Tooth pulp tissue promotes neurite outgrowth from rat trigeminal ganglia in vitro.

The mammalian tooth pulp becomes innervated by nociceptive and sympathetic axons relatively late during development, when part of the root has formed. In the adult, regenerating axons from an injured tooth nerve or sprouting axons from uninjured nerves in the vicinity rapidly reinnervate denervated tooth pulps. These observations indicate that tooth pulp tissue can use molecular factors to attract pulpal axons from local nerve trunks. The present study examines the hypothesis that these factors include nerve growth factor (NGF), brain derived neurotrophic factor (BDNF) and glial cell line derived neurotrophic factor (GDNF). Explants of trigeminal ganglia from neonatal rat pups showed a distinct neurite outgrowth when co-cultured with pulpal explants collected from molar teeth of 12-day old pups, or after application of a pulpal extract. Control cultures, containing single ganglionic explants, or explants co-cultured with heat-treated pulpal tissue, exhibited a sparse neurite outgrowth. Exogenous NGF and/or GDNF, but not exogenous BDNF, stimulated neurite outgrowth from ganglionic explants. Unexpectedly, application of antibodies against NGF, BDNF and/or GDNF to co-cultures of ganglionic and pulpal explants did not inhibit neuritogenesis. Control experiments showed that IgG molecules readily penetrate the gel used for culture and that even very high concentrations of NGF and GDNF antibodies in combination failed to block neurite growth. On the basis of these data we suggest that other as yet unknown neurite-promoting factors might be present and active in TG/pulpal co-cultures.

Target influence on aging of myelinated sensory nerve fibres.

Large myelinated fibres in the rat inferior alveolar nerve (IAN) exhibit age-related aberrations partly similar to alterations in target-deprived nerves. Shortly after entering the mandibular canal the rat IAN splits into a mental (MN) and an inferior dental (IDN) portion. This offers a possibility to compare the occurrence of age-related aberrations in the MN and the IDN, which project to different target types. Hence, we examined the relation between internodal length (L) and fibre diameter (D) in teased fibre preparations of MNs and IDNs from adult rats of different ages. The results show that the relation L/D is similar in MNs and IDNs of 6- to 24-month-old rats and that it is significantly more irregular in IDNs than in MNs of 26- to 30-month-old rats. This difference may be related to the fact that the IDN-innervated mandibular dentition is deteriorating in rats older than 2 years, whereas the muco-cutaneous target of the MN is not. We suggest that target deterioration may be one major factor behind the increasing incidence of structural aberrations in large myelinated peripheral nerve fibres.

Maleimide-mediated protein conjugates of a nucleoside triphosphate gamma-S and an internucleotide phosphorothioate diester.

The purpose of this study was to determine whether the gamma-S of nucleoside thiotriphosphates and the non-bridging sulfur of internucleotide phosphorothioate diesters possess sufficient thiol character to form adducts with maleimides. Adenosine triphosphate gamma-S (ATPS) and thymidyl-PS-thymidine (TPST) were each reacted with the reporter molecule N-1 pyrene maleimide (PM) and the fluorescence intensity was recorded. The observed reactivity of the phosphorothioate nucleotides towards maleimide was used as a basis for preparing covalent protein-nucleotide conjugates of ATPS and of the internucleotide phosphorothioate diester, deoxyadenylyl-PS-deoxy-adenylyl-PS-deoxyadenosine (dA3(PS)2). The absorbance spectra of bovine serum albumin (BSA) conjugates of ATPS and of dA3(PS)2 showed the formation of protein-nucleotide conjugates, with absorbance maxima near 260 nm. The degree of conjugation was 1.69 nucleotides (nt)/BSA molecule for ATPS and 0.44 nt/BSA molecule for dA3(PS)2. The extent of conjugation of the gamma-S of the nucleoside thiotriphosphate and of the non-bridging sulfur of the internucleotide phosphorothioate diester with maleimide-derivatized protein agreed with their relative reactivity towards PM. Both the gamma-S of the nucleoside thiotriphosphate and the internucleotide phosphorothioate diester were found to possess sufficient thiol character to permit formation of maleimide-mediated protein conjugates.

