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Temporal expression of immunoreactivity for heat shock protein 25 (Hsp25) in the rat periodontal ligament following transection of the inferior alveolar nerve
Brain Research 979 (2003) 146–152 www.elsevier.com / locate / brainres
Research report
Temporal expression of immunoreactivity for heat shock protein 25 (Hsp25) in the rat periodontal ligament following transection of the inferior alveolar nerve Kenji Iijima a,b , Fumiko Harada a,b , Kooji Hanada b , Kayoko Nozawa-Inoue a , Megumi Aita a , Yukako Atsumi c , Satoshi Wakisaka c , Takeyasu Maeda a , * a
Division of Oral Anatomy, Department of Biological Science, Niigata University Graduate School of Medical and Dental Sciences, 2 -5274 Gakkocho-dori, Niigata 951 -8514, Japan b Division of Orthodontics, Department of Biological Science, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan c Department of Oral Anatomy and Developmental Biology, Osaka University Graduate School of Dentistry, Suita, Japan Accepted 23 April 2003
Abstract The present study examined the immunohistochemical localization of heat shock protein 25 (Hsp25) during the regeneration of nerve fibers and Schwann cells in the periodontal ligament of the rat lower incisor following transection of the inferior alveolar nerve. In the untreated control group, the periodontal ligament of rat incisor did not contain any Hsp25-immunoreaction. On postoperative day 3 (PO 3d), a small number of Schwann cells with slender cytoplasmic processes exhibited Hsp25-immunoreactivity. From PO 5d to PO 21d, Hsp25-positive nerve fibers and Schwann cells drastically increased in number in the alveolar half of the ligament. Although the axons of some regenerating Ruffini-like endings also showed Hsp25-immunoreactions, the migrated Schwann cells were devoid of Hsp25immunoreaction. Thereafter, Hsp25-positive structures decreased in number gradually to disappear from the periodontal ligament by PO 56d. This temporal expression of Hsp25 in the periodontal ligament well-reflected the regeneration process of the nerve fibers. Hsp25 in the regenerating nerve fibers and denervated Schwann cells most likely serves in modulating actin dynamics and as a cellular inhibitor of apoptosis, respectively. 2003 Elsevier B.V. All rights reserved. Theme: Sensory systems Topic: Somatic and visceral afferents Keywords: Hsp25; Ruffini Ending; Regeneration; Periodontal ligament
1. Introduction The heat shock proteins (Hsps) are highly conserved proteins which are induced by heat shock and other kinds of deleterious environmental and pathophysiologic stresses [28]. The upregulation of these proteins enables the cell to survive and recover from the stressful conditions. Hsps have been classified into several families according to their molecular weights [2,3,10]. Since murine Hsp25 is a
*Corresponding author. Tel.: 181-25-227-2815; fax: 181-25-2236499. E-mail address: [email protected] (T. Maeda). 0006-8993 / 03 / $ – see front matter 2003 Elsevier B.V. All rights reserved. doi:10.1016 / S0006-8993(03)02889-0
homologue of human and rat Hsp27 / 28, the term Hsp25 is applied to its homologue in this paper. Like other Hsps, Hsp25, one of the small Hsps (20–30 kDa), works as a molecular chaperone [19], or enzyme modulating actin dynamic [16,25] to protect cells under stressful conditions. Furthermore, Hsp25 is involved in the suppression of apoptosis [32,33,42]. Hsp25 has been reported to be constitutively expressed at low levels by a subpopulation of adult primary sensory neurons [11,40]. In vivo and in vitro studies have demonstrated that the expression of Hsp25 mRNA and protein are dramatically upregulated in the dorsal root ganglion after sciatic nerve injury [11,15,27]. The Ruffini ending, a low-threshold slowly adapting
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type II stretch receptor, is the primary mechanoreceptor in the periodontal ligament [29,48]. They are characterized by elaborate ramifications of the expanded axon terminals and by an association with a peculiar Schwann cell called the terminal Schwann cell [6,20,21,30,45]. A series of our experimental studies have shown that the denervated periodontal ligament of rat incisor could be reinnervated by the periodontal Ruffini endings [4,14,53]. Our experimental studies have demonstrated the regeneration process of the periodontal nerve fibers in the rat incisors following transection of inferior alveolar nerve (IAN) [4,14,53]; the nerve fibers disappeared from the periodontal ligament at postoperative 3 days (PO 3d), and began to regenerate at PO 5d. Through PO 10d to PO 21d, the regeneration of the periodontal nerve fibers actively took place to recover to normal levels by PO 28d. These data indicate the possibility that the periodontal Ruffini endings have a high potential for neural plasticity. Schwann cells play important roles during the regeneration of peripheral nerves; denervated Schwann cells proliferate and promote the regeneration of axons following nerve injury. During the regeneration of the periodontal Ruffini endings, the terminal Schwann cells also showed a characteristic behavior: they migrate into regions where they are usually not found and their distribution and number return to normal levels following the completion of regeneration [4]. In the central nervous system, the expression of Hsp25 has been reported in glial components under some pathological conditions [5,17,18,22,23,38,39,41,43]. In spite of many reports on the wide distribution and diverse function of Hsp25 in non-neural cells, little information is available on the functional roles of Hsp25 in the Schwann cell, a peripheral glial element. Thus, the present study was undertaken to examine immunohistochemically the expression of Hsp25 in the periodontal nerves of the rat incisors.
