Transient expression of heat shock protein (Hsp)25 in the dental pulp and enamel organ during odontogenesis in the rat incisor

The expression of heat shock protein (Hsp) 25 during odontogenesis in the dental pulp and enamel organ of rat incisors was investigated by immunocytochemistry and confocal microscopy. In the process of dentin formation, immature odontoblasts first exhibited Hsp 25-immunoreactivity, and increased in immunointensity with the advance of their differentiation. In the dental pulp, in contrast, intense immunoreaction in the mesenchymal cells became weak or negative in parallel with the progress of cell differentiation. The immunoreaction for Hsp 25 in the enamel organ revealed a characteristic stage-related alteration during amelogenesis. In secretory ameloblasts, the immunoreaction for Hsp 25 was found throughout their cell bodies, intense reactivity being located near the proximal and distal terminal webs. At the maturation stage, ruffle-ended ameloblasts (RA) consistently showed Hsp 25-immunoreactivity throughout the cell bodies, whereas smooth-ended ameloblasts (SA) lacking a ruffled border were weak in immunoreaction at the distal cytoplasm. Other cellular elements of the enamel organ were negative. The subcellular localization of Hsp 25-immunoreactivity in this study appeared essentially identical to that of actin filaments as demonstrated by confocal microscopy using rhodamine-labeled phalloidin. These immunocytochemical data suggest that the Hsp 25 molecule is involved in reinforcement of the cell layer following cell movement during odontogenesis and in the formation and maintenance of the ruffled border of RA.     

Responses of odontoblasts to cavity preparation in rat molars as demonstrated by immunocytochemistry for heat shock protein (Hsp) 25

Responses of odontoblasts to cavity preparation in rat molars were investigated by immunocytochemistry for heat shock protein (Hsp) 25. In untreated control teeth, intense Hsp 25-immunoreactivity was found in the cell bodies of odontoblasts and their processes within the predentin. Confocal microscopy of Hsp 25-immunostained and rhodamine-labeled sections revealed that the immunoreactive odontoblasts were intensely labeled for phalloidin at the periphery of their cytoplasm and throughout their processes, but the reaction for phalloidin was limited within the inner half of the dentin. Cavity preparation caused an edematous reaction between the injured odontoblasts and predentin as well as a beaded swelling and successive destruction of the odontoblast processes. Immediately after cavity preparation, the odontoblasts beneath the edematous lesion showed an immunoreactivity for Hsp 25, which subsequently disappeared completely from the pulp-dentin border by 12 h after the operation. However, round cells without apparent cytoplasmic processes continued to be immunoreactive, suggesting the survival of a part of the odontoblasts against preparation stimuli. Numerous phalloidin-reactive but Hsp 25-immunonegative cells appeared along the pulp-dentin border and extended their processes deep into the exposed dentinal tubules, probably categorized in a lineage of immunocompetent cells. By postoperative 72 h, newly differentiated odontoblasts with Hsp 25-immunoreactivity were arranged at the pulp-dentin border. These findings indicate that the time course of changes in the expression of Hsp 25-immunoreactivity reflects the regeneration process of odontoblasts, and suggest that this protein is a useful marker substance for differentiated odontoblasts.     

Possible role of heat shock protein (Hsp) 25 in the enamel organ during amelogenesis in the rat molar.

The postnatal expression of heat shock protein (Hsp) 25 during the amelogenesis of rat molars was investigated by immunocytochemistry and confocal microscopy. The localization pattern of Hsp 25-immunoreactivity in the inner enamel epithelium and ameloblast cell layer of the rat molars was almost identical to that in the rat incisors which we have previously reported: an intense Hsp25-immunoreactivity, which first appeared in the preameloblasts, was recognized in secretory ameloblasts and ruffle-ended ameloblasts with stage-specific immunointensity. Confocal microscopy with Hsp 25-antibody and rhodamine-labeled phalloidin clearly demonstrated the co-localization of Hsp 25 and actin filaments in the ameloblast layer, supporting our hypothesis that this molecule might serve to reinforce the ameloblast layer during enamel formation as well as the formation and maintenance of the ruffled border in ruffle-ended ameloblasts. Interestingly, the enamel free area cells, which essentially lack the ability for enamel formation, showed the Hsp 25-immunoreactivity during 4-11 days when they developed a ruffled border, but decreased in that immunoreactivity after postnatal 15 days following apoptosis. Since Hsp 25 has been shown to be a specific inhibitor of apoptosis, the enamel-free area cells contribute to determine the outline of dentin at the cusped area. These data support our previous hypothesis on the diverse functions of Hsp 25 in amelogenesis.     

