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Storage of human retinal pigment epithelium (hRPE) can contribute to the advancement of cell-based RPE replacement therapies. The present study aimed to improve the quality of stored hRPE cultures by identifying storage medium additives that, alone or in combination, contribute to enhancing cell viability while preserving morphology and phenotype. hRPE cells were cultured in the presence of the silk protein sericin until pigmentation. Cells were then stored for 10 days in storage medium plus sericin and either one of 46 different additives. Individual effects of each additive on cell viability were assessed using epifluorescence microscopy. Factorial design identified promising additive combinations by extrapolating their individual effects. Supplementing the storage medium with sericin combined with adenosine, L-ascorbic acid and allopurinol resulted in the highest cell viability (98.6 ± 0.5%) after storage for three days, as measured by epifluorescence microscopy. Flow cytometry validated the findings. Proteomics identified 61 upregulated and 65 downregulated proteins in this storage group compared to the unstored control. Transmission electron microscopy demonstrated the presence of melanosomes after storage in the optimized medium. We conclude that the combination of adenosine, L-ascorbic acid, allopurinol and sericin in minimal essential medium preserves RPE pigmentation while maintaining cell viability during storage.

The purpose of this work was to determine whether the morphology of the oral mucosa epithelium (OME) of patients with xerostomia differ from patients without xerostomia. In total, 34 patients with dry eye disease (DED) with or without xerostomia were examined at The Norwegian Dry Eye Disease Clinic with in vivo confocal microscopy of the lower lip. In addition, age- and gender-matched healthy controls (HC) were included. DED patients with xerostomia had a higher superficial to deep backscatter ratio compared with DED patients without xerostomia (p=0.002) and HC (p=0.001). Regression analysis demonstrated that this ratio was related to xerostomia independently of gender and age (p<0.001). Sensitivity and specificity of detecting xerostomia were 0.78 and 0.85, respectively, when using a superficial to deep backscatter ratio cut-off value of 0.995 (p=0.004). The mean nucleus to cytosol backscatter ratio in the superficial OME was lower in patients with xerostomia than in those without xerostomia (p=0.034). In vivo confocal microscopy is a potential tool for evaluating the oral cavity and to assess changes in the OME associated with xerostomia, objectively and quantitatively. The cause of the increased backscatter in the superficial OME in xerostomia, however, remains to be elucidated.

The pressure-volume relationship in the ascending aorta ("windkessel") of the hooded seal was determined and the morphology of its vasa vasorum described in some detail. We found that the ascending aorta has a high compliance and can easily accommodate the entire stroke volume when the peripheral vascular resistance becomes much increased and maintain perfusion pressure during the much extended diastole and thereby reduce cardiac stroke work during diving. We also found that the 3- to 5-mm thick wall of the ascending aorta had a very elaborate vasa vasorum interna with a hitherto undescribed vascular structure that penetrates the entire vascular wall. If similar structures with similar importance for the nutrition of the wall of the vessel are found in humans, important implications for the understanding of pathological conditions, such as aneurisms, may be indicated., (Copyright © 2016 the American Physiological Society.)

The purpose of the study was to investigate if xerostomia (dry mouth) is associated with symptoms and signs of dry eye disease (DED). At the Norwegian Dry Eye Clinic, patients with symptomatic DED with different etiologies were consecutively included in the study. The patients underwent a comprehensive ophthalmological work-up and completed self-questionnaires on symptoms of ocular dryness (Ocular Surface Disease Index [OSDI] and McMonnies Dry Eye Questionnaire) and the Sjögren's syndrome (SS) questionnaire (SSQ). Three hundred and eighteen patients (52% women and 48% men) with DED were included. Patient demographics were: 0 to 19 years (1%), 20 to 39 (25%), 40 to 59 (34%), 60 to 79 (35%) and 80 to 99 (5%). Xerostomia, defined as "daily symptoms of dry mouth the last three months" (as presented in SSQ) was reported by 23% of the patients. Female sex was more common among patients with xerostomia (81%) than among non-xerostomia patients (44%; P<0.001). Patients with xerostomia (60 ± 15 years) were older than those without xerostomia (51 ± 17; P<0.001). The use of prescription drugs was more prevalent among xerostomia patients (65%) than among non-xerostomia patients (35%; P<0.021; adjusted for age and sex). Patients with xerostomia had a higher OSDI score (19.0 ± 10.0) than those without xerostomia (12.9 ± 8.0; P<0.001). Moreover, xerostomia patients had more pathological meibum expressibility (0.9 ± 0.7) than those without xerostomia (0.7 ± 0.8; P = 0.046). Comparisons of OSDI and ocular signs were performed after controlling for the effects of sex, age and the number of systemic prescription drugs used. In conclusion, xerostomia patients demonstrated a higher DED symptom load and had poorer meibum expressibility than non-xerostomia patients.

