Dorsal horn lesion resulting from spinal root avulsion leads to the accumulation of stress-responsive proteins

Brain Research 893 (2001) 84–94 www.elsevier.com / locate / bres

Research report

Dorsal horn lesion resulting from spinal root avulsion leads to the accumulation of stress-responsive proteins Hiroshi Nomura*, Akiko Furuta, Satoshi O. Suzuki, Toru Iwaki Department of Neuropathology, Neurological Institute, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812 -8582, Japan Accepted 28 November 2000

Abstract The aim of this study was to demonstrate acute to subacute molecular episodes in the dorsal horn following root avulsion using immunohistochemical methods with the markers for synapses, astrocytes and such stress-responsive molecules as heat shock proteins (Hsps) and p38 MAP kinase (p38). Among them, Hsp27 was accumulated selectively in the injured substantia gelatinosa 24 h after avulsion injury. The localization of Hsp27 in astrocytes within the substantia gelatinosa was confirmed by the double immunofluorescence method using anti-Hsp27 antibody and either anti-synaptophysin antibody or anti-glutamine synthetase antibody and by immunoelectron microscopy for Hsp27. The pattern of Hsp27 expression subsequently changed from glial pattern to punctate pattern by 7 days. Immunoelectron microscopy revealed that the punctate pattern in the subacute stage corresponded to distal parts of the astrocytic processes. Hsp27 immunoreaction was decreased 21 days after root avulsion. In the distal axotomy model, Hsp27 was accumulated later in the ipsilateral dorsal horn in a punctate pattern from 7 days after the axotomy. Phosphorylation of p38 was detected in microglia in the dorsal horn following both avulsion and axotomy. Substance P was slightly decreased in the injured substantia gelatinosa in both the avulsion and axotomy models around 14–21 days. We conclude that Hsp27 is a useful marker for demonstrating dorsal horn lesions following avulsion injury and that avulsion injury may induce Hsp27 in the dorsal horn more rapidly than distal axotomy.  2001 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Trauma Keywords: Root avulsion; Root axotomy; Rat; Heat shock protein 27; p38 MAP kinase; Substantia gelatinosa

1. Introduction It is common knowledge that some patients with avulsion injuries to the brachial plexus suffer from severe intractable pain in the deafferentiated limb [4,20]. This intractable pain appears as a shooting incidental pain lasting a few seconds at a time, and this makes it distinguishable from causalgia arising from peripheral nerve damage [53]. Recently, surgical treatment to induce coagulation from the dorsal root entry zone to the substantia gelatinosa, such as thermocoagulation, cordotomy

*Corresponding author. Tel.: 181-92-642-5536; fax: 181-92-6425540. E-mail address: h [email protected] (H. Nomura). ]

and so on, has been applied for phantom pain following brachial plexus avulsion injury and this has been reported to provide initial relief from pain [9,34,47,48,51,53]. This would suggest that phantom pain could arise following any episode of injury to the dorsal horn. To clarify this in the dorsal areas of the avulsed side of the spinal cord, several histopathological studies have been performed. Ovelmen-Levitt et al. showed chronic degeneration of Lissauer’s tract and gliosis in the substantia gelatinosa, as well as shrinkage of the interneurons in the dorsal horn of the avulsed side following root avulsion [38]. McNeill et al. provided evidence that chronic spinal degeneration following dorsal rhizotomy led to synaptogenesis from the surviving primary afferent fibers in the injured dorsal horn [32]. In addition, an examination of acute events in the avulsed dorsal horn was carried out by Zhao et al. [60]. They showed that nerve root avulsion led

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H. Nomura et al. / Brain Research 893 (2001) 84 – 94

to an increased and prolonged expression of c-fos in excess of that seen following root axotomy. Their results were compatible with the direct effects of avulsion on spinal neurons. However, no stress-related proteins had been investigated to assess the effects of the acute spinal lesion immunohistochemically. Recently, heat shock proteins were demonstrated as proteins that are induced by various forms of stress, such as heat shock or oxidative injury, in order to protect injured cells [8,15,18]. In particular, heat shock protein 27 (Hsp27), one of the low molecular weight heat shock proteins, is known as a molecular chaperone and is rapidly induced in the nervous system following oxidative and cellular metabolic stress [23,28,29,40]. Costigan et al. reported that Hsp27 expression was increased in the ipsilateral dorsal horn after peripheral axotomy, but not after rhizotomy, and they suggested that the increased Hsp27 expression after the axotomy contributed towards the prevention of cell death [10]. Moreover, some previous studies have shown differences in damage between rhizotomy and avulsion denervation [38]. The aim of the present study is to describe temporal molecular episodes in the avulsed dorsal horn by immunohistochemical methods. Immunohistochemistry for Hsp27 was useful for demonstrating the molecular dynamics in the affected dorsal horn in our study. Here, we show temporal, yet distinct, molecular changes in reactive astrocytes, microglia and synapses, and these alterations are compared with the peripheral nerve transection model.