2. Materials and methods All animal experiments were done under the Guidelines of Intramural Animal Use and Care Committee of the Niigata University Faculty of Dentistry.
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postoperative treatment such as antibiotics administration was given to the operated rats.
2.2. Tissue preparation By use of this experimental model, our previous studies [4,14,53] have classified the regeneration process of the periodontal Ruffini endings after transection of the IAN into four stages as previously reported [4]; a degeneration stage (PO 1–3d); a commencement of regeneration stage (PO 5–7d); a regeneration stage (PO 10–21d), and the completion of regeneration (PO 28–56d). Thus, the animals were allowed to survive for 3, 7, 10, 14, 21, 28 and 56 days (n55 each), respectively. After an appropriate survival period, they were deeply anesthetized by an intraperitoneal injection of chloral hydrate (400 mg / kg), and perfused transcardially with 4% paraformaldehyde in a 0.1 M phosphate buffer (pH 7.4). The mandibles, including the incisors, were removed en bloc and decalcified with a 10% ethylene diamine tetraacetic acid (EDTA)-2Na solution for 3–4weeks at 4 8C. Serial frozen sections of incisors were cut at a thickness of 35 mm with a freezing microtome, collected in 0.01 M phosphate-buffered saline (PBS; pH 7.4), and treated as free-floating sections.
2.3. Immunohistochemistry Our immunocytochemical protocol has been reported previously [36]. Briefly, after an inhibition of endogenous peroxidase, frozen sections were primarily incubated with a rabbit polyclonal antisera against murine Hsp25 (1: 5000: StressGen Biotechnologies, Victoria, Canada) for 12 h at 4 8C. According to the manufacturer’s instructions, this antiserum against Hsp25 can recognize Hsp25 / 27 from mouse, hamster, guinea pig, rat, bovine and canine cell lysates. As murine Hsp25 is a homologue of human and rat Hsp 27 / 28, we used the term Hsp25. The sections were then incubated with biotinylated goat anti-rabbit IgG (1:1000, Vector Labs., Burlingame, CA, USA), and subsequently with an avidin–peroxidase complex (ABC kit, Vector Labs.) for 90 min each at room temperature. Final visualization used 0.04% 3,39-diaminobenzidine (DAB) and 0.003% H 2 O 2 in a 0.05 M Tris–HCl buffer (pH 7.6) with nickel ammonium sulfate (0.1%) intensification.
2.1. Animals and surgery
2.4. Immuno-control experiments
A total of 43 Wistar rats (60-day-old, weighing 180–200 g at the time of surgery) were used in this experimental study. The surgical transection of the IAN was applied to 35 animals (experimental group) according to a protocol by Atsumi et al. [4]. Animals without an IAN-transection were used as a sham group (n55). Three rats without any surgical treatment further served as a control group. No
Immunohistochemical controls were performed by: (1) replacing the primary antiserum with nonimmune rabbit serum or PBS; (2) omitting the anti-rabbit IgG or the avidin–peroxidase complex. The specificity of antisera against Hsp25 was also checked by an absorption test and has been reported elsewhere [36,40]. These control sections did not show any specific immunoreactions.
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mic processes, appearing as slender profiles (Fig. 2b). However, Hsp25-immunohistochemistry failed to demonstrate any terminal Schwann cells, round in shape, associated with the periodontal Ruffini endings. Hsp25-immunoreaction was also found in the ordinary Schwann cells in the nerve bundles and some endothelial cells (Fig. 2a).
3.3. PO 5 – 7 d: commencement of regeneration stage
Fig. 1. Hsp25-immunoreactivity in the lower periodontal ligament of the untreated control rat. No cellular element immunoreactive to Hsp25 is found in the periodontal ligament while the odontoblasts (OB) show an intense immunoreaction. AB, alveolar bone; T, tooth. Scale bar550 mm.