Purpose: Storage of cultured human oral keratinocytes (HOK) allows for transportation of cultured transplants to eye clinics worldwide. In a previous study, one-week storage of cultured HOK was found to be superior with regard to viability and morphology at 12°C compared to 4°C and 37°C. To understand more of how storage temperature affects cell phenotype, gene expression of HOK before and after storage at 4°C, 12°C, and 37°C was assessed., Materials and Methods: Cultured HOK were stored in HEPES- and sodium bicarbonate-buffered Minimum Essential Medium at 4°C, 12°C, and 37°C for one week. Total RNA was isolated and the gene expression profile was determined using DNA microarrays and analyzed with Partek Genomics Suite software and Ingenuity Pathway Analysis. Differentially expressed genes (fold change > 1.5 and P < 0.05) were identified by one-way ANOVA. Key genes were validated using qPCR., Results: Gene expression of cultures stored at 4°C and 12°C clustered close to the unstored control cultures. Cultures stored at 37°C displayed substantial change in gene expression compared to the other groups. In comparison with 12°C, 2,981 genes were differentially expressed at 37°C. In contrast, only 67 genes were differentially expressed between the unstored control and the cells stored at 12°C. The 12°C and 37°C culture groups differed most significantly with regard to the expression of differentiation markers. The Hedgehog signaling pathway was significantly downregulated at 37°C compared to 12°C., Conclusion: HOK cultures stored at 37°C showed considerably larger changes in gene expression compared to unstored cells than cultured HOK stored at 4°C and 12°C. The changes observed at 37°C consisted of differentiation of the cells towards a squamous epithelium-specific phenotype. Storing cultured ocular surface transplants at 37°C is therefore not recommended. This is particularly interesting as 37°C is the standard incubation temperature used for cell culture.

Purpose/aims: To assess the effect of storage temperature on the viability, phenotype, metabolism, and morphology of cultured human oral keratinocytes (HOK)., Materials and Methods: Cultured HOK cells were stored in HEPES- and sodium bicarbonate-buffered Minimum Essential Medium (MEM) at nine temperatures in approximately 4 °C increments from 4 °C to 37 °C for seven days. Cells were characterized for viability by calcein fluorescence, phenotype retention by immunocytochemistry, metabolic parameters (pH, glucose, lactate, and O2) within the storage medium by blood gas analysis, and morphology by scanning electron microscopy and light microscopy., Results: Relative to the cultured, but non-stored control cells, a high percentage of viable cells were retained only in the 12 °C and 16 °C storage groups (85% ± 13% and 68% ± 10%, respectively). Expression of ABCG2, Bmi1, C/EBPδ, PCNA, cytokeratin 18, and caspase-3 were preserved after storage in the 5 groups between 4 °C and 20 °C, compared to the non-stored control. Glucose, pH and pO2 in the storage medium declined, whereas lactate increased with increasing storage temperature. Morphology was best preserved following storage of the three groups between 12 °C, 16 °C, and 20 °C., Conclusion: We conclude that storage temperatures of 12 °C and 16 °C were optimal for maintenance of cell viability, phenotype, and morphology of cultured HOK. The storage method described in the present study may be applicable for other cell types and tissues; thus its significance may extend beyond HOK and the field of ophthalmology.