Teeth and tooth nerves.

(1) Although our knowledge on teeth and tooth nerves has increased substantially during the past 25 years, several important issues remain to be fully elucidated. As a result of the work now going on at many laboratories over the world, we can expect exciting new findings and major break-throughs in these and other areas in a near future. (2) Dentin-like and enamel-like hard tissues evolved as components of the exoskeletal bony armor of early vertebrates, 500 million years ago, long before the first appearance of teeth. It is possible that teeth developed from tubercles (odontodes) in the bony armor. The presence of a canal system in the bony plates, of tubular dentin, of external pores in the enamel layer and of a link to the lateral line system promoted hypotheses that the bony plates and tooth precursors may have had a sensory function. The evolution of an efficient brain, of a head with paired sense organs and of toothed jaws concurred with a shift from a sessile filter-feeding life to active prey hunting. (3) The wide spectrum of feeding behaviors exhibited by modern vertebrates is reflected by a variety of dentition types. While the teeth are continuously renewed in toothed non-mammalian vertebrates, tooth turnover is highly restricted in mammals. As a rule, one set of primary teeth is replaced by one set of permanent teeth. Since teeth are richly innervated, the turnover necessitates a local neural plasticity. Another factor calling for a local plasticity is the relatively frequent occurrence of age-related and pathological dental changes. (4) Tooth development is initiated through interactions between the oral epithelium and underlying neural crest-derived mesenchymal cells. The interactions are mediated by cell surface molecules, extracellular matrix molecules and soluble molecules. The possibility that the initiating events might involve a neural component has been much discussed. With respect to mammals, the experimental evidence available does not support this hypothesis. In the teleost Tilapia mariae, on the other hand, tooth germ formation is interrupted, and tooth turnover ceases after local denervation. (5) Prospective dental nerves enter the jaws well before onset of tooth development. When a dental lamina has formed, a plexus of nerve branches is seen in the subepithelial mesenchyme. Shortly thereafter, specific branches to individual tooth primordia can be distinguished. In bud stage tooth germs, axon terminals surround the condensed mesenchyme and in cap stage primordia axons grow into the dental follicle.(ABSTRACT TRUNCATED AT 400 WORDS)

Functional properties and nodal spacing of myelinated fibers in developing rat mental and sural nerves.

While the postnatal length growth of the largest internodes in the rat sural nerve (SN) is proportional to nerve elongation, in the developing inferior alveolar nerve (IAN), early postnatal myelin sheath remodelling allows internodal lengthening to exceed the growth rate of the whole nerve. To assess the functional consequences of ongoing myelin sheath remodelling in a developing nerve, we examined the physiological properties of the mental nerve (MN), a cutaneous IAN branch and the SN during maturation. In addition, the nodal spacing and the microscopic anatomy of the nodes in the two nerves were studied. The youngest MNs and SNs (2 weeks) exhibited comparable sensitivities to K(+)-channel blockade with 4-aminopyridine (4-AP), although myelin sheath remodelling was more frequent in the MNs. Subsequently, myelin sheath remodelling ceased in both nerves but the MNs exhibited a greater sensitivity to 4-AP. Large fibers in adult MNs and SNs had a similar nodal anatomy but the former had shorter internodes. Thus, myelin sheath remodeling, per se, does not appear to be a determinant of 4-AP sensitivity in mammalian myelinated fibers. Rather, sensitivity to potassium channel blockade is more likely mediated at the internodal or molecular level.

Transfusion-associated Serratia marcescens infection: studies of the mechanism of action.