2. Materials and methods

2.1. Lower lumbar nerve avulsion Our methods of nerve avulsion are based on the protocol of He et al. [21]. A total of 21 female Wistar rats (28–35 days old, 60–100 g) were deeply anesthetized with intraperitoneal injections of 20 mg / kg of sodium pentobarbitol. The right lower lumbar nerves were exposed at the intervertebral foramen by resection of the transverse vertebral process under an operating microscope and the nerve was avulsed carefully with forceps. In this procedure, both the ventral root and the dorsal root with the dorsal root ganglion (DRG) were pulled out, accompanied by minor bleeding from the intervertebral foramen. The wound was then closed in layers with sutures and the animals were allowed to survive for between 6 h and 21 days after the operation. At each time point (6 h, 12 h, 24 h, 48 h, 7 days, 14 days and 21 days) three rats were perfused through the ascending aorta with 200 ml saline followed by 200 ml 4% paraformaldehyde under deep anesthesia. Then, the spinal cord was carefully removed and postfixed at 48C overnight. The avulsed point on the spinal cord was confirmed under microscopy.

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2.2. Lower lumbar spinal nerve axotomy Seven female Wistar rats (28–35 days old, 60–100 g) were anesthetized as described above. The right lower lumbar nerve was exposed as described above, and then axotomized at a point distal to the DRG, at the same distance as in the avulsed model. After survival for 48 h, 7 days or 21 days, the rats were killed under deep anesthesia and perfused as described above. The axotomized level of the spinal cord was determined by careful observation of the motion of the incomplete paralytic lower limb.

2.3. Immunohistochemistry The spinal cords were immersed in chilled 30% sucrose in phosphate-buffered saline for 4–7 days. Then, the spinal cords were embedded in O.C.T. compound (Tissue-Tek, Torrance, CA) and frozen instantly in chilled 2methylbutane. A total of 10 mm-thick transverse spinal cord sections were cut from caudal to rostral on a cryostat. The sections were routinely stained with hemotoxylin– eosin. Immunohistochemistry was performed using the indirect immunoperoxidase method. The following antibodies were used in this study: rabbit anti-heat shock protein 25 polyclonal antibody which reacts with both mouse Hsp25 and rat Hsp27 (1:2000 dilution; StressGen, Canada), rabbit anti-glial fibrillary acidic protein (GFAP) polyclonal antibody (1:2000 dilution; DAKO, Denmark), rabbit anti-phospho-p38 MAP kinase (p38) polyclonal antibody (1:200 dilution; BioLabs, UK) and rabbit antisubstance P (SP) polyclonal antibody (1:2500 dilution; Chemicon, Temecula, CA). After being washed twice with Tris–HCl (50 mM, pH 7.6), the sections were pretreated with 0.3% H 2 O 2 in absolute methanol for 30 min at room temperature to reduce non-specific staining by endogenous peroxidase. After being rinsed twice with Tris–HCl containing 0.1% Triton X-100 and washed with Tris–HCl, the sections were incubated in a solution of primary antibody diluted in TBST (25 mM Tris–HCl pH 7.6, 0.5 M NaCl, 0.05% NaN 3 and 0.05% Tween 20) containing 5% non-fat milk or PBS containing 5% normal goat serum at 48C overnight. After being washed with Tris–HCl, the specimens were incubated either with horseradish peroxidaseconjugated anti-rabbit IgG antibody (Vector, Burlingame, CA) or Envision (DAKO). Diaminobenzidine tetrahydrochloride (DAB) containing H2O2 was used to develop the color. Scores were assigned as follows: no increased staining (2), detectable increased staining (1) and strongly increased staining (11), by comparing the lesion side with the contralateral (control) side.