3. Results
3.1. Control group No Hsp25-immunoreactive structure was recognized in the periodontal ligament of the untreated control group (Fig. 1), though the ameloblasts and odontoblasts in the same sections showed an intense Hsp25-immunoreactivity as reported previously [36,37]. The distribution and terminal morphology of Hsp25 in animals of the sham group were comparable to those of the control group.
3.2. PO 3 d: degeneration stage Hsp25-immunoreactive cellular elements occurred in the periodontal ligament on PO 3d (Fig. 2a and b). The alveolar half of the periodontal ligament (alveolus-related part: ARP) contained many Schwann cells with intense Hsp25-immunoreactivity. They possessed long cytoplas-
A dense distribution of Hsp25-positive structures was found in the periodontal ligament at this stage (Fig. 2c). The immuno-intensity of Schwann cells in the nerve bundles became stronger. The cells with cytoplasmic extensions (Fig. 2d) and some round cells with an indented nucleus in the ARP showed an Hsp25-immunoreactivity. Furthermore, thin nerve fibers, frequently beaded in appearance, were also positive in Hsp25-immunoreaction. A major population of these fibers was distributed at the border between tooth half of the ligament (tooth related part; TRP) and ARP (shear zone) as well as the ARP, and showed various running courses (Fig. 2e). The Schwann cells associated with the nerve bundles were also positive in Hsp25-immunoreaction (Fig. 2d).
3.4. PO 10 – 21 d: regeneration stage On PO 14d, the dendritic structures in the ARP and thin nerve fibers became immunopositive for Hsp25 (Fig. 2f). At higher magnification, these positive structures exhibited thick, ramified nerves (Fig. 2g). However, the slender- or satellite-shaped cells, which appeared in the TRP and shear zone with intense S-100 immunoreactivity, were devoid of Hsp25-immunoreaction. Some Schwann cells in the nerve bundles retained Hsp25-immunoreaction (Fig. 2f). On PO 21d, Hsp25-positive structures decreased in number. Thin nerve fibers taking a straight course and thick nerve terminals showing a tree-like ramification were found in the ARP of the periodontal ligament (Fig. 2h). A few
Fig. 2. Changes in expression of Hsp25-immunoreactivity in the periodontal ligament on PO 3d (a, b), PO 5d (c–e), PO 14d (f, g), PO 21d (h), PO 28d (i) and PO 56d (j). (a) Hsp25-immunoreactive structures appear in the periodontal ligament. In addition, Hsp25-immunoreactions are found in some endothelial cells and the Schwann cells in the nerve bundles (NB). (b) Higher magnification of the boxed area in (a). Hsp25-immunoreactions are localized in the slender cytoplasmic processes and cell bodies of Schwann cells, but not in the nerve fibers. (c) On PO 5d, the number of the Hsp25-positive structures has increased compared with that on PO 3d. Note the absence of Hsp-25 positive cells in the tooth related part (asterisks). (d) Highly magnified image of the boxed area in (c). Hsp25-immunoreactivities are found in the slender Schwann cells and thin nerve fibers. These thin nerve fibers frequently run parallel to the tooth axis (arrows). (e) The round cells in the alveolus-related part show Hsp25 immunoreactivity. (f) The Hsp25 positive nerve fibers remain in the periodontal ligament in contrast to a decrease in the number of cellular elements. At this stage, thick nerve fibers show Hsp25immunoreactivity. (g) Highly magnified view of the boxed area in (f). Thick nerve fibers frequently ramify to display a dendritic fashion, appearing as the Ruffini endings. (h) Hsp25-immunoreaction shows a tendency to disappear on PO 21d. Note the intense immunoreaction in the odontoblasts (OB). (i, j) Hsp25-immunoreactive structures in the periodontal ligament drastically have decreased in number and immuno-intensity daily. No positive element exists in the tooth related part (asterisks). Note intense immunoreaction in the odontoblasts (OB) in (j). AB, alveolar bone; BM, bone marrow; BV, blood vessel; NB, nerve bundle; T, tooth. Scale bars550 mm in (a–d, f, h–j), 10 mm in (e), 20 mm in (g).
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Schwann cells in the ARP exhibited Hsp25-immunoreaction. The odontoblasts and ameloblasts retained intense Hsp25-immunoreaction (Fig. 2h).