Purpose: To evaluate the effect of storage temperature on the morphology, viability, cell number and metabolism of cultured human conjunctival epithelial cells (HCjEs)., Materials and Methods: Three-day cultured HCjEs were stored at nine different temperatures between 4 °C and 37 °C for four and seven days. Phenotype was assessed by immunofluorescence microscopy, morphology by scanning electron microscopy, viability and cell number by a microplate fluorometer and glucose metabolism by a blood gas analyzer., Results: Cultured cells not subjected to storage expressed the conjunctival cytokeratins 7 and 19 and the proliferation marker proliferating cell nuclear antigen. Cell morphology was best maintained following four-day storage between 12 °C and 28 °C and following 12 °C storage after seven days. Assessed by propidium iodide uptake, the percentage of viable cells after four-day storage was maintained only between 12 °C and 28 °C, whereas it had decreased in all other groups (p < 0.05; n = 4). After seven days this percentage was maintained in the 12 °C group, but it had decreased in all other groups, compared to the control (p < 0.05; n = 4). The total number of cells remaining in the cultures after four-day storage, compared to the control, had declined in all groups (p < 0.05; n = 4), except 12 °C and 20 °C groups. Following seven days this number had decreased in all groups (p < 0.01; n = 4), except 12 °C storage. Four-day storage at 12 °C demonstrated superior preservation of the number of calcein-stained viable cells (p < 0.05) and the least accumulation of ethidium homodimer 1-stained dead cells (p < 0.001), compared to storage at 4 °C and 24 °C (n = 6). The total metabolism of glucose to lactate after four-day storage was higher in the 24 °C group compared to 4 °C and 12 °C groups, as well as the control (p < 0.001; n = 3)., Conclusions: Storage at 12 °C appears optimal for preserving the morphology, viability and total cell number in stored HCjE cultures. The superior cell preservation at 12 °C may be related to temperature-associated effects on cell metabolism.

Introduction: There is increasing evidence that retinal pigment epithelium (RPE) can be used to treat age-related macular degeneration, one of the leading causes of blindness worldwide. However, the best way to store RPE to enable worldwide distribution is unknown. We investigated the effects of supplementing our previously published storage method with seven additives, attempting to improve the number of viable adult retinal pigment epithelial (ARPE)-19 cells after storage., Materials and Methods: ARPE-19 cells were cultured on multiwell plates before being stored for 1 week at 16 °C. Unsupplemented Minimal Essential Medium (MEM) (control) and a total of seven individual additives (DADLE ([D-Ala(2), D-Leu(5)]-encephalin), capsazepine, docosahexaenoic acid (DHA), resveratrol, quercetin, simvastatin and sulforaphane) at three to four concentrations in MEM were tested. The individual effect of each additive on cell viability was analyzed with a microplate fluorometer. Cell phenotype was investigated by both microplate fluorometer and epifluorescence microscopy, and morphology by scanning electron microscopy., Results: Supplementation of the storage medium with DADLE, capsazepine, DHA or resveratrol significantly increased the number of viable cells by 86.1% ± 41.9%, 67.9% ± 24.7%, 36.5% ± 10.3% and 21.1% ± 6.4%, respectively, compared to cells stored in unsupplemented MEM. DHA and resveratrol significantly reduced caspase-3 expression, while expression of RPE65 was maintained across groups., Conclusion: The number of viable ARPE-19 cells can be increased by the addition of DADLE, capsazepine, DHA or resveratrol to the storage medium without perturbing apoptosis or differentiation.