Immunohistochemical localization of heat shock protein 25 (HSP 25) during root formation of the rat molar

The present study investigated the immunohistochemical localization of heat shock protein 25 (HSP 25) of rat molar teeth during root formation. Most, probably all, cells of the epithelial rest of Malassez (ERM cells) had immunoreaction for laminin, a marker protein for basement membrane. During root formation, HSP 25 immunoreactivity was observed in odontoblasts, cells at the subodontoblastic layer, and those in close proximity to the acellular cementum. HSP 25-immunopositive cells at the subodontoblastic layer were present only at the apical region. Most HSP 25-immunoreactive cells in close proximity to the cementum lacked laminin immunoreactivity. However, at postnatal day 28 a small number of cells showed immunoreaction for both HSP 25 and laminin at the cervical and bifurcational regions. Under the electron microscope, most HSP 25-immunoreactive cells along the surface of the cementum were round and contained rich organelles such as mitochondria and rough endoplasmic reticulum. They lay between fiber bundles of the periodontal ligament. The localization and morphological features of these HSP 25-immunoreactive cells resemble those of cementoblasts. On the other hand, HSP 25-immunoreactive cells at the cervical region were oval and contained few cell organelles. They were closely apposed to each other, and separated from the surrounding tissues with basal lamina. These features were similar to those of mature ERM cells. In contrast, cells with microvillus-like processes and relatively rich mitochondria, which were similar to immature ERM cells, had no immunoreaction for HSP 25. These results suggest that HSP 25 may be involved in shape alterations of ERM cells, cementoblasts, and odontoblasts during differentiation.     

Extracellular heat shock protein 60 (Hsp60) levels in children with septic shock

Objective and design: Recent data suggest that extracellular Hsp60 modulates the host innate immune response. We analyzed plasma Hsp60 levels in children admitted to a level III tertiary care PICU with septic shock. Materials and subjects: Blood samples were obtained from children meeting criteria for septic shock (n = 63), critically ill children without septic shock (n = 10), and healthy controls (n = 24). Treatment: Not applicable. Methods: Hsp60 levels were measured in the plasma using a commercially available ELISA. Differences between groups were analyzed with a Kruskal-Wallis one way ANOVA due to the non-parametric nature of the data. A p value ≤ 0.05 was considered significant. Results: Extracellular Hsp60 levels were significantly higher in children with septic shock (median, 16.7 ng/mL) compared to both critically ill children without septic shock (median, 0 ng/mL) and healthy controls (median, 0 ng/mL, p <0.001). Conclusions: Extracellular Hsp60 levels are significantly elevated in children with septic shock compared with both healthy controls and critically ill children without sepsis. Extracellular Hsp60 may play a role in the pathogenesis of sepsis in children. Received 3 July 2006; returned for revision 18 October 2006; accepted by K. Visvanathan 6 December 2006     

Immunoelectron microscopic observation of calcitonin gene-related peptide (CGRP)-positive nerves in the dental pulp of rat molars.

The ultrastructure of nerves containing immunoreactivity for calcitonin gene-related peptide (CGRP) was investigated in the dental pulp of rat molars. The immunoreactivity was recognized predominantly in unmyelinated nerve fibers, and sparsely in a few myelinated fibers. It was localized throughout the axoplasm, as well as in the large cored vesicles. Small clear vesicles and mitochondria were free of the immunoreaction. The CGRP-immunoreactive nerves were frequently observed to terminate, being devoid of Schwann cell investment, in the vicinity of blood vessels in the coronal pulp, suggesting that CGRP may be involved in the regulation of pulpal blood flow. Moreover, CGRP-immunoreactive axon terminals containing numerous small clear vesicles, a few large cored vesicles and mitochondria were recognized in contact with the cell bodies of odontoblasts and their processes in the dentinal tubules. Although specialized synaptic ultrastructures were not recognizable, a functional association of CGRP nerves and odontoblasts was suggested. Thus, CGRP in the dental pulp appears to have multiple functions, including vascular regulation and sensory transduction.     