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3.5. PO 28 – 56 d: completion of regeneration stage Between PO 28d and PO 56d, Hsp25-positive structures
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decreased in number and immuno-intensity gradually, though the odontoblasts and ameloblasts showed a constant intense Hsp25-immunoreactivity (Fig. 2i and j). A few Hsp25 positive structures were restricted to the ARP.
4. Discussion The present experimental study was able to demonstrate the temporal expression of Hsp25 in the regenerating nerve fibers and Schwann cell elements in the periodontal ligament following transection of the IAN. There has been controversy over Hsp25-expression in sensory neurons under normal conditions; normal adult brains express little or no Hsp25-immunoreactivity [9,13,23,24,46,47,49]. In contrast, some investigators have pointed out the constitutive expression of Hsp25 in a subset of primary sensory neurons in both cranial sensory ganglia and dorsal root ganglia [11,40,51]. In the present study, however, no Hsp25-immunopositive structure was recognizable in the periodontal ligament under normal conditions, consistent with previous reports on the rat upper incisor [36,37]. There are possible two explanations for this discrepancy. First, since previous studies have demonstrated that approximately 60–70% of neurons in the trigeminal ganglion (TG) [51] and 45% of neurons in the trigeminal mesencephalic nucleus (MeV) [39,40]— both of which innervate the periodontal ligament—showed Hsp25-immunoreactivity, the immunonegative neurons in TG and MeV may innervate the lower periodontal ligament of rat incisors. Second, the antiserum against Hsp25 used in this study was a polyclonal one raised against a short amino acid sequence of the mouse Hsp25. Plumier et al. [40] reported a discrepancy between detection of Hsp25 by Western blot analysis and the absence of Hsp25-positive staining with immunohistochemistry, and considered the presence of three phosphorylation states of Hsp25 in rat spinal cord samples. Thus, the periodontal nerve fibers might be in a functionally different state, even under normal conditions. This immunohistochemical study demonstrated the temporal expression of Hsp25-immunoreactivity in the thin and thick nerve fibers, predominantly from PO 5d to PO 21d, which has been regarded as the regeneration stage because of a drastic increase in the regenerating nerve fibers [4,14,53]. Since previous studies have revealed the upregulation of Hsp25 protein and mRNA in injured neurons in vivo [11,15] and in vitro [27] and since Hsp is transported in nerve fibers, it is likely that the presence of Hsp25 in the regenerating nerve fibers is indicative of the synthesis of Hsp25 in the cell body and the transport of this protein to the periodontal nerves. An intriguing characteristic of Hsp25 is its involvement in actin filament dynamics; Hsp25 has been proposed to act as an actin filament capping protein, potentially either preventing or permitting actin filament synthesis depending on its phos-
phorylation states [26]. Taken together, these findings would seem to indicate that the temporal expression of Hsp25 reflects on actin dynamics in the regenerating nerve fibers. There is no question of the temporal expression of Hsp25-immunoreaction in Schwann cells due to their serving to protect themselves against nerve injury. Similar to Hsp25-expression in the nerve fibers, Schwann cells in the denervated periodontal ligament temporally exhibited an immunoreaction. In general, the glial components—in particular astrocytes—become positive in Hsp25-immunoreaction in certain neurological diseases [5,18,22,23,41,43] and experimentally damaged brains [17,23,38–40]; Hsp25expression is found in peripheral Schwann cells under normal conditions ([51]; this study). Like other Hsps, Hsp25 functions as a molecular chaperone, conserving the conformation of proteins [19], or as an enzyme modulating actin dynamic [26] which is considered to affect cell motility and shape [25,31]. This protein also has been shown to act as a specific cellular inhibitor of apoptosis [32,33,42]. Since the cells migrated to the TRP lacked an Hsp25-immunoreactivity in the experimental model, it would seem that Hsp25 functions as an inhibitor of apoptosis in denervated Schwann cells. On the other hand, damage to neurons induces an increase in the glial fibrillary acidic protein (GFAP) of the glia [1,8,12,35,44,50]. GFAP-immunoreaction has been demonstrated in some ordinary Schwann cells and terminal Schwann cells associated with the Ruffini endings in specific regions [7,52]. Since Hsp25 can affect GFAP assembly [34] in addition to serving as molecular chaperone, Hsp25 in denervated Schwann cells may also serve in the remodeling of intermediate filaments during development and cell differentiation as well as regeneration.
Acknowledgements The authors thank Messers Kiichi Takeuchi and Masaaki Hoshino, Division of Oral Biological Science, Niigata University Graduate School of Medical and Dental Science, for their technical assistance. This study was supported by Grants-in-Aid for Scientific Research ([13470383 to SW and [14370580 to TM) from Japan Society for the Promotion of Science (JSPS).