Cultured epidermal cell sheets (CECS) are used in regenerative medicine in patients with burns, and have potential to treat limbal stem cell deficiency (LSCD), as demonstrated in animal models. Despite widespread use, short-term storage options for CECS are limited. Advantages of storage include: flexibility in scheduling surgery, reserve sheets for repeat operations, more opportunity for quality control, and improved transportation to allow wider distribution. Studies on storage of CECS have thus far focused on cryopreservation, whereas refrigeration is a convenient method commonly used for whole skin graft storage in burns clinics. It has been shown that preservation of viable cells using these methods is variable. This study evaluated the effect of different temperatures spanning 4°C to 37°C, on the cell viability, morphology, proliferation and metabolic status of CECS stored over a two week period in a xenobiotic-free system. Compared to non-stored control, best cell viability was obtained at 24°C (95.2±9.9%); reduced cell viability, at approximately 60%, was demonstrated at several of the temperatures (12°C, 28°C, 32°C and 37°C). Metabolic activity was significantly higher between 24°C and 37°C, where glucose, lactate, lactate/glucose ratios, and oxygen tension indicated increased activation of the glycolytic pathway under aerobic conditions. Preservation of morphology as shown by phase contrast and scanning electron micrographs was best at 12°C and 16°C. PCNA immunocytochemistry indicated that only 12°C and 20°C allowed maintenance of proliferative function at a similar level to non-stored control. In conclusion, results indicate that 12°C and 24°C merit further investigation as the prospective optimum temperature for short-term storage of cultured epidermal cell sheets.

Whales are unique in that the supply of blood to the brain is not by the internal carotid arteries, but by way of thoracic and intra-vertebral arterial retia. We found in the harbor porpoise (Phocoena phocoena) that these retia split up into smaller anastomosing vessels and thin-walled sinusoid structures that are embedded in fat. The solubility of nitrogen is at least six times larger in fat than in water, and we suggest that nitrogen in supersaturated blood will be absorbed in the fat, by diffusion, during the very slow passage of the blood through the arterial retia. Formation of nitrogen bubbles that may reach the brain is thereby avoided. We also suggest that mass stranding of whales may be due to disturbances to their normal dive profiles, resulting in extra release of nitrogen that may overburden the nitrogen 'trap' and allow bubbles to reach the brain and cause abnormal behavior.

Purpose. The establishment of future retinal pigment epithelium (RPE) replacement therapy is partly dependent on the availability of tissue-engineered RPE cells, which may be enhanced by the development of suitable storage methods for RPE. This study investigates the effect of different storage temperatures on the viability, morphology, and phenotype of cultured RPE. Methods. ARPE-19 cells were cultured under standard conditions and stored in HEPES-buffered MEM at nine temperatures (4°C, 8°C, 12°C, 16°C, 20°C, 24°C, 28°C, 32°C, and 37°C) for seven days. Viability and phenotype were assessed by a microplate fluorometer and epifluorescence microscopy, while morphology was analyzed by scanning electron microscopy. Results. The percentage of viable cells preserved after storage was highest in the 16°C group (48.7% ± 9.8%; P < 0.01 compared to 4°C, 8°C, and 24°C-37°C; P < 0.05 compared to 12°C). Ultrastructure was best preserved at 12°C, 16°C, and 20°C. Expression of actin, ZO-1, PCNA, caspase-3, and RPE65 was maintained after storage at 16°C compared to control cells that were not stored. Conclusion. Out of nine temperatures tested between 4°C and 37°C, storage at 12°C, 16°C, and 20°C was optimal for maintenance of RPE cell viability, morphology, and phenotype. The preservation of RPE cells is critically dependent on storage temperature.