Immunocytochemical demonstration of heat shock protein 25 in the rat temporomandibular joint

The expression of heat shock protein 25 (Hsp 25) was investigated in the rat temporomandibular joint by immunocytochemistry combined with confocal and electron microscopy. Immunostaining with an antibody to Hsp25 was able to demonstrate various cellular elements in the synovial membrane of the joint. Intense immunoreaction for Hsp25 was recognized in certain cells comprising the synovial lining layer. Confocal microscopic observation revealed two characteristic profiles of the Hsp25-positive cells with cytoplasmic processes: one extended thick and long processes towards the articular cavity, and the other prejected horizontally slender processes which covered the synovial membrane. Under the electron microscope, the immunoreactive synovial lining cells were characterized by a well-developed rough endoplasmic reticulum and secretory granules, suggesting that they can be categorized as fibroblastic type B cells. The covering by the cytoplasmic extensions was confirmed by immuno-electron microscopic observations. This cytoplasmic covering presumably performs a barrier function and expedites the effective secretion/resorption of synovial fluids. Since it has been proposed that Hsp 25 is associated with an estrogen receptor, the immunopositive synovial lining cells were considered estrogen-target cells. Immunoreactivity for Hsp25 was also observed in the chondrocytes of the maturative and hypertrophic cell layers as well as in the cells of the articular disk. A suggestion was made that Hsp25 might be involved in the inhibition of apoptosis of those cells.     

Effects of heat shock protein 70 (Hsp70) on arsenite-induced genotoxicity

Arsenic, a human carcinogen, is genotoxic, although its mechanism(s) of action for tumorigenesis is not well understood. Among the toxicity-related properties of this chemical are its clastogenic and aneugenic activities, as well as its capacity for inducing stress-response in the form of elevated heat shock protein (HSP) expression. In the present study, we evaluated the effects of Hsp70 expression on arsenite (As)-induced structural and numerical chromosome anomalies in human cells. Human MCF-7 Tet-off cells stably transfected with a pTRE/Hsp70-1 transgene construct were used to regulate Hsp70 levels prior to in vitro As exposures. Separate cultures of relatively high vs. low Hsp70-expressing cells were established. A cytokinesis block micronucleus assay with kinetochore immunostaining was used to detect micronuclei (MN) derived from chromosome breakage (K-MN) or loss (K+MN). These studies demonstrated significant increases in micronucleus frequencies in response to As following either a long exposure (5 or 10 microM for 46 hr), or short exposure (10 or 40 microM for 8 hr) protocol. Overall, the long protocol was more efficient in producing K+MN and cells with multiple MN. Overexpressing Hsp70 resulted in significant reductions in the percent of cells positive for MN for both the long and short As exposure protocols. Both K+ and K- types of As-induced MN were lower in cells with elevated Hsp70 as compared to cells without overexpression of Hsp70. We conclude that the dose and duration of As exposure influence the type as well as amount of chromosomal alteration produced and that inducible Hsp70 protects against both the clastogenic and aneugenic effects of this chemical.     

Injury to retinal ganglion cells induces expression of the small heat shock protein Hsp27 in the rat visual system

Optic nerve transection results in apoptotic cell death of most adult rat retinal ganglion cells that begins at 4 days and leaves few surviving neurons at 14 days post-injury [Berkelaar et al. (1994) J. Neurosci. 14, 4368–4374]. The small heat shock protein Hsp27 has recently been shown to play a role in sensory neuron survival following peripheral nerve axotomy [Lewis et al. (1999) J. Neurosci. 19, 8945–8953]. To investigate the role of Hsp27 in injured CNS sensory neurons, we have studied the induction and cell-specific expression of Hsp27 in rat retinal ganglion cells 1–28 days after optic nerve transection. Immunohistochemical results indicate that Hsp27 is not present at detectable levels in the ganglion cell layer of control (uninjured) or sham-operated control rats. In contrast, Hsp27 is detected in retinal ganglion cells from 4 to 28 days following axotomy. Furthermore, the percentage of surviving retinal ganglion cells that are Hsp27-positive increased over the same time period. Hsp27 is also detected in glial fibrillary acidic protein-positive astrocytes in the optic layer of the superior colliculus from 4 to 28 days after optic nerve transection.

These experiments demonstrate that transection of the optic nerve results in the expression of Hsp27 in three distinct regions of the rat visual system: sensory retinal ganglion cells in the eye, glial cells of the optic tract, and astrocytes in the optic layer of the superior colliculus. Hsp27 may be associated with enhanced survival of a subset of retinal ganglion cells, providing evidence of a protective role for Hsp27 in CNS neuronal injury.     