The growth of two strains of Serratia marcescens in blood components was tested in this study. One of the strains had been implicated in the epidemic of transfusion-associated sepsis experienced in Denmark and Sweden in 1991. In whole blood with a final concentration of 100 colony-forming units per mL of S. marcescens, there was an immediate reduction of more than 95 percent of colony-forming units, but no reduction of the bacterial concentration if the blood had been white cell-reduced before inoculation. This is interpreted as an effect due to phagocytosis by white cells and as a lack of bactericidal effect of the plasma. A reduction to 10 percent of the original concentration, observed if the blood had a nominal content of white cells, was most likely due to phagocytosis. White cell reduction by filtration after inoculation further reduced the bacterial concentration of one of the strains tested, but, after a 1-week lag phase, growth accelerated to high concentrations by 6 weeks. In platelet-rich plasma prepared from S. marcescens-inoculated units, abundant growth was found after 24 hours, increasing to very high concentrations (10(12) colony-forming units/mL) during 10-day storage at 22 +/- 2 degrees C. Keeping the whole blood at ambient temperature for 20 hours before preparation of platelet-rich plasma caused only temporary reduction of bacterial concentration in the S. marcescens experiments, but resulted in a complete absence of bacteria in the platelet-rich plasma for 10 days in control experiments performed with Staphylococcus epidermidis.(ABSTRACT TRUNCATED AT 250 WORDS)

Transfusion-transmitted Yersinia enterocolitica infection. Protection through buffy coat removal and failure of the bacteria to grow in platelet-rich or platelet-poor plasma.

In a previous study, removal of white blood cells (WBC), by filtration 5 h after deliberate contamination of whole blood with a type 0:3 strain of Yersinia enterocolitica, was shown to be an effective way of avoiding bacterial growth in red blood cells (RBC) during storage. In the present study the Opti-System technique was used to remove the buffy coat from whole blood, leaving 10-20% of the original number of WBC in the RBC preparation. In one series of experiments, all of 4 units of RBC suspension, from which buffy coats were removed 2 h after inoculation of 112 colony-forming units (cfu) per ml of Y. enterocolitica, became Yersinia-free, while abundant bacterial growth occurred in all of 4 units where RBC suspension and buffy coat had been recombined. In a second series of 10 experiments, with an inoculum of 80 cfu/ml, no growth was found in platelet-poor plasma stored for 42 days at 4 degrees C. Five out of 10 RBC suspensions in SAGM additive solution remained Yersinia-free throughout a 6-week storage period; 4 of these 10 units showing growth of Yersinia after 4 weeks and 5 after 6 weeks. In the buffy coats bacterial growth was found in 1 out of 10 units after 1 week, 4 after 2 weeks, and in all of 10 units after 4 weeks. In 2 control experiments with WBC-reduced RBC inoculated with the same bacterial dose, growth started within 24 h and was abundant after 1 week.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of white cells in the transmission of Yersinia enterocolitica in blood components.

The mechanism for the transmission of Yersinia enterocolitica in blood components has been studied experimentally. One hypothesis is that, during a Yersinia infection in the blood donor, bacteria are phagocytosed by white cells (WBCs), but are not killed. After collection of blood from such a donor and component production, the bacteria are present in WBCs for some time, during which the unit appears sterile. Later, when the WBCs disintegrate, the bacteria are released and multiply in the unit. Aliquots of whole blood and buffy coat were inoculated with 100 colony-forming units (CFU) per mL of a Y. enterocolitica strain of type O:3 and left at room temperature for 5 hours. Some aliquots were then WBC-reduced by filtration, while others retained their WBC contents. All aliquots were kept at 4 degrees C for 6 weeks. Meat extract broth culture medium was used as a control. Growth in the range of 2000 CFU per mL was obtained in the broth control by 24 hours, whereas the whole blood and buffy coat units appeared sterile for the first days of storage. After 1 week, a trace of bacteria and, after 4 weeks, massive growth were found in the WBC-containing units but not in the WBC-reduced units. The likely explanation is that the bacteria had been phagocytosed by the WBCs and were thereby hidden and not available for bacterial culture during the first phase of storage. When the WBCs spontaneously disintegrated, bacteria were released and multiplied in the blood units.(ABSTRACT TRUNCATED AT 250 WORDS)

Anatomy and developmental chronology of the rat inferior alveolar nerve.