2.4. Double immunofluorescence For identification of the the cellular localization of Hsp27, a panel of antibodies was used for double immunofluorescence: rabbit anti-Hsp27 antibody (1:50 dilution),

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mouse anti-glutamine synthetase monoclonal antibody (1:5 dilution, clone 6, Transduction Laboratories, Lexington, KY), mouse anti-GFAP monoclonal antibody (1:50 dilution, clone G-A-5, Novocastra, UK), mouse anti-synaptophysin monoclonal antibody (1:20 dilution, clone SY-38, DAKO) or anti-human neurofilament protein monoclonal antibody (1:50 dilution, clone 2F11, DAKO) overnight at 48C. After being rinsed twice with Tris–HCl containing 0.1% Triton X-100, the sections were incubated in a mixture of Texas red-labeled donkey anti-rabbit and fluorescein isothiocyanate-labeled sheep anti-mouse IgG (1:50, Amersham, UK) for 2 h at room temperature. The sections were rinsed with Tris–HCl containing 0.1% Triton X-100 covered with glycerol supplemented with 2.3% DABCO (1,4-diazabicyclo-2,2,2-octane). Doublestained sections were observed under a laser scan confocal microscope (LSM-GB200, Olympus, Japan).

in resin. Ultrathin sections without staining as well as those stained with uranyl acetate for 15 min and then lead citrate for 15 min, were observed under an electron microscope (JEM-100CX, JEOL, Japan).

3. Results

3.1. Substantia gelatinosa on the lesion side 3.1.1. Avulsion model In normal controls, weak Hsp27 immunoreactivity (Hsp27 IR) was present in laminae I, III and IV, axons of the gracile fasciculus, somatic motorneurons and their processes of the ventral horn, occasional astrocytes, and ependymal cells and their processes beneath the arachnoid layer. However, laminae II of the dorsal horn was immunonegative for Hsp27. Avulsion did not significantly alter this pattern in the contralateral side of the lumbar spinal cord [40]. Therefore, we assessed Hsp27 expression in the avulsion model by comparing the lesion side with the contralateral side of the spinal cord (Table 1). Hsp27 IR was detected in the substantia gelatinosa on the ipsilateral side of the avulsion model at 24 h after avulsion (Fig. 1A and B). Hsp27 immunostaining appeared to be glial (Fig. 1C). The accumulation of Hsp27 continued 48 h after root avulsion showing a pattern of glial staining (Fig. 2A and B). However, the expression pattern of Hsp27 IR changed to a punctate pattern at 7 days and although the intensity continued for around 14 days (Fig. 2C and D), it had decreased by 21 days after the operation (Fig. 2E and F). To characterize the Hsp27-positive cells, the double immunofluorescence method was carried out. Hsp27-IR

2.5. Immunoelectron microscopy For identification of ultrastructural localization of Hsp27 on the avulsed substantia gelatinosa, the immunoperoxidase method for electron microscopy was performed. For electron microscopy, rats which had been allowed to survive for 24 h and 14 days after avulsion were perfused with 2% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer. The spinal cords were removed, rinsed in cold PBS, and cut transversely at a 50-mm thickness on a vibratome. After treatment with 1% sodium borohydride for 10 min, the vibratome sections were prepared for immunohistochemistry of Hsp27 (1:2500 dilution) using the same method as for light microscopy, except that Triton-X was omitted. Thick sections, which were immunostained for Hsp27, were osmicated, dehydrated by graded concentrations of ethanol, and embedded

Table 1 Immunohistochemistry of Hsp27 and phosphorylated p38 in the spinal card lesions in both rool avulsion and axotomy models a SG