The use of amniotic membrane (AM) represents one of the major developments in ocular surface reconstruction. However, in a study on patients with primary pterygium, transplantation of AM with ex vivo expanded human conjunctival epithelial cells (HCjE) promoted earlier epithelialization than AM alone. We previously showed that cultured human limbal epithelial cells maintain their morphology, phenotype, and viability for one week when stored at 23°C. The current study investigates the feasibility of storing HCjE in HEPES-MEM and Optisol-GS at 23°C for 4 and 7 days, respectively. The five experimental groups were analyzed by light microscopy, immunohistochemistry, transmission electron microscopy, and a viability assay. The ultrastructural integrity of cultured HCjE was well preserved following 4 days of storage, however, 7 days of storage resulted in some loss of cell-cell contacts and epithelial detachment from the amniotic membrane. The number of microvilli in cultured HCjE not subjected to storage was 2.03±0.38 microvilli/μm. In comparison, after 4 and 7 days of HEPES-MEM storage this number was 1.69±0.54 microvilli/μm; P=0.98 and 0.89±1.0 microvilli/μm; P=0.28, respectively. After Optisol-GS storage for 4 and 7 days, the mean number of microvilli was 1.07±1.0 microvilli/μm; P=0.47 and 0.07±0.07 microvilli/μm; P=0.03, respectively. The number of cell layers in cultured HCjE not subjected to storage was 4.4±0.3 cell layers, as opposed to 4.0±0.9 cell layers; P=0.89 after 4 days of HEPES-MEM storage and 2.8±0.6 cell layers; P=0.01 after 7 days of storage in HEPES-MEM. The number of cell layers after 4 and 7 days of storage in Optisol-GS was 3.7±0.2 cell layers; P=0.46 and 3.4±0.4 cell layers; P=0.18, respectively. The expression of markers for undifferentiated cells (ΔNp63α, ABCG2 and p63), proliferating cells (Ki67 and PCNA), goblet cells (Ck7 and MUC5AC), stratified squamous epithelial cells (Ck4), and apoptotic cells (caspase-3) in cultured HCjE appeared to be unchanged after 4 and 7 days of HEPES-MEM and Optisol-GS storage. The percentage of viable cells in cultured HCjE not subjected to storage (91.4%±3.2%) was sustained after 4 and 7 days of storage in HEPES-MEM (94.1%±4.5%; P=0.99 and 85.1%±13.7%; P=0.87, respectively) as well as after 4 and 7 days of storage in Optisol-GS (87.7%±15.2%; P=0.97 and 79.8%±15.7%; P=0.48, respectively). We conclude that cultured HCjE may be stored for at least 4 days in serum-free conditions at 23°C while maintaining the phenotype and viability. HEPES-MEM appears to be comparable to Optisol-GS for serum-free storage with preservation of the ultrastructure for at least 4 days., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Aquaporin 5 (AQP5) is known to be central for salivary fluid secretion. A study of the temporal-spatial distribution of AQP5 during submandibular gland (SMG) development and in adult tissues might offer further clues to its unknown role during development. In the present work, SMGs from embryonic day (E) 14.5-18.5 and postnatal days (P) 0, 2, 5, 25, and 60 were immunostained for AQP5 and analyzed using light microscopy. Additional confocal and transmission electron microscopy were performed on P60 glands. Our results show that AQP5 expression first occurs in a scattered pattern in the late canalicular stage and becomes more prominent and organized in the terminal tubuli/pro-acinar cells towards birth. Additional apical membrane staining in the entire intralobular duct is found just prior to birth. During postnatal development, AQP5 is expressed in both the luminal and lateral membrane of pro-acinar/acinar cells. AQP5 is also detected in the basal membrane of acinar cells at P25 and P60. In the intercalated ducts at P60, the male glands show apical staining in the entire segment, while only the proximal region is positive in the female glands. These results demonstrate an evolving distribution of AQP5 during pre- and postnatal development in the mouse SMGs.

Brain (T(brain)), intra-aorta (T(aorta)), latissimus dorsi muscle (T(m)) and rectal temperature (T(r)) were measured in harp (Pagophilus groenlandicus) and hooded (Cystophora cristata) seals during experimental dives in 4 degrees C water. The median brain cooling was about 1 degrees C during 15 min diving, but in some cases it was as much as 2.5 degrees C. Cooling rates were slow for the first couple of minutes, but increased significantly after about 5 min of diving. The onset of cooling sometimes occurred before the start of the dive, confirming that the cooling is under cortical control, like the rest of the diving responses. T(aorta) also fell significantly, and was always lower than T(brain), while T(m) was fairly stable during dives. Detailed studies of the vascular anatomy of front flippers revealed that brachial arterial blood can be routed either through flipper skin capillaries for nutritive purposes and return through sophisticated vascular heat exchangers to avoid heat loss to the environment, or, alternatively, through numerous arterio-venous shunts in the skin and return by way of large superficial veins, which then carry cold blood to the heart. In the latter situation the extent to which the brain is cooled is determined by the ratio of carotid to brachial arterial blood flow, and water temperature, and the cooling is selective in that only those organs that are circulated will be cooled. It is concluded that T(brain) is actively down-regulated during diving, sometimes by as much as 2.5 degrees C, whereby cerebral oxygen requirements may be reduced by as much as 25% during extended dives.

