The effect of buffer molarity on axonal exposure and axoaxonal apposition in the rat molar pulp.код для вставкиСкачать
T H E ANATOMICAL RECORD 201:471-476 (1981) The Effect of Buffer Molarity on Axonal Exposure and Axoaxonal Apposition in the Rat Molar Pulp G. R. HOLLAND Departments of Anatomy and Oral Biology, Uniuersity of Manitoba, Winnipeg, Manitoba, Canada R3E OW3 Axons in the rat molar pulp have been examined morphometriABSTRACT cally to determine axonal size and the degree of axonal exposure and axoaxonal apposition in tissue fixed by perfusion using 2% glutaraldehyde in cacodylate buffers ranging in molarity from 0.025 M to 0.4 M. Between 31.2% and 45.0% of the axons were incompletely ensheathed. This proportion of axons exposed was linearly related to the buffer molarity ( P < 0.05) and was approximately double that found in more central axons. Between 32.3% and 45.0% of the axons were in contact with other axons. This proportion was not linearly related to buffer molarity but was at least ten times higher than that observed in more centrally positioned nerve fibers in the inferior alveolar nerves. Increasing buffer molarity reduced the size of the axons, a relationship not found in the more central axons. It is suggested that axonal exposure and axoaxonal apposition are constant, significant features of pulpal nerve fibers that may be related to the onset and spread of nociceptive activity. The permeability properties of pulpal axons may differ from those of more centrally placed axons. Axons in Raschkow's subodontoblastic plexus show two structural features which may be related to their role in nociception (Holland, 1980a).One is their incomplete ensheathment and resultant exposure of the axolemma to the extracellular space and the other is the close contact between many of the axons. These features are not unique to the terminal portion of nerve fibers (Gasser, 1955; Ochoa, 1976). Our recent study of these arrangements in the inferior alveolar nerve (Holland, 1981) showed that while up to a third of the axons may be partially exposed, less than 4% were apposed to another axon with no intervening tongue of Schwann cell cytoplasm. The number of axons exposed was related to the molarity of the solution in which they were fixed, although even in high molarity solutions which allow the least exposure 10% or more of the axons remained incompletely ensheathed. The hypothesis that axonal exposure and axoaxonal apposition are particularly extensive in the terminal innervation of the dental pulp has been tested by subjecting the unmyelinated nerve fibers of the dental pulps from the animals used in our earlier study to a similar quantitative analysis. MATERIALS AND METHODS Tissue preparation Eighteen rats from three litters, all 5-6 weeks old and weighing 200-250 gm were used in random order. Each was anesthetized with sodium pentobarbital and then perfused via the ascending aorta for 1 minute with a prewash of normal saline containing 1%procaine hydrochloride and 0.08 gm of heparin1 liter and then for 15 minutes with a fixative mixture. In each case the perfusate was maintained at 4OC in a water bath. The fixative mixture used consisted of 2% glutaraldehyde, all from the same batch and manufacturer (TAAB Laboratories, U.K.), dissolved in cacodylate buffer. Three animals were perfused with fixture mixtures containing each of the following buffer concentrations: 0.025 M, 0.05 M, 0.1 M, 0.2 M, 0.3 M, and 0.4 M. The osmolarity of the buffers and the final Received March 9, 1981; accepted May 7. 1981 G.R. Holland's current address is Department of Endodontics, College of Dentistry, University of Iowa, Iowa City, Iowa 52242. 0003-276X/81/2013-0471$02.00 0 1981 ALAN R. LISS, INC. 472 G.R. HOLLAND fixative mixtures were all measured using a osmomiter (Wescor 5100 B, vapor pressure type). The values are recorded in Table 1. The pH of each mixture was measured and ranged between 7.0 and 7.3. The mandibular molar teeth were removed from each animal and left overnight at 4°C in the same fixative mixture as was used for the perfusion. The teeth were then decalcified in EDTA (Warshawsky and Moore, 1967) and processed according to the following schedule: Two 10-minute washes in buffer identical to that used in the perfusion; 2 hours in 2% osmium tetroxide in distilled water; ethanolic dehydration with 2% uranyl acetate in each of the alcohols; and infiltration and embedment in Araldite. Sections in the gold-silver range were cut from the pulps of the molar crowns and mounted on uncoated 100-mesh copper grids. Microscopy The sections were examined in a Hitachi HUl2B electron microscope at X 15,000 magnification. Micrographs were taken of at least 100 nonmyelinated nerve fibers from each group. Each negative was printed at X 2.7 magnification to give a final magnification of X 40,500. Measurements Measurements were made using a "Smart One Magic System" (Talos Systems, Inc., Scottsdale, AZ) which consists of a high-resolution graphics tablet and a microprocessor programmed such that irregular perimeters and distances can be measured reproducibly and accurately by tracing with a cross-hair sensor. The data may be fed directly into a larger computer for