Localization of heat shock protein 27 (hsp27) in the rat gingiva and its changes with tooth eruption

Heat shock protein 27 kDa (Hsp27) functions as a molecular chaperon to prevent apoptosis as well as to contribute to the regulation of cell proliferation and differentiation during development. In the present study, the localization of Hsp27 in the oral epithelium of rats and its expression change during formation of the gingiva with the tooth eruption were examined immunohistochemically to elucidate the roles of Hsp27 in the oral mucosa.In adult rats, Hsp27-immunoreactivity was localized in the prickle and granular layers but absent in the basal and horny layers of the oral epithelium. On the other hand, in the outer and sulcular epithelia of the free gingival, Hsp27-immunoreactivity was detected in the whole layers, while it was not found in the proliferation zone of the junctional epithelium immunoreactive for Ki67. In immature rats on 10th postnatal day, Hsp27-immunoreactivity was intense in the prickle and granular layers of the oral epithelium, but was not detected in its basal layer. In rats at the eruptive phase on 15th postnatal day, Hsp27-immunoreactivity was detected in sites of the basal layer adjacent to where the dental cusps penetrated through the oral epithelium. Although the immunoreactivity for Ki67 was found in the basal layer of the oral epithelium, it was not localized in the Hsp27-immunopositive sites of tooth-penetration in the basal layer. Just after the tooth-eruption on 20th postnatal day, Hsp27-immunoreactivity was not found in the stratified squamous epithelium at the dentogingival junction, whereas it was intense in a single layer of cuboidal epithelial cells attached to the tooth neck. Ki67-positive cells were scattered in the stratified squamous epithelium at the dentogingival junction, whereas no positive cells were found in the portion of a single layer of cuboidal epithelial cells.These findings suggest that the outer and sulcular epithelia of the free gingiva have a relatively slower rate of proliferation than other gingival and oral epithelia, and that Hsp27 might inhibit the proliferation of the basal cells. Such specific phenomenon in the free gingiva occurred immediately after the dental cusps were exposed to the oral cavity.     

Ultrastructural changes in odontoblasts and pulp capillaries following cavity preparation in rat molars.

Responses of odontoblasts and pulp capillaries to cavity preparation were investigated in the upper first molar teeth of rats, using light and transmission electron microscopy. At 100 days of age, the blood vessels of the pulp formed a subodontoblastic network consisting of continuous capillaries at a short distance from the odontoblast layer. Cavity preparation caused the displacement of some odontoblasts into the dentinal tubules, while others were separated from the predentin by rapid inflammatory exudation after drilling. The subodontoblastic capillary network under the injured dentin was shifted inwards together with the separated odontoblasts. The endothelium of the shifted capillaries showed a remarkable increase of pinocytotic vesicles, an event thought to be closely related to the formation of the exudative lesion. By one day after cavity preparation, most of the damaged odontoblasts had degenerated. Many cells with high nucleus/cytoplasm (N/C) ratios and prominent nucleoli accumulated around the subodontoblastic capillaries, some of which had many endothelial fenestrae facing these cells. These cells were suggestive of newly differentiating odontoblasts receiving nutritional supply from the capillaries. Three days after cavity preparation, newly differentiating odontoblasts took the place of the degenerated odontoblasts. They began to produce reparative dentin by five days after cavity preparation. Capillaries were located beneath the newly differentiating odontoblasts, but endothelial fenestrae gradually decreased in number. During the active reparative dentin formation, capillaries remained closely beneath the new odontoblast layer. Although the rate of reparative dentin deposition was not significantly lower than that in the primary dentin formation, one could not recognize an invasion of capillaries into the odontoblast layer nor a remarkable increase of endothelial fenestrae, both of which are common in active primary dentin formation. The results suggest that the function of capillaries differs between primary and reparative dentin formation.     

Expression patterns of heat shock protein 25 in carbon tetrachloride-induced rat liver injury