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Research report
Temporal expression of immunoreactivity for heat shock protein 25 (Hsp25) in the rat periodontal ligament following transection of the inferior alveolar nerve Kenji Iijima a,b , Fumiko Harada a,b , Kooji Hanada b , Kayoko Nozawa-Inoue a , Megumi Aita a , Yukako Atsumi c , Satoshi Wakisaka c , Takeyasu Maeda a , * a
Division of Oral Anatomy, Department of Biological Science, Niigata University Graduate School of Medical and Dental Sciences, 2 -5274 Gakkocho-dori, Niigata 951 -8514, Japan b Division of Orthodontics, Department of Biological Science, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan c Department of Oral Anatomy and Developmental Biology, Osaka University Graduate School of Dentistry, Suita, Japan Accepted 23 April 2003
Abstract The present study examined the immunohistochemical localization of heat shock protein 25 (Hsp25) during the regeneration of nerve fibers and Schwann cells in the periodontal ligament of the rat lower incisor following transection of the inferior alveolar nerve. In the untreated control group, the periodontal ligament of rat incisor did not contain any Hsp25-immunoreaction. On postoperative day 3 (PO 3d), a small number of Schwann cells with slender cytoplasmic processes exhibited Hsp25-immunoreactivity. From PO 5d to PO 21d, Hsp25-positive nerve fibers and Schwann cells drastically increased in number in the alveolar half of the ligament. Although the axons of some regenerating Ruffini-like endings also showed Hsp25-immunoreactions, the migrated Schwann cells were devoid of Hsp25immunoreaction. Thereafter, Hsp25-positive structures decreased in number gradually to disappear from the periodontal ligament by PO 56d. This temporal expression of Hsp25 in the periodontal ligament well-reflected the regeneration process of the nerve fibers. Hsp25 in the regenerating nerve fibers and denervated Schwann cells most likely serves in modulating actin dynamics and as a cellular inhibitor of apoptosis, respectively. 2003 Elsevier B.V. All rights reserved. Theme: Sensory systems Topic: Somatic and visceral afferents Keywords: Hsp25; Ruffini Ending; Regeneration; Periodontal ligament
1. Introduction The heat shock proteins (Hsps) are highly conserved proteins which are induced by heat shock and other kinds of deleterious environmental and pathophysiologic stresses [28]. The upregulation of these proteins enables the cell to survive and recover from the stressful conditions. Hsps have been classified into several families according to their molecular weights [2,3,10]. Since murine Hsp25 is a
*Corresponding author. Tel.: 181-25-227-2815; fax: 181-25-2236499. E-mail address: [email protected] (T. Maeda). 0006-8993 / 03 / $ – see front matter 2003 Elsevier B.V. All rights reserved. doi:10.1016 / S0006-8993(03)02889-0
homologue of human and rat Hsp27 / 28, the term Hsp25 is applied to its homologue in this paper. Like other Hsps, Hsp25, one of the small Hsps (20–30 kDa), works as a molecular chaperone [19], or enzyme modulating actin dynamic [16,25] to protect cells under stressful conditions. Furthermore, Hsp25 is involved in the suppression of apoptosis [32,33,42]. Hsp25 has been reported to be constitutively expressed at low levels by a subpopulation of adult primary sensory neurons [11,40]. In vivo and in vitro studies have demonstrated that the expression of Hsp25 mRNA and protein are dramatically upregulated in the dorsal root ganglion after sciatic nerve injury [11,15,27]. The Ruffini ending, a low-threshold slowly adapting
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type II stretch receptor, is the primary mechanoreceptor in the periodontal ligament [29,48]. They are characterized by elaborate ramifications of the expanded axon terminals and by an association with a peculiar Schwann cell called the terminal Schwann cell [6,20,21,30,45]. A series of our experimental studies have shown that the denervated periodontal ligament of rat incisor could be reinnervated by the periodontal Ruffini endings [4,14,53]. Our experimental studies have demonstrated the regeneration process of the periodontal nerve fibers in the rat incisors following transection of inferior alveolar nerve (IAN) [4,14,53]; the nerve fibers disappeared from the periodontal ligament at postoperative 3 days (PO 3d), and began to regenerate at PO 5d. Through PO 10d to PO 21d, the regeneration of the periodontal nerve fibers actively took place to recover to normal levels by PO 28d. These data indicate the possibility that the periodontal Ruffini endings have a high potential for neural plasticity. Schwann cells play important roles during the regeneration of peripheral nerves; denervated Schwann cells proliferate and promote the regeneration of axons following nerve injury. During the regeneration of the periodontal Ruffini endings, the terminal Schwann cells also showed a characteristic behavior: they migrate into regions where they are usually not found and their distribution and number return to normal levels following the completion of regeneration [4]. In the central nervous system, the expression of Hsp25 has been reported in glial components under some pathological conditions [5,17,18,22,23,38,39,41,43]. In spite of many reports on the wide distribution and diverse function of Hsp25 in non-neural cells, little information is available on the functional roles of Hsp25 in the Schwann cell, a peripheral glial element. Thus, the present study was undertaken to examine immunohistochemically the expression of Hsp25 in the periodontal nerves of the rat incisors.