storage and subsequent processing. The following parameters were measured: The circumference of each axon. The length of axolemma exposed to the extracellular space. The length of the axolemma in contact between apposed axons. For each group of animals the following data were either derived from the above or did not require the use of the digitizing system. The mean axon circumference. Proportion of the axons that were "exposed." Proportion of the total axolemma that was exposed. The mean circumference of the exposed axons. BUFFER MOLARITY EFFECT ON AXONS IN RAT MOLAR PULP 473 same animals is also shown on the same axes (lower line). When the points for the inferior alveolar nerve are compared with those for the pulpal axons in a paired T-test the difference is statistically significant ( P< 0.01). The proportion of pulpal axons exposed was always greater than 30% - the maximum proportion reached by axons in the inferior alveolar nerve. RESULTS The relationship between buffer molarity Figures 1-4 show typical nonmyelinated and exposure was even stronger when the denerve fibers fixed in mixtures containing four gree of exposure was expressed as the percendifferent buffer molarities. In all mixtures the tage of the total axolemma exposed ( P < plasma membranes were intact and the basic 0.01). On average between 6.8% and 15.4% of organelles readily recognizable. The cyto- the total axolemma was exposed. At all plasm of some axons fixed in the lower molari- molarities the mean circumference of exposed ty solutions was less electron dense than other axons was significantly lower than of the total cytoplasmic matrices, giving the axons a rela- population of axons ( P < 0.05). Many axons were apposed to one another tively empty appearance (arrow, Fig. 1). This did not occur in solutions containing buffer at (between 32.3% and 45%). The proportion of 0.2 M (Fig. 2) or above (Figs. 3, 4). Mitochon- axons apposed was not related linearly to the dria were often disrupted at very low molarity buffer molarity. The mean circumference of (0.05 M or less, Fig. 1)but were well preserved the apposed axons was significantly less than at 0.2 M or above. Microtubules and microfila- the mean circumference of the total populaments were well preserved in all mixtures. The tion of axons ( P < 0.01) and also significantly gap between axons and the surrounding less than the mean circumference of the exSchwann cell cytoplasm appeared to widen as posed axons ( P < 0.01). There was no signifibuffer molarity increased. The electron densi- cant linear relationship between the size of the ty of the Schwann cell cytoplasm increased in apposed axons and buffer molarity when the same direction though no similar effect on tested by regression analysis. the axoplasm was discernible. The axons in DISCUSSION low buffer molarity were commonly circular in I t is reassuring that reasonable ultrastrucprofile, assuming a more irregular, flattened tural detail can be preserved over such a wide outline in the stronger mixtures. The numerical data for each parameter is range of buffer molarities. Without knowing summarized in Table 1. Table 2 shows the what the living ultrastructure is it is imposresults of a regression analysis by which the sible to suggest which fixed image most closelinearity of the relationship between each par- ly resembles it. A high proportion of the pulpal axons are inameter and buffer molarity was tested. The mean axon size measured as circumfer- completely ensheathed. Although this is deence was always between 1.5 and 2.1 pm pendent on buffer molarity it does not fall (Table 1).The mean size resulting from pool- below 30% and is always much higher than ing the measurements for the three animals in that found in the inferior alveolar nerve. We each group became lower as buffer molarity in- may conclude that axonal exposure is a concreased. The relationship between buffer mo- stant and significant feature of the terminal larity and mean axon circumference tested by innervation of the pulp and speculate that in regression analysis was statistically signifi- the absence of any more specialized receptor (Holland, 1980a) this exposed membrane may cant ( P < 0.05). More axons were lacking a Schwann cell in- be the site at which nociception is initiated. vestment in tissue fixed with low molarity Axon to axon contact is low in the inferior albuffer. The proportion of axons exposed veolar nerve (Holland, 1981) but more than a declined linearly with increasing buffer molar- third of pulpal axons are apposed, a proportion ity ( P < 0.05). The regression plot of this par- apparently unaffected by the molarity of the ameter is shown in Figure 4 (upper line). For buffer in the fixative mixture. Coupling becomparison the data derived from our earlier tween pulpal axons has been demonstrated study on the inferior alveolar nerve of the electrophysiologically (Matthews and Hol- The proportions of the axons that were closely apposed to other axons. The proportions of the axolemma that was apposed to other axons. The data obtained was examined statistically. The relationship of each measured parameter to the buffer molarity was examined by regression analysis. 