Heat shock protein 25 (Hsp25) is a molecular chaperone playing roles in cytoprotection. We investigated the distribution and localization of Hsp25 expression in CCl₄-induced rat hepatic lesions; liver samples were obtained from 3 h to 10 days after a single oral administration of CCl₄. Immunohistochemically, Hsp25-positive hepatocytes started to appear in the perivenular area at 6 h after CCl₄ administration. Their number and strength increased till day 1. Expression of Hsp25 mRNA significantly increased after 3 h and proceeded to increase with time till day 1. Apoptotic hepatocytes were detected around the perivenular area after 6 h. The area where Hsp25-positive hepatocytes were observed till day 1 corresponded to the area where apoptotic hepatocytes were seen. On days 2 and 3, degenerative and/or necrotic hepatocytes in the perivenular area were replaced by macrophages reacting to ED1 (for CD68) and ED2 (for CD163); Hsp25 expression was seen in hepatocytes around the perivenular area and there was a close relationship of reactive macrophages with Hsp25-positive hepatocytes, suggesting a potential role for Hsp25 in suppressing injury by inflammation. The mRNA expression of tumor necrosis factor-α, monocyte chemoattractant protein-1 and osteopontin, which can be produced by infiltrating macrophages, corresponded to that of Hsp25 from day 1 to day 3; these factors might be related to the induction of Hsp25 expression. The shift of the Hsp25 expression pattern in the liver lesion might have depended on microenvironmental conditions evoked by interactions between necrobiotic hepatocytes and infiltrating macrophages. Thus, Hsp25 expression analyses should be beneficial for evaluations of hepatotoxicants.     

Different expression of 25-kDa heat-shock protein (Hsp25) in Meckel's cartilage compared with other cartilages in the mouse

The 25-kDa heat-shock protein (Hsp25) is expressed in the cartilage of the growth plate and suggested to function in chondrocyte differentiation and degeneration. Using immunohistochemistry, we examined the temporal and spatial occurrence of Hsp25 in Meckel's cartilage in embryonic mice mandibles, and in other types of cartilage in both embryonic and adult mice. In adults, Hsp25 immunoreactivity was detected in the hypertrophic chondrocytes located in growth plates of long bones and in non-osteogenic laryngeal and tracheal cartilages. No chondrocytes in the resting or proliferating phase exhibited Hsp25 immunoreactivity. In the embryonic mandibles, resting and proliferating chondrocytes in the anterior and intermediate portions of Meckel's cartilage showed Hsp25 immunoreactivity from the 12th day of gestation (E12) through E15, whereas those in the posterior portion showed little or no immunoreactivity. After E16, the overall Hsp25 immunoreactivity in Meckel's cartilage substantially reduced in intensity, and little or no immunoreactivity was detected in the hypertrophic chondrocytes located in the degenerating portions of Meckel's cartilage. The antisense oligonucleotide for Hsp25 mRNA applied to the culture media of the mandibular explants from E10 embryos caused significant inhibition of the development of the anterior and middle portions of Meckel's cartilage. These results suggested that Hsp25 is essential for the development of Meckel's cartilage and plays different roles in Meckel's cartilage from those in the permanent cartilages and the cartilages undergoing endochondral ossification.     

Relationship of heat shock protein 25 with reactive macrophages in thioacetamide-induced rat liver injury

Heat shock protein 25 (Hsp25), which has anti-inflammatory activity, was examined for the relationship of its expression to macrophage appearance in thioacetoamide (TAA)-induced rat acute hepatic lesions. TAA-induced lesions, consisting of hepatocyte coagulation necrosis and reactive macrophages, developed in the centrilobular areas. Macrophages immuno-reacting to ED1 (CD68; exudative macrophages) were mainly seen within the lesions, whereas macrophages reacting to ED2 (CD163; resident macrophages and Kupffer cells), which have abundant cytoplasm, appeared mainly in the periphery of the lesions. Hsp25-immunopositivity was seen in hepatocytes around the lesions in relation to ED1- and ED2-positive macrophages in and around the centrilobular lesions, respectively. Because macrophages appearing in early stages of hepatic lesions produce various pro-inflammatory factors, mRNA expressions of tumor necrosis factor-α (TNF-α), monocyte chemoattractant factor-1 (MCP-1) and osteopontin (OPN) were examined in relation to Hsp25 mRNA expression. Hsp25 mRNA expression generally was correlated with TNF-α, MCP-1 and OPN expressions, suggesting their direct or indirect association with Hsp25 expression. Thus, Hsp25 might have a cytoprotection function against macrophages appearing in hepatic lesions, and factors produced by macrophages in the very early stages of hepatic lesions may influence Hsp25 expression. Hsp25 expression should be useful as an index of anti-inflammatory action for evaluation of hepatotoxicants in vivo.     

