with bronchial asthma, which was not confirmed for patients with CRS without asthma.

[Indications for tonsillectomy in children aged under 16 years in ENT Department of Sestre Milosrdnice Clinical Hospital]. / Indikacije za tonzilektomiju u djece do 16 godina u Klinici za otorinolaringologiju i kirurgiju glave i vrata KB "Sestre milosrdnice".

AIM OF THE STUDY: To study frequency and type of indications for tonsillectomy in ENT Department of "Sestre milosrdnice" Clinical Hospital, distribution of operations according to the age and sex of the patients. To present history of tonsillectomies. METHODS: Retrospective study, information collected by clinical documentation review (anamnesis, physical findings). RESULTS: In the nine-year period 1995 - 2003 the total number of operations was 4704, 2527 male, 2177 female patients. There were 2692 tonsillectomies, 2011 adenotomies and 1 tonsillectomy "a chaud". The most frequent indication for tonsillectomy was recurrent tonsillitis (72%), followed by adenotonsillar hypertrophy (13%), chronic tonsillitis (12%), focaloses (1%) and peritonsillar abscess (2%). Distribution according to the surgeon: 80% of the operations were performed by specialists, 20% by residents. According to the age of patient: operations were most often performed at the age of 4 (800 patients). CONCLUSION: According to this study the most frequent indication for tonsillectomy was recurrent tonsillitis. Indications for tonsillectomy are clearly defined and as such should be followed.

Local IgE and inflammation in chronic rhinosinusitis of asthmatics and non-asthmatics.

The aim of the study was to show the difference in the pattern of inflammation, and Th1/Th2 polarization between asthmatic and non-asthmatic patients with CRS, specifically eosinophil activation, local IgE levels in the sinus fluid and tissue, and the severity of inflammation were measured. The maxillary sinus lavages, mucosal biopsies and bacteriological swabs were taken in 17 asthmatic and 36 non-asthmatic adult patients with CRS. The concentrations of IgE, eosinophil cationic protein (ECP), myeloperoxidase (MPO), and tryptase were analyzed and IgE+ cells, eosinophils, lymphocytes and plasma cells were counted. The granulocyte activation markers and IgE in sinus lavages, and the inflammatory and IgE+ cells counts were significantly higher in the asthmatics with the greatest difference in ECP and IgE concentrations. The tryptase concentrations did not differ, but only in the asthmatics they correlated significantly with the IgE concentrations and IgE+ cells count. Asthmatic patients present a distinct subgroup among the patients with chronic rhinosinusitis (CRS). The levels of the cellular markers and IgE in the sinus fluid differ from those of non-asthmatic patients with CRS. The activation of granulocytes (especially eosinophils), local IgE concentrations and the inflammatory cells infiltration are significantly higher in the asthmatics.

Etiology of deafness in children cochlear implant candidates in Croatia.

The aim of this study was to provide more information on the causes of sensorineural hearing loss (SNHL) in children cochlear implant candidates in Croatia. The retrospective study included 270 children candidates for cochlear implantation between January 1997 and January 2005 at our institution. The medical assessment of the candidates included the history, physical examination, radiologic evaluation of the temporal bone and audiologic assessment. A family history of SNHL had 82 (30.4%) candidates. The prematurity and/or complicated perinatal course was found in 35 (12.9%) of candidates. Computerized tomography (CT) scan analysis identified 44 (16.3%) candidates presenting with an inner ear malformation. Overall, a definite or probable cause of SNHL was identified in 58.9% of candidates and 41.1% had no obvious cause. The results of the study might give us better insight into the potential causes of SNHL and allow more timely intervention, allowing children with SNHL to reach their potential.

Prolonged esophagitis after primary dysfunction of the pyloric sphincter in the rat and therapeutic potential of the gastric pentadecapeptide BPC 157.