2. Materials and methods All animal experiments were done under the Guidelines of Intramural Animal Use and Care Committee of the Niigata University Faculty of Dentistry.
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postoperative treatment such as antibiotics administration was given to the operated rats.
2.2. Tissue preparation By use of this experimental model, our previous studies [4,14,53] have classified the regeneration process of the periodontal Ruffini endings after transection of the IAN into four stages as previously reported [4]; a degeneration stage (PO 1–3d); a commencement of regeneration stage (PO 5–7d); a regeneration stage (PO 10–21d), and the completion of regeneration (PO 28–56d). Thus, the animals were allowed to survive for 3, 7, 10, 14, 21, 28 and 56 days (n55 each), respectively. After an appropriate survival period, they were deeply anesthetized by an intraperitoneal injection of chloral hydrate (400 mg / kg), and perfused transcardially with 4% paraformaldehyde in a 0.1 M phosphate buffer (pH 7.4). The mandibles, including the incisors, were removed en bloc and decalcified with a 10% ethylene diamine tetraacetic acid (EDTA)-2Na solution for 3–4weeks at 4 8C. Serial frozen sections of incisors were cut at a thickness of 35 mm with a freezing microtome, collected in 0.01 M phosphate-buffered saline (PBS; pH 7.4), and treated as free-floating sections.
2.3. Immunohistochemistry Our immunocytochemical protocol has been reported previously [36]. Briefly, after an inhibition of endogenous peroxidase, frozen sections were primarily incubated with a rabbit polyclonal antisera against murine Hsp25 (1: 5000: StressGen Biotechnologies, Victoria, Canada) for 12 h at 4 8C. According to the manufacturer’s instructions, this antiserum against Hsp25 can recognize Hsp25 / 27 from mouse, hamster, guinea pig, rat, bovine and canine cell lysates. As murine Hsp25 is a homologue of human and rat Hsp 27 / 28, we used the term Hsp25. The sections were then incubated with biotinylated goat anti-rabbit IgG (1:1000, Vector Labs., Burlingame, CA, USA), and subsequently with an avidin–peroxidase complex (ABC kit, Vector Labs.) for 90 min each at room temperature. Final visualization used 0.04% 3,39-diaminobenzidine (DAB) and 0.003% H 2 O 2 in a 0.05 M Tris–HCl buffer (pH 7.6) with nickel ammonium sulfate (0.1%) intensification.
2.1. Animals and surgery
2.4. Immuno-control experiments
A total of 43 Wistar rats (60-day-old, weighing 180–200 g at the time of surgery) were used in this experimental study. The surgical transection of the IAN was applied to 35 animals (experimental group) according to a protocol by Atsumi et al. [4]. Animals without an IAN-transection were used as a sham group (n55). Three rats without any surgical treatment further served as a control group. No
Immunohistochemical controls were performed by: (1) replacing the primary antiserum with nonimmune rabbit serum or PBS; (2) omitting the anti-rabbit IgG or the avidin–peroxidase complex. The specificity of antisera against Hsp25 was also checked by an absorption test and has been reported elsewhere [36,40]. These control sections did not show any specific immunoreactions.
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mic processes, appearing as slender profiles (Fig. 2b). However, Hsp25-immunohistochemistry failed to demonstrate any terminal Schwann cells, round in shape, associated with the periodontal Ruffini endings. Hsp25-immunoreaction was also found in the ordinary Schwann cells in the nerve bundles and some endothelial cells (Fig. 2a).
3.3. PO 5 – 7 d: commencement of regeneration stage
Fig. 1. Hsp25-immunoreactivity in the lower periodontal ligament of the untreated control rat. No cellular element immunoreactive to Hsp25 is found in the periodontal ligament while the odontoblasts (OB) show an intense immunoreaction. AB, alveolar bone; T, tooth. Scale bar550 mm.