Expression of heat-shock protein 25 immunoreactivity in the dental pulp and enamel organ during odontogenesis in the rat molar

The present immunocytochemical study reports on the expression of heat-shock protein (Hsp) 25 during odontogenesis in rat molars from postnatal 1 to 100 days. Hsp 25 immunoreactivity (IR) appeared in the immature dental mesenchymal cells and the differentiating and differentiated odontoblasts. At 30 days, the coronal odontoblasts retained intense Hsp25-IR, whereas the odontoblasts in the root and floor pulp were initially weak or negative but increased in IR in the later stages, indicating that the expression of Hsp 25 reflects the differentiation status of odontoblasts. During amelogenesis, the secretory ameloblasts were Hsp 25 immunopositive and the enamel free area (EFA) cells showed intense Hsp 25-IR when they developed a ruffled border. Ruffle-ended ameloblasts (RA) also consistently showed intense Hsp 25-IR, but smooth ended ameloblasts (SA) showed weak IR. These data suggest that Hsp 25 is related to the formation and maintenance of the ruffled border of RA and EFA cells.     

Administration of brain-derived neurotrophic factor suppresses the expression of heat shock protein 27 in rat retinal ganglion cells following axotomy

Optic nerve transection results in the apoptotic cell death of the majority of retinal ganglion cells by 14 days. The neurotrophin brain-derived neurotrophic factor (BDNF) enhances survival of retinal ganglion cells. In addition, the small heat shock protein Hsp27, with its anti-apoptotic effects, may be important for neuron survival following axotomy or trophic factor withdrawal. We recently reported the induction and expression of Hsp27 in a subset of retinal ganglion cells following axotomy. Here we have examined the effect of BDNF administration on the expression of Hsp27 in axotomized adult rodent retinal ganglion cells. Retinal ganglion cells were pre-labeled with Fluorogold prior to optic nerve transection and concomitant intraocular injection of BDNF or vehicle. Hsp27 immunofluorescence was examined in retinal sections from 4 to 28 days following injury. Consistent with previous survival studies, the number of Fluorogold-labeled retinal ganglion cells declined from 100% at 4 days to approximately 15% by 14 days following axotomy and vehicle injection. In contrast, with BDNF administration, retinal ganglion cell survival was maintained at 100% to 7 days following axotomy. We report that the number of Hsp27-positive injured retinal ganglion cells, as detected by immunohistochemical staining, was decreased by 50% in BDNF-treated retinas, when compared with vehicle-treated controls. This decreased expression of Hsp27 in response to BDNF treatment was seen both at early (4 days) and delayed (14 days) times. BDNF following optic nerve transection significantly reduced the expression of Hsp27 in retinal ganglion cells. These results indicate that BDNF may down-regulate alternate cell survival pathways, including the stress-induced expression of Hsp27, and may help to explain the failure of chronic neurotrophin treatment to maintain long-term retinal ganglion cell survival.     

Photo-acoustic stimulation increases the amount of 70 kDa heat shock protein (Hsp70) in human whole saliva. A pilot study.

Long-term photo-acoustic stimulation leads to changes in the composition of saliva, which may have a key contribution to the effectivity of this technique in easing mucosal symptoms of psychosomatic patients. In the present study a significant (P 相似文献   

Re-innervation of rat molar tooth pulp following transection of the inferior alveolar nerve

The inferior alveolar nerves (IAN) of young male Wistar rats (b.wt. greater than or equal to 200 g) were transected unilaterally, slightly proximal to the mandibular foramen under anesthesia with chloral hydrate (0.4 g/kg, i.p.). After various postoperative periods of time, the animals received horseradish peroxidase (HRP) applications to 3 mandibular molar tooth pulps on both sides and were fixed by transvascular perfusion 24 h later. Horizontal 60 microns sections of the trigeminal ganglion were incubated with tetramethylbenzidine hydrochloride and the cross-sectional areas of all the labeled neuronal cell bodies were measured. The average number of labeled cells on the untransected (control) side was 148 (n = 26), with cross-sectional areas ranging between 131.9 and 2129.6 microns 2. Of these, 42.5% fell between 300 and 600 microns 2. About 13.5% (n = 7) of the primary neurons innervating the tooth pulps escaped the ipsilateral neurotomy and were labeled by HRP application on postoperative day 0. With HRP application on postoperative day 3, the number of labeled neurons recovered to 56.8% (n = 7) that of the control and maintained this level up to postoperative day 75. From postoperative days 3 through 75, the cell size spectrum of labeled neurons on the transection side was similar to that of the control and no consistent tendency of alteration was observed; i.e. they were distributed between 134.4 and 2214.3 microns 2, with the mode being 41.5% in the range between 300 and 600 microns 2 (n = 19).