ZL

EZ

FD

FA

VH

Hsp27

p38

Hsp27

p38

Hsp27

p38

Hsp27

p38

Hsp27

p38

Hsp27

p38

Root avulsion 6h 12 h 24 h 48 h 7 days 14 days 21 days

2 2 1|11g 11g 1|11p 11p 2|1p

2 2|1r 2|1r 2|1r,a 2|1a.r 11r5a 11r5a

1 2|1 1g 2 1|11 1|11 11

2|1r,a 2 1r 1a.r 2|1a.r 11a.r 1|11a.r

2 1 1 2 11 11 1

2|1r 2 1r 2|1r 2 2|1r.a 1r5a

2 2 2|1 2|1 1|11 1|11 11

2|1r 1r 2 1a.r 2|1r.a 11a5r 1|11a.r

2 2 2|1 1 1|11 11 1|11

2 2|1r 1r 2|1r 1r.a 11r.a 11

2|1 1 1|11 11 11 11 1|11r.a

2 2|1r 1r 1a.r 11a.r 11r.a

Nerve axotomy 48 h 7 days 21 days

2|1p 11p 2|11p

1r 11r,a 1|11r

2|1 1|11 11

1|11r 2|11r 11a.r

2|1 1|11 11

2|1r 2|11r 1a.r

2 2 11

2 1r.a 1|11r

1|11 1 1|11

1r 1|11r.a 1|11r

1|11 11 11

1r 2|11 2|11r

a

2, no laterality compared with contralteral side; 1, small to moderate increase on the ipsilateral side; 11 marked increase on the ipsilateral side; g, glial pattern; p, punctate pattern; r, ramified microglia; a, ameboid microglia; SG, insilateral side of the substantia gelationsa; ZL, ipsilatral side of the zone of Lissauer; EZ ipsilateral side of the entry zone; FD, ipsilateral side of the funiculus dorsalis; FA, ipsilateral side of the funiculus anterior; VH, ipsilateral side of the ventral horn.

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Fig. 1. Immunohistochemistry of the lumbar cord 24 h after root avulsion probed with anti-Hsp27 antibody. Whole transverse section at a thickness of 10 mm shows strong Hsp27 immunoreactivity in the unilateral substantia gelatinosa. Lumbar spinal cord 24 h after root avulsion (A). Hsp27 immunostaining in the ipsilateral dorsal horn is demonstrated (B). The area shown by the arrow in B is magnified in C. Higher magnification reveals the glial profiles of Hsp27-positive structures (C). Confocal laser scanning micrographs of the ipsilateral substantia gelatinosa of the lumbar spinal cord at 24 h after root avulsion (D–F). (D): glutamine synthetase, green. (E): Hsp27, red. (F): a merged image for glutamine synthetase and Hsp27. Note that Hsp27-positive cells at 24 h are generally overlapping with glutamine synthetase. Bars in (A)5500 mm, (B)5100 mm, (C–F)520 mm.

was generally overlapped with glutamine synthetase (Fig. 1D–F), an astrocytic marker at 24–48 h. However, most of the Hsp27 IR was not colocalized for GFAP at the dorsolateral part on the avulsed substantia gelatinosa. Immunoelectron microscopy for Hsp27 verified Hsp27 IR within the astrocytic cytoplasm at the avulsed substantia gelatinosa 24 h after the operation (Fig. 3). The Hsp27 IR at 7, 14 and 21 days after avulsion was closely associated with immunoreactivity for synaptophysin (Fig. 4A–C). However, Hsp27 IR was hardly colocalized with glutamine synthetase, GFAP or neurofilament. Immunoelectron microscopy for Hsp27 verified that most of the Hsp27 IR lay within the distal processes of the astrocytes at the avulsed substantia gelatinosa 14 days after the operation (Fig. 5). GFAP IR was also upregulated on the lesion side of the avulsion model from 6 h postoperatively in the fibrillary astrocytes, becoming more intense after 48 h in a timedependent manner (Fig. 8A–C). Phosphorylated p38 MAPK in normal spinal cords was localized in the microglia as previously reported [5,43,58]. Phosphorylated p38 IR was enhanced in the ramified microglia, especially at the medial part, on the ipsilateral substantia gelatinosa of the avulsion model from 12 to 24 h. Some microglia became transformed into ameboid microglia after 48 h (Fig. 7A). Substance P was decreased slightly on the ipsilateral side of the avulsion model from 14 days, but it did not disappear completely (Fig. 9A).