3. Results
3.1. Control group No Hsp25-immunoreactive structure was recognized in the periodontal ligament of the untreated control group (Fig. 1), though the ameloblasts and odontoblasts in the same sections showed an intense Hsp25-immunoreactivity as reported previously [36,37]. The distribution and terminal morphology of Hsp25 in animals of the sham group were comparable to those of the control group.
3.2. PO 3 d: degeneration stage Hsp25-immunoreactive cellular elements occurred in the periodontal ligament on PO 3d (Fig. 2a and b). The alveolar half of the periodontal ligament (alveolus-related part: ARP) contained many Schwann cells with intense Hsp25-immunoreactivity. They possessed long cytoplas-
A dense distribution of Hsp25-positive structures was found in the periodontal ligament at this stage (Fig. 2c). The immuno-intensity of Schwann cells in the nerve bundles became stronger. The cells with cytoplasmic extensions (Fig. 2d) and some round cells with an indented nucleus in the ARP showed an Hsp25-immunoreactivity. Furthermore, thin nerve fibers, frequently beaded in appearance, were also positive in Hsp25-immunoreaction. A major population of these fibers was distributed at the border between tooth half of the ligament (tooth related part; TRP) and ARP (shear zone) as well as the ARP, and showed various running courses (Fig. 2e). The Schwann cells associated with the nerve bundles were also positive in Hsp25-immunoreaction (Fig. 2d).
3.4. PO 10 – 21 d: regeneration stage On PO 14d, the dendritic structures in the ARP and thin nerve fibers became immunopositive for Hsp25 (Fig. 2f). At higher magnification, these positive structures exhibited thick, ramified nerves (Fig. 2g). However, the slender- or satellite-shaped cells, which appeared in the TRP and shear zone with intense S-100 immunoreactivity, were devoid of Hsp25-immunoreaction. Some Schwann cells in the nerve bundles retained Hsp25-immunoreaction (Fig. 2f). On PO 21d, Hsp25-positive structures decreased in number. Thin nerve fibers taking a straight course and thick nerve terminals showing a tree-like ramification were found in the ARP of the periodontal ligament (Fig. 2h). A few
Fig. 2. Changes in expression of Hsp25-immunoreactivity in the periodontal ligament on PO 3d (a, b), PO 5d (c–e), PO 14d (f, g), PO 21d (h), PO 28d (i) and PO 56d (j). (a) Hsp25-immunoreactive structures appear in the periodontal ligament. In addition, Hsp25-immunoreactions are found in some endothelial cells and the Schwann cells in the nerve bundles (NB). (b) Higher magnification of the boxed area in (a). Hsp25-immunoreactions are localized in the slender cytoplasmic processes and cell bodies of Schwann cells, but not in the nerve fibers. (c) On PO 5d, the number of the Hsp25-positive structures has increased compared with that on PO 3d. Note the absence of Hsp-25 positive cells in the tooth related part (asterisks). (d) Highly magnified image of the boxed area in (c). Hsp25-immunoreactivities are found in the slender Schwann cells and thin nerve fibers. These thin nerve fibers frequently run parallel to the tooth axis (arrows). (e) The round cells in the alveolus-related part show Hsp25 immunoreactivity. (f) The Hsp25 positive nerve fibers remain in the periodontal ligament in contrast to a decrease in the number of cellular elements. At this stage, thick nerve fibers show Hsp25immunoreactivity. (g) Highly magnified view of the boxed area in (f). Thick nerve fibers frequently ramify to display a dendritic fashion, appearing as the Ruffini endings. (h) Hsp25-immunoreaction shows a tendency to disappear on PO 21d. Note the intense immunoreaction in the odontoblasts (OB). (i, j) Hsp25-immunoreactive structures in the periodontal ligament drastically have decreased in number and immuno-intensity daily. No positive element exists in the tooth related part (asterisks). Note intense immunoreaction in the odontoblasts (OB) in (j). AB, alveolar bone; BM, bone marrow; BV, blood vessel; NB, nerve bundle; T, tooth. Scale bars550 mm in (a–d, f, h–j), 10 mm in (e), 20 mm in (g).
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Schwann cells in the ARP exhibited Hsp25-immunoreaction. The odontoblasts and ameloblasts retained intense Hsp25-immunoreaction (Fig. 2h).
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3.5. PO 28 – 56 d: completion of regeneration stage Between PO 28d and PO 56d, Hsp25-positive structures
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decreased in number and immuno-intensity gradually, though the odontoblasts and ameloblasts showed a constant intense Hsp25-immunoreactivity (Fig. 2i and j). A few Hsp25 positive structures were restricted to the ARP.