Seven or fourteen days or twelve months after suturing one tube into the pyloric sphincter (removed by peristalsis by the seventh day), rats exhibit prolonged esophagitis with a constantly lowered pressure not only in the pyloric, but also in the lower esophageal sphincter and a failure of both sphincters. Throughout the esophagitis experiment, gastric pentadecapeptide BPC 157 (PL 14736) is given intraperitoneally once a day (10 microg/kg, 10 ng/kg, last application 24 h before assessment), or continuously in drinking water at 0.16 microg/ml, 0.16 ng/ml (12 ml/rat per day), or directly into the stomach 5 min before pressure assessment (a water manometer connected to the drainage port of a Foley catheter implanted into the stomach either through an esophageal or duodenal incision). This treatment alleviates i) the esophagitis (macroscopically and microscopically, at either region or interval), ii) the pressure in the pyloric sphincter, and iii) the pressure in the lower esophageal sphincter (cmH2O). In the normal rats it increases lower esophageal sphincter pressure, but decreases the pyloric sphincter pressure. Ranitidine, given using the same protocol (50 mg/kg, intraperitoneally, once daily; 0.83 mg/ml in drinking water; 50 mg/kg directly into the stomach) does not have an effect in either rats with esophagitis or in normal rats.

An experimental model of prolonged esophagitis with sphincter failure in the rat and the therapeutic potential of gastric pentadecapeptide BPC 157.

We report a simple novel rat model that combines prolonged esophagitis and parallel sphincters failure. The anti-ulcer gastric pentadecapeptide BPC 157, which was found to be stable in gastric juice, and is being evaluated in inflammatory bowel disease trials, is an anti-esophagitis therapy that recovers failed sphincters. Twelve or twenty months after the initial challenge (tubes sutured into sphincters for one week and then spontaneously removed by peristalsis), rats exhibit prolonged esophagitis (confluent hemorrhagic and yellowish lesions, thinner epithelium and superficial corneal layer, with stratification derangement); constantly lowered pressure of both sphincters (assessed by using a water manometer connected to the drainage port of a Foley catheter implanted into the stomach either through esophageal or duodenal incision); and both lower esophageal and pyloric sphincter failure. Throughout the esophagitis experiment, BPC 157 was given at either 10 micro g/kg, i.p., once a day (last application 24 h before assessment) or alternatively, it was given continuously in drinking water at 0.16 micro g/ml (12 ml/rat). This treatment recovers i) esophagitis (macroscopically and microscopically, at either region or investigated time period) and ii) pressure in both sphincters (cmH2O). In addition, BPC 157 (10 micro g/kg) or saline (1 ml/rat, 5 ml/kg) was specifically given directly into the stomach; pressure assessment was performed at 5 min thereafter. The effect of BPC 157 is specific because in normal rats, it increases lower esophageal sphincter-pressure, but decreases pyloric sphincter-pressure. Ranitidine, given as the standard drug using the same protocol (50 mg/kg, i.p., once daily; 0.83 mg/ml in drinking water; or 50 mg/kg directly into the stomach) had no effect.

Different effect of antiulcer agents on rat cysteamine-induced duodenal ulcer after sialoadenectomy, but not gastrectomy.

The focus was on salivary glands in cysteamine-induced duodenal ulcer and the different effects of antiulcer agents on cysteamine-induced duodenal ulcer in sialoadenectomized but not gastrectomized rats. We tested antiulcer agents on cysteamine-induced duodenal ulcer in rats (agents/kg i.p.) simultaneously with cysteamine 400 mg/kg s.c., rat killed 24 h thereafter subjected to no surgery (normal), to gastrectomy (24 h before) or sialoadenectomy, acute (24 h before) or chronic (21 days before). (i) Ulcerogenesis: cysteamine-induced duodenal ulcer had the same severity and incidence in normal, gastrectomized or acutely or chronically sialoadenectomized rats. (ii) Antiulcer effect under normal conditions or following gastrectomy: in normal or gastrectomized rats all agents tested, gastric pentadecapeptide BPC 157 [currently in clinical trials for inflammatory bowel disease (PL-10, PLD-116, PL-14736, Pliva) (10.0 microg or 10.0 ng), ranitidine (10 mg), atropine (10 mg), omeprazole (10 mg)] inhibited cysteamine-induced duodenal ulcers, acting through gastric acid-independent mechanisms. Following sialoadenectomy, acute or chronic: ranitidine, omeprazole and atropine were completely ineffective, while pentadecapeptide BPC 157 could protect. Thus, we found that contrary to stomach, salivary glands are implicated in cytoprotective agent activity (standard agents were ineffective after sialoadenectomy). Also, gastric pentadecapeptide BPC 157 was consistently associated with a cytoprotective effect, suggesting a beneficial activity distinctive from that of H2-receptor blockers, proton-pump inhibitors and anticholinergics; but probably replacing missing salivary glands factors.