3.1.2. Axotomy model Immunostaining for Hsp27 showed a punctate pattern in the substantia gelatinosa on the ipsilateral side of the axotomy model from 7 days (Fig. 6C and D). However, Hsp27 IR was only slightly detected at 48 h, this being much weaker than that in the avulsion model (Fig. 6A and B). To characterize the Hsp27-positive cells on the ipsilateral substantia gelatinosa at 48 h and 21 days after axotomy, the double immunofluorescence method was performed. In the 48-h model, Hsp27-IR was overlapped mainly with glutamine synthetase on the ipsilateral substantia gelatinosa, although the Hsp27 IR was punctate fashion, which was distinct from the glial pattern of the avulsion model. In the 21-day model, Hsp27-IR was markedly increased and was partly associated with immunoreactivity for synaptophysin, but they were not identical (data not shown). GFAP IR was similar to the avulsion model, which showed a persistent increase in astrocytes on the ipsilateral substantia gelatinosa of the axotomy model. A marked increase in microglia immunopositive to activated p38 was noted in the dorsal horn on the axotomized side from 48 h to 21 days postoperatively. The morphology of the microglia demonstrated mainly ramified microglia rather than ameboid one (Fig. 7B). A decrease in SP IR was observed in the substantia gelatinosa on the ipsilateral side at 21 days (Fig. 9B). The degree of decreased intensity was almost the same as that seen in the avulsion model.

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Fig. 2. Photomicrographs showing Hsp27 immunoreactivity (Hsp27 IR) in the substantia gelatinosa of the ipsilateral dorsal horn of the spinal cords at 48 h (A, B),14 days (C, D) and 21 days after avulsion (E, F). Each area shown by the arrow in A, C and E is magnified in B, D and F, respectively. At 48 h after root avulsion, Hsp27 IR is detected in the ipsilateral substantia gelatinosa as a glial pattern (A, B). At 14 days postoperatively, Hsp27 IR is observed in the ipsilateral substantia gelatinosa, but the pattern has changed to a punctate pattern (C, D). By 21 days after root avulsion, Hsp27 IR is decreased in the avulsed substantia gelatinosa, and is only detectable in the lateral part of the substantia gelatinosa (E, F). Bars in (A, C, E)550 mm, (B, D, F)520 mm.

3.2. Zone of Lissauer on the ipsilateral side The zone of Lissauer was affected by the direct impact of the injury. Hsp27 IR was apparently upregulated at the zone of Lissauer on the avulsed side from 7 days, and was persistently increased by 21 days (Fig. 2C and E). On the other hand, Hsp27 IR in the axotomized model was

detected from 7 days and continued to be upregulated until 21 days (Fig. 6C). GFAP IR was increased in the fibrillary astrocytes from 6 h to 21 days postoperatively on the ipsilateral side of the zone of Lissauer in the avulsion model (Fig. 8A and B). On the other hand, GFAP IR was increased at a later point, from 7 days after the axotomy.

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Fig. 3. Immunoelectron microscopy for Hsp27 in the substantia gelatinosa of the ipsilateral dorsal horn 24 h after root avulsion. Hsp27 is localized within the astrocytic cytoplasm. Bar51 mm.

Phosphorylated p38 IR was increased in both the ramified and ameboid microglia from 24 h to 21 days on the avulsed side of the zone of Lissauer (Fig. 7A). In the axotomy model, a temporal increase in ramified microglia was observed by immunohistochemistry for phosphorylated p38 from 48 h to 21 days on the ipsilateral side of the zone.

3.3. Funiculus dorsalis on the ipsilateral side Axonal staining of Hsp27 IR was upregulated at the ipsilateral funiculus dorsalis both in the avulsion and axotomy models from around 48 h to 7 days (Fig. 2C and E). Marked accumulation of GFAP or phosphorylated p38 in the reactive astrocytes and in the microglia, respectively, was observed after 14 days or 21 days at the funiculus dorsalis on the ipsilateral side in both injury models.

3.4. Ventral horn and funiculus anterior Hsp27 IR was augmented within the cytoplasm of the motorneurons and their processes on the avulsed side of the ventral horn when compared with the contralateral side after 24–48 h (Fig. 1A). The increase in Hsp27 IR was observed until 21 days. In the axotomized model, Hsp27 IR was also elevated from 48 h until 21 days on the ipsilateral side in the same pattern as the avulsion model. In addition, Hsp27 IR was upregulated in the astrocytic processes surrounding the efferent axons on the avulsed or axotomized side of the funiculus anterior from 48 h and was further increased by 21 days. GFAP IR was increased in the fibrillary astrocytes around the motor neurons on the ipsilateral ventral horn in both the avulsion and the axotomy models from 48 h to 21 days (Fig. 8A). Phosphorylated p38 IR was increased in the microglia

Fig. 4. Confocal laser scanning micrographs showing double-labeled sections of the ipsilateral substantia gelatinosa of the lumbar spinal cord at 14 days after root avulsion. (A): synaptophysin, green (B): Hsp27, red (C): a merged image for synaptophysin and Hsp27. Colocalized signals are in yellow. Note that Hsp27-positive structures at 14 days are closely related with synaptophysin, but they are not identical (C). Bar in (C)520 mm.

from 24 to 48 h to 21 days on the avulsed or axotomized side of the ventral horn.