This study examined the expression, in oral keratinocytes and in the major and minor salivary glands, of Trefoil factor family 3 (TFF3) peptide. Trefoil factor family 3 messenger RNA (mRNA) and peptide were detected in cultures of normal oral keratinocytes by quantitative real-time polymerase chain reaction (PCR) and western blotting, respectively. Trefoil factor family 3 was found, by immunohistochemical analyses, to be expressed in the basal layers of the oral mucosal epithelium. In salivary glands, immunohistochemical staining showed that TFF3 peptide expression was strongest in the mucous acini of the submandibular and the small salivary glands. Serous cells in the same glands showed weak staining. In the parotid gland, many serous acini showed weak positive staining, while other areas did not. In all glands examined, the intercalated, striated, and collecting ducts were moderately TFF3-positive. Double immunostaining confirmed that mucous (MUC5B positive) cells were moderately or strongly positive for TFF3 and that some serous (MUC7 positive) cells showed restricted TFF3 expression, mostly in a granular pattern. The prevalence of the TFF3 peptide in the salivary secretions of healthy volunteers was detected by western blotting of saliva from minor salivary glands (four of five) and the parotid gland (one of five) and of mixed submandibular/sublingual saliva (five of five). In conclusion, the submandibular and small salivary glands appear to be the major producers of oral TFF3, but duct cells of all glands and keratinocytes of the oral mucosa may also contribute as sources of TFF3 in the oral cavity.

Objectives: This in vitro study was aimed to investigate the attachment, spreading and proliferation of human gingival fibroblasts to milled and polished non-veneered ceramic surfaces in alumina and zirconia and to ceramic surfaces veneered by two different types of porcelain baseliners., Materials and Methods: Fibroblasts were cultured on discs of pressed alumina or zirconia, on discs which had been milled, on discs comprising alumina or zirconia which had been polished, on discs of alumina veneered with NobelRondo baseliner Al, on discs of zirconia veneered with Cercon-S baseliner, and on alumina or zirconia discs veneered with the above baseliners and then polished. The surfaces were analyzed using an optical interferometer and scanning electron microscopy (SEM). Cell profile areas were measured using SEM and an image analyzer. Cell attachment was determined after 3 and 24 h as a ratio of the cell profiles and the total micrograph area and was expressed as percent of attachment. MTT analyses were undertaken to determine cellular attachment after 3 h of incubation and cellular proliferation after 7 days., Results: The polished zirconia specimens had the smoothest surface in terms of average height deviation (S(a)=0.03 microm): the roughest were the zirconia specimens with milled surfaces (S(a)=0.36 microm). The application of the baseliners resulted in surfaces smoother than those of the non-veneered discs. The milled surfaces of both alumina and zirconia had significantly higher percentages of cell attachment and proliferation than the other surfaces whereas the milled surfaces in zirconia demonstrated better cellular attachment after 3 and 24 h of culture than the one in alumina. Fibroblasts attached and grew effectively on the surfaces veneered with NobelRondo throughout the experiments, whereas the zirconia surfaces veneered with Cercon-S had the lowest percentage of cell attachment and proliferation., Conclusions: Although the roughness of all surfaces investigated was <0.4 mum, the study disclosed significant differences in cellular attachment and proliferation associated with the various surface modifications.