This report describes the anatomy of the inferior alveolar neurovascular bundle in the adult rat and provides a quantitative analysis of the developing inferior alveolar nerve (IAN). Soon after its entrance in the mandibular canal, the IAN splits into a mental nerve (MN) and an inferior dental nerve (IDN), which course in separate bony compartments. The MN passes unbranched through the mandibular canal. The IDN sends branches to the incisor, the first molar, and the second molar. The third molar (M3) is supplied by a separate IAN branch. The adult rat IAN contains 8,000-10,000 axons, 70% of which are myelinated. The MN accounts for 70% of all IAN axons, the IDN 26%, and 4% form the M3 branch. The proportion of large myelinated axons is lower in the MN than in the IDN. Following chemical sympathectomy, the IAN axon number does not change in a statistically significant way. The total number of IAN axons, which is high prenatally and neonatally, has decreased to the adult level about 1 week after birth. De novo myelination commences at birth and is complete 3-4 weeks later. The size spectrum of the myelinated fibres is narrow and unimodal during the first postnatal weeks. By 1 month, the largest fibres reach diameters of approximately 6 microns, and a bimodal pattern is emerging. From 3 months and on, the size range reaches up to 10-12 microns, and the distribution is bimodal. These data provide a basis for further studies on developmental tooth-nerve interactions.

Nodal spacing in the developing, young adult and aging rat inferior alveolar nerve.

This study examines the nodal spacing (L) in teased preparations of developing, young adult and aging rat inferior alveolar nerves. In nerves from rats aged 1-2 weeks, most internodes show L-values, which increase from 150 microns to 400 microns, as fiber diameter (D) increases. Other internodes are very short (L = 20-150 microns), and exhibit distorted or fragmented myelin sheaths. In nerves from 2-3 week old rats such very short internodes are rare. By 3-4 weeks, and in young adult animals, very short internodes are lacking. The young adult relation L/D is regular and rectilinear. While D ranges from 2 microns to 10 microns, L ranges from 200 microns to 700-800 microns. In nerves from 1-2.5 year old adult rats some internodes are greater than 1000 microns long. These old nerves show signs of of nodal widening and segmental de- and remyelination. Some newly formed internodes are very short. We suggest that the occurrence of very short internodes in the developing rat inferior alveolar nerve reflects a myelin sheath remodelling, that allows the growing sheaths to elongate more than the nerve. Similarly, a scattered segmental de- and remyelination or a contraction of some internodes might enable other internodes in the old adult IAN to elongate, although the animal is fully grown.

White cells protect donor blood against bacterial contamination.

The possible beneficial role of white cells (WBCs) in donor blood has been investigated with respect to their capacity to remove bacteria. Preparations of buffy coat and whole blood, containing as well as reduced of WBCs, were inoculated with Staphylococcus epidermidis, S. aureus, Escherichia coli, Pseudomonas aeruginosa, and Propionibacterium species. Upon storage at room temperature, the presence of WBCs resulted in a reduction of the bacterial content. Units inoculated with S. epidermidis and E. coli were completely cleared of bacteria within 5 to 24 hours. On the other hand, S. aureus, after an initial reduction in number, started to multiply. In WBC-reduced units, the initial bacterial content remained unchanged for 5 hours, but the bacteria then exhibited vigorous growth within 48 hours in buffy coat and slower growth in whole blood. Propionibacterium sp. did not grow with or without WBCs. P. aeruginosa did not grow in buffy coat but showed a growth pattern similar to that of S. aureus in whole blood. The presence of WBCs in the donor blood during the first hours after collection thus seems to rid the blood of at least some species of bacteria. These results indicate that it would be favorable not to perform WBC reduction during blood collection and that several hours of contact can be needed to obtain sterility.