4. Discussion Hsp27 is a member of the group of small heat shock proteins, and is widely known as one of the low molecular weight chaperones which increase cell resistance to several types of injury [10,23,27]. This study shows that Hsp27 is

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Fig. 5. Immunoelectron microscopy for Hsp27 in the avulsed side of the substantia gelatinosa 14 days after the operation. Hsp27 IR can be detected in the fine processes of the astrocytes. Bar50.5 mm.

the most useful immunohistochemical marker for revealing dorsal horn lesions caused by root avulsion. We also identified the cellular localization of Hsp27 immunoreactivity by using a panel of antibodies for different cellular markers [5]. During the early period, Hsp27 IR was augmented within the cytoplasm of astrocytes and their processes in

the ipsilateral substantia gelatinosa following root avulsion. Hsp27 has been shown to accumulate mainly in reactive astrocytes following several types of stress stimuli [12,16,25,28,41,49]. Wagstaff et al. demonstrated that Hsp27 mRNA was raised in the glial cells in the subregions of the hippocampus within 2 h after focal cerebral ischemia in rats, whereas Hsp27 protein was not detected until after 24 h [57]. We also detected the accumulation of Hsp27 at the affected substantia gelatinosa from 24 h after avulsion, this time point of detection being largely consistent with that report. Furthermore, we observed that Hsp27-positive astrocytes were overlapped with glutamine synthetase, which is generally labels protoplasmic astrocytes [14,36,37]. Hsp27-positive astrocytes were also frequently noted in the dorsolateral part on the avulsed substantia gelatinosa, in which many sensory neurons exist. This means that astrocytes surrounding the sensory neurons are exposed to stress by the root avulsion. In a time-dependent manner, Hsp27 IR after root avulsion was closely associated with the synaptic component at the ipsilateral substantia gelatinosa, and it was localized within the processes of the astrocytes. This phenomenon suggests that the stress-related site at the avulsed substantia gelatinosa temporally shifts from the cytoplasm of the astrocytes to any of the glial parts of the synaptic element. Bechtold and Brown demonstrated that

Fig. 6. Photomicrographs showing Hsp27 IR in the ipsilateral substantia gelatinosa of the spinal cords at 48 h (A, B) and 14 days (C, D) after distal axotomy. At 48 h after distal axotomy, Hsp27 IR is slightly upregulated in the ipsilateral substantia gelatinosa (A, B). 21 days after distal axotomy, Hsp27 IR can be detected clearly in the ipsilateral substantia gelatinosa showing a punctate pattern (C, D). Bars in (A, C)550 mm, (B, D)520 mm.

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Fig. 7. Photomicrographs showing phosphorylated p38 IR, which is localized in the microglia, at the medial part of the ipsilateral dorsal horn in the lumbar spinal cord 14 days after root avulsion (A) and 21 days after distal axotomy (B). A marked increase in both ameboid and ramified microglia is observed at 14 days after root avulsion by immunohistochemistry for activated p38 from the zone of Lissauer to the medial part of the ipsilateral dorsal horn (A). An increase in microglia with phosphorylated p38 is also evident in the medial part of the dorsal horn on the axotomized side at 21 days, the morphology of which is mainly demonstrated by ramified microglia (B). Bars in (A, B)550 mm.

Hsp27 and Hsp32 were strongly induced in Bergmann glial cells in the rat brain and transported into their radial fibers, and that these hyperthermia-induced Hsp27 and Hsp32 were detected in both postsynaptic structures and perisynaptic glial processes [3]. In our results, Hsp27 IR is only detected in the glial processes at the avulsed substantia gelatinosa under the immunoelectron microscopy. However, we can not deny the possibility of the transportation of Hsp27 from the perisynaptic glial processes to the postsynaptic elements. Hsp27 IR from 7 days after root avulsion was not colocalized with glutamine synthetase. Norenberg and Martinez-Hernandez showed that glutamine synthetase was not found in the synaptic endings of glutamine synthetase-positive astrocytes by ultrastructural immunocytochemistry [35]. Costigan et al. reported that Hsp27 expression was increased in the ipsilateral dorsal horn after peripheral nerve axotomy but not after rhizotomy due to axonal