Purpose: A previous report has described the use of eye bank storage of cultured human limbal epithelial cells (HLECs) to provide a reliable source of tissue for treating limbal stem cell deficiency. In the present study, conventional organ culture (OC) storage and Optisol-GS (Bausch & Lomb, Irvine, CA) storage of cultured HLECs were compared., Methods: Three-week HLEC cultures were either organ cultured at 31 degrees C or 23 degrees C or stored in Optisol-GS at 5 degrees C in a closed container for 1 week. Morphology was studied by light microscopy and transmission electron microscopy, and phenotypic characterization was assessed by immunohistochemistry. Apoptosis was evaluated by real-time RT-PCR microarray analysis, caspase-3 immunohistochemistry, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)., Results: The ultrastructure was preserved at 23 degrees C, while storage at 31 degrees C and 5 degrees C was associated with enlarged intercellular spaces, separation of desmosomes, and detachment of epithelial cells. Cultured HLECs remained undifferentiated in all storage conditions. The expression of the antiapoptotic gene BCL2 was prominently upregulated in storage at 23 degrees C and 5 degrees C. Downregulation of BCL2A1, BIRC1, and TNF and upregulation of CARD6 in 23 degrees C and 5 degrees C storage conditions suggests a reduction in nuclear factor-kappaB activity. No significant increase in cleaved caspase-3 and TUNEL staining was observed in response to eye bank storage, and the labeling indices of cleaved caspase-3 (range, 0.0%-4.7%) and TUNEL (range, 0.0%-7.8%) were low., Conclusions: These data indicate that OC storage of cultured HLECs at ambient temperature is superior to OC storage at 31 degrees C and Optisol-GS storage at 5 degrees C and that apoptosis is minimal after eye bank storage of cultured HLECs.

An animal study is presented examining the effect of low level laser (LLL) treatment on nerve regeneration following axonotmesis. Twenty animals received a standardised injury to the right sciatic nerve using a time, load and length sequence (10 min, 150 N, 5 mm) known to cause extensive axonal degeneration of the rat sciatic nerve. The LLL treatment was administered using a hand-held laser probe in light contact with the skin on the dorsal aspect of the hind leg overlying the site of the axonotmesis injury to the sciatic nerve. A group of 10 animals were treated with 6J of LLL (GaAlAs 830 nm) daily for a period of 28 d. Ten more animals were treated daily with a sham exposure setting and served as controls. Nerve function was assessed by a recognised method of walking tract print analysis; the "Sciatic Functional Index" (SFI), and nerve regeneration was assessed by recording the evoked compound action potentials (cAP) in the common peroneal nerve. At 21 d post-injury, the laser-treated group had a significantly lower median SFI than the sham laser-treated group, indicating that the real laser treatment had improved functional recovery in the nerve. However, no differences were found between the evoked cAP parameters that were measured in the laser-treated and sham laser-treated groups. Histological examination reiterated the lack of difference between the two groups. Consequently, the effects of LLL on recovery must have occurred more peripherally to the point measured.

The present study describes an in vivo model for the collection of the subgingival pellicle adsorbed to tooth surface, and the identification of some serum proteins within this layer. Clean dentin slabs were prepared from freshly extracted teeth, and then placed subgingivally for 2 h. The dentin slabs with their adsorbed pellicle layer were processed for transmission electron microscopy. Thin sections were made from the specimens, and treated with antisera to human immunoglobulins and albumin. The reactions were visualized by means of protein A-gold complex, which allowed semiquantification of the serum proteins. The indicator proteins were all identified within the pellicle material, but their amounts and distribution varied. Albumin demonstrated higher amounts in the pellicle layer than other proteins, followed by IgA, IgG, and IgM in descending order. The model described seems useful for studying the acquired subgingival pellicle under varying degrees of disease and health.

The separating zone of rat submandibular gland striated duct cells were studied in detail by electron microscopy. In order to get optimal stimulation of the glands the animals were starved for 24 h followed by feeding 15 min before sacrifice, and tissue removal. The separating zone is morphologically defined by numerous filaments which are located within the apical part of cell. Some of the filaments had a parallel arrangement, running horizontally and being attached to belt desmosomes at the cell periphery. Following the shedding of apical blebs, these filaments appeared to constitute the luminal boundary of striated duct cells. It was observed that the separating zones showed different degrees of density. This is supposed to reflect different stages in the reconstruction of the apical cell surface. It is thus considered that the separating zone, with numerous filaments, is of importance both of the apical secretion per se, and the formation of the new apical cell membrane.