474 G.R. HOLLAND 475 BUFFER MOLARITY EFFECT ON AXONS I N RAT MOLAR PULP TABLE 2. The relationship between buffer molarity and axon size, exposure, and contact, a s determined bv remession analysis Buffer molarity vs. Mean axon circumference Proportion of axons exposed Proportion of total axolemma circumference exposed Proportion of axons apposed Proportion of total axolemma circumference apposed Slope -1.4 -40.2 -21.4 -1.60 -2.01 Standard error of slope 0.55 4.88 1.82 15.95 16.76 Significance < 0.05 <0.05 <0.01 N.S. N.S. land, 1975)and it has been suggested that this may occur via gap junctions that link axons either directly or via an intermediate odontoblast (Matthews and Holland, 1975; Holland, 1975, 1976, 1977, 1980b). Attempts using autoradiography (Byers, 1977)and serial sectioning at the ultrastructural level (Holland, 1980a) have failed to confirm this hypothesis. The suggestion that mere apposition may result in the spread of nervous activity in this situation (Holland, 1980a)is supported by the findings of this study. Although some apposi- M tion does occur in the more central nerve fibers it would appear to be relatively insignificant in amount. Gasser (1955)computed that apposition would have to be very extensive to allow nervous activity to cross from one axon to another. I t would seem that this degree of con0 1 2 3 4 tact may be reached in the dental pulp, alBuffer Molarily though, as Bennet (1977) has pointed out, Fig. 5. Regression analysis of the proportion of axons exmuch would depend on the permeability propposed against buffer molarity. The upper line is for pulpal erties of the apposed membranes. As axonal nerve fibers, the lower line is for fibers in the inferior alveosize in the dental pulp but not in the inferior lar nerve. Both slopes are significant a t P < 0.05. The data alveolar nerve (Holland, 1981)is related to buf- for the lower line are from an earlier study (Holland. 1980). fer molarity, it may be that the membrane properties of axons in these two sites do differ potent source of intensely painful sensory exconsiderably. In the dental pulp there are two anatomical periences. characteristics - extensive divestment of axACKNOWLEDGMENTS ons by their Schwann cells and widespread axI would like to thank Susan Pylypas and Linoaxonal apposition - that could form the basis for the initiation and rapid spread of ner- da Nahnybida for their expert technical assistance, Carolyn Klassen for typing the manuvous activity. As the predominant innervation script, and Roy Simpson and Glenn Reid for of the pulp is the A delta and C fiber range (Beasley and Holland, 1978) and these fiber preparing the illustrations. Cindy Wong advissizes play a special role in pain perception (Mel- ed on the statistical treatment of results. This study was supported by grant No. MA6468 zack and Wall, 1965),this arrangement could from the Medical Research Council of Canada. in part explain why the dental pulp is such a t Figs. 1-4. Nonmyelinated pulpal nerve fibers fixed in mixtures containing buffers of different molarity Fig. 1. 0.05 M, arrow indicates axon of low electron density. Fig. 2. 0.2 M. Fig. 3. 0.3 M. Fig. 4. 0.4 M. G.R. HOLLAND 476 LITERATURE CITED Beasley, W.L., and G.R. Holland (1978) A quantitative analysis of the innervation of the pulp of the cat’s canine tooth. J . Comp. Neurol., 178:487-494. Bennet. M.V.L. (1977)Electrical transmission: A functional analysis and comparison of chemical transmission. In: Handbook of Physiology. Section I. The Nervous System. J.M. Brookhart and V.B. Mountcastle. eds. American Physiological Society, Bethesda, pp. 357-416. Byers, M.R. (1977)Fine structure of trigeminal receptors in rat molars. In: Pain in the Trigeminal Region. D.J. Anderson and B. Matthews, eds. Elsevier, New York, pp 37-48. Gasser, H.S. (1955) Properties of dorsal root unmedullated fibers on two sides of the ganglion. J. Gen. Physiol.. 32: 709-728. Holland. G.R. (1975) Membrane junctions on cat odontoblasts. Arch Oral Biol.. 20:551-552. Holland, G.R. (1976) Lanthanum hydroxide labelling of gap junctions in the odontoblast layer. Anat. Rec., 186:121126. Holland, G.R. (1977) Structural relationships in the odonto- blast layer. In: Pain in the Trigeminal Region. D.J. Anderson and B. Matthews, eds. Elsevier, New York. pp. 25-35. Holland. G.R. (1980a)Non-myelinated fibers and their terminals in the sub-odontoblasticplexus of the feline dental pulp. J. Anat., 130:457-467. Holland, G.R. (1980b)Microtubule and microfilaments populations of cell processes in the dental pulp. Anat. Rec., 198.421-426. Holland, G.R. (1981)The extent of axonal exposure and axoaxonal apposition in the non-myelinated nerve fibers of peripheral nerve trunks and their dependence on buffer molarity. J. Anat., in press. Matthews, B., and G.R. Holland (1975) Coupling between nerves in teeth. Brain Res., 98t354-358. Melzack, R., and P.D. Wall (1965)Pain mechanisms: A new theory. Science, 150r971-979. Ochoa. J. (1976) The unmyelinated nerve-fiber. In: The Peripheral Nerve. D.N. Landon, ed. Chapman and Hall, London, pp. 106-158. Warshawsky, H., and G. Moore (1967) A technique for the fixation and decalcification of rat incisors for electron microscopy. J . Histochem. Cytochem., 15:542-549.