Fig. 8. Photomicrographs showing GFAP IR in the lumbar spinal cord 14 days after root avulsion (A, B, C). GFAP IR is increased in the astrocytes in the right dorsal horn of the avulsion model at 14 days (B) when compared with the contralateral dorsal horn (C). Bars in (A)5500 mm, (B, C)5100 mm.

transport to the central terminals from the injured sensory neurons in the DRG, and they suggested that increased Hsp27 expression following axotomy contributed to the prevention of cell death [10]. On the other hand, OvelmenLevitt demonstrated differences in damage between rhizotomy and avulsion denervation [38]. Our results also propose that stress-associated sites at the avulsed substantia gelatinosa are due to different pathological events, compared with the sites of axotomy or rhizotomy. We demonstrated that Hsp27 IR was only slightly detected on the axotomized substantia gelatinosa at 48 h, and this staining pattern of Hsp27 IR was unlike that seen in the avulsion model. However, Hsp27 IR at 48 h after axotomy

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Fig. 9. Photomicrographs showing substance P IR in the lumbar spinal cord 14 days after root avulsion (A) and 21 days after distal axotomy (B). Note that substance P is slightly decreased in the ipsilateral side in both the avulsion and axotomy models. Bars5500 mm.

was also detected in several glial cells on the axomized substantia gelatinosa. This finding suggests that glial cells at the ipsilateral substantia gelatinosa of the axotomy model also show mild stress reaction in the early stage. Hsp27 IR was upregulated as axonal staining, and it seemed to be localized within the axoplasm and glial sheath surrounding the axon, at the avulsed funiculus dorsalis. However, the axoplasm does not contain the constituents required for classical protein synthesis. We propose one possibility that the Hsp27 may be transferred from the astrocytic sheath to the axon at the avulsed funiculus dorsalis [22,50,54]. Tytell and Lasek described how Hsp95 was transferred from the glia to the axon in the heat-treated sheath and suggested that the process of gliaaxon transfer of stress protein was a common feature of the response of the nervous system to acute stress and that it may be an important factor in the reaction of neurons to trauma [54]. We also investigated astrocytic gliosis at the avulsed substantia gelatinosa using GFAP immunohistochemistry and compared it with the nerve axotomy model. In both models, astrocytic gliosis at the lesioned substantia gelatinosa was evident. Various conflicting opinions have been proposed by many investigators regarding the role of reactive astrogliosis, whether it is the regeneration of

injured neurons or the inhibition of their regrowth [1,2,17,26,42,46,56]. Ribet et al. showed that reactive gliosis varies qualitatively and quantitatively depending on both the nature of the injury and the microenvironment of the injury site [45]. In contrast, Tacconi suggested that astrocytes contribute to a decline in neurologic function, for example through the accumulation and release of excitotoxic amino acids following ischemia or oxidative stress, through the formation of epileptogenic scars in response to CNS injury, or through the metabolism of protoxins to potent toxins [52]. To assess the pathologic changes of the synaptic terminals of the sensory neurons, we examined synaptic density at the avulsed substantia gelatinosa with immunohistochemistry for SP, which is known to be a potential transmitter substance of pain pathways [6,11,30,31,55], and we compared it with that following nerve axotomy. In both models, SP IR was slightly decreased in the injured substantia gelatinosa, however, its immunoreactivity never completely disappeared. Blumenkopf reported that SP was similarly reduced on the ipsilateral side of laminae I and II at 1 week after avulsion injury, that it continued to show a relative decline in the appropriate laminae through the 6th week, but that it recovered to a normal level by the 10th week in laminae I and V, thus suggesting a possible mechanism for deafferentation pain through this change in distribution of SP [7]. Our results which showed a slight decrease in SP IR on the injured substantia gelatinosa were, thus, consistent with this previous report. Furthermore, we observed pre-synaptic terminals of the sensory neurons on the avulsed substantia gelatinosa using synaptophysin expression. However, no visible change in synaptophysin immunoreactivity was detected (data not shown). We showed that phosphorylation of p38 was clearly detected in the microglia on the dorsal horn lesioned by both avulsion and axotomy. P38 is known to be one of the mitogen-activated protein kinase family members, which plays