Enzyme variants may serve an adaptive role in providing the correct vectorial properties for the metabolism of a tissue, or broadening its environmental tolerance range. To determine if the LDH isoenzyme pattern of human dental pulp changes during inflammation, supernatants from normal and inflamed dental pulp homogenates were separated by polyacrylamide gel electrophoresis. Inflamed pulps had a higher M subunit content and a markedly increased enzyme activity. These results might reflect adaptive changes at the enzyme level associated with a partial shift towards anaerobic metabolism during inflammation of the pulp.

The homeothermic seal possesses a heterothermic skin, while the skin of the sheep behaves as a truly homeothermic tissue. A new method for skin homogenization is described. The effect of temperature on the catalytic behaviour of seal and sheep skin LDH has been investigated by fluorimetric activity measurements, with results presented as Arrhenius plots. The seal skin enzyme had ten times higher activity at low temperatures as compared to the sheep skin. The mechanisms responsible for this different behaviour are discussed.

Previous investigations have indicated that striated-duct cells react to stimulation with an apocrine secretion, morphologically demonstrated by bleb-like projections of the apical cytoplasm. Since bleb formation as an ultrastructural feature also has been debated and sometimes interpreted as a fixation artifact, it was considered essential to extend the studies by exposing the submandibular gland to X rays to establish whether such treatment would have any influence on the formation of blebs. The material used in the present study consisted of rat submandibular glands exposed to X rays in the range of 200-1800 rad. The glands were examined by both SEM and TEM. The duct cells exposed to 200 rad appeared normal, with no sign of alteration in their ability to produce blebs, whereas duct cells exposed to 750 rad showed no sign of bleb formation. Some of the duct cells exposed to 1800 rad showed considerable morphological changes, consistent with oncotic transformation. The results support the conclusion that bleb formation is a normal morphological feature and not an artifact. This study also indicates that the functional activity of the cells is reduced after exposure to X rays.

Apical blebs of rat submandibular gland striated ducts were studied by electron microscopy. The glands were stimulated by starvation of the animals for 24 h whereafter they were fed 15 min before death and tissue removal. After stimulation most striated duct cells developed apical blebs, with various shapes and usually without granules and vesicles. A semi-separating filamentous structure separates the cell from the content of the bleb. Apparently only the vesicular and granular content can pass through this structure and enter the bleb. Following the appearance of small ruptures in the bleb membrane, the bleb is discarded into the lumen of the duct where it disintegrates. Typical wall-like protrusions on the luminal surface of the duct cell, are probably, rudiments of the ruptured bleb membrane. It is suggested that the apical blebs are manifestations of apocrine secretion.

For the present study Seal (Phoca vitulina) and rat submandibular gland striated ducts were investigated by electron microscopy. Both normal and stimulated animals (starved for 24 h and then fed 2 h before the tissues were removed) were examined. Basal invaginations of the cell membrane with areas heavily loaded with mitochondria were typical features of both animals. Secretory granules were especially numerous in the apical part of stimulated duct cells. The granules were separated from the luminal membrane of the cells by a condensed area called the spearating zone. Apical protrusions or blebs, which were frequently occurring in striated ducts of both animals, are interpreted as manifestations of apocrine secretion. It was concluded that this way of apocrine secretion is a fundamental function, since seals, which have salivary glands of a rather simple composition with acini which are functionally reduced, have retained the ability to form blebs.

Several previous investigations have shown that blebs form on the apical surface of the striated duct cells of the rat submandibular gland on feeding after starvation. In the present report the influence of autonomic nerve stimulation on bleb formation was studied by electron microscopy. Both parasympathetic and sympathetic stimulation were performed, using electric nerve stimulation. In addition, sympathetic nerve stimulation in combination with alpha- or beta-adrenergic blockers was used. Massive bleb formation took place in response to sympathetic nerve stimulation. This response was almost completely abolished by the administration of alpha- but not by beta-adrenergic blocker. Bleb formation was not seen after parasympathetic nerve stimulation.