4. Discussion The present experimental study was able to demonstrate the temporal expression of Hsp25 in the regenerating nerve fibers and Schwann cell elements in the periodontal ligament following transection of the IAN. There has been controversy over Hsp25-expression in sensory neurons under normal conditions; normal adult brains express little or no Hsp25-immunoreactivity [9,13,23,24,46,47,49]. In contrast, some investigators have pointed out the constitutive expression of Hsp25 in a subset of primary sensory neurons in both cranial sensory ganglia and dorsal root ganglia [11,40,51]. In the present study, however, no Hsp25-immunopositive structure was recognizable in the periodontal ligament under normal conditions, consistent with previous reports on the rat upper incisor [36,37]. There are possible two explanations for this discrepancy. First, since previous studies have demonstrated that approximately 60–70% of neurons in the trigeminal ganglion (TG) [51] and 45% of neurons in the trigeminal mesencephalic nucleus (MeV) [39,40]— both of which innervate the periodontal ligament—showed Hsp25-immunoreactivity, the immunonegative neurons in TG and MeV may innervate the lower periodontal ligament of rat incisors. Second, the antiserum against Hsp25 used in this study was a polyclonal one raised against a short amino acid sequence of the mouse Hsp25. Plumier et al. [40] reported a discrepancy between detection of Hsp25 by Western blot analysis and the absence of Hsp25-positive staining with immunohistochemistry, and considered the presence of three phosphorylation states of Hsp25 in rat spinal cord samples. Thus, the periodontal nerve fibers might be in a functionally different state, even under normal conditions. This immunohistochemical study demonstrated the temporal expression of Hsp25-immunoreactivity in the thin and thick nerve fibers, predominantly from PO 5d to PO 21d, which has been regarded as the regeneration stage because of a drastic increase in the regenerating nerve fibers [4,14,53]. Since previous studies have revealed the upregulation of Hsp25 protein and mRNA in injured neurons in vivo [11,15] and in vitro [27] and since Hsp is transported in nerve fibers, it is likely that the presence of Hsp25 in the regenerating nerve fibers is indicative of the synthesis of Hsp25 in the cell body and the transport of this protein to the periodontal nerves. An intriguing characteristic of Hsp25 is its involvement in actin filament dynamics; Hsp25 has been proposed to act as an actin filament capping protein, potentially either preventing or permitting actin filament synthesis depending on its phos-
phorylation states [26]. Taken together, these findings would seem to indicate that the temporal expression of Hsp25 reflects on actin dynamics in the regenerating nerve fibers. There is no question of the temporal expression of Hsp25-immunoreaction in Schwann cells due to their serving to protect themselves against nerve injury. Similar to Hsp25-expression in the nerve fibers, Schwann cells in the denervated periodontal ligament temporally exhibited an immunoreaction. In general, the glial components—in particular astrocytes—become positive in Hsp25-immunoreaction in certain neurological diseases [5,18,22,23,41,43] and experimentally damaged brains [17,23,38–40]; Hsp25expression is found in peripheral Schwann cells under normal conditions ([51]; this study). Like other Hsps, Hsp25 functions as a molecular chaperone, conserving the conformation of proteins [19], or as an enzyme modulating actin dynamic [26] which is considered to affect cell motility and shape [25,31]. This protein also has been shown to act as a specific cellular inhibitor of apoptosis [32,33,42]. Since the cells migrated to the TRP lacked an Hsp25-immunoreactivity in the experimental model, it would seem that Hsp25 functions as an inhibitor of apoptosis in denervated Schwann cells. On the other hand, damage to neurons induces an increase in the glial fibrillary acidic protein (GFAP) of the glia [1,8,12,35,44,50]. GFAP-immunoreaction has been demonstrated in some ordinary Schwann cells and terminal Schwann cells associated with the Ruffini endings in specific regions [7,52]. Since Hsp25 can affect GFAP assembly [34] in addition to serving as molecular chaperone, Hsp25 in denervated Schwann cells may also serve in the remodeling of intermediate filaments during development and cell differentiation as well as regeneration.
Acknowledgements The authors thank Messers Kiichi Takeuchi and Masaaki Hoshino, Division of Oral Biological Science, Niigata University Graduate School of Medical and Dental Science, for their technical assistance. This study was supported by Grants-in-Aid for Scientific Research ([13470383 to SW and [14370580 to TM) from Japan Society for the Promotion of Science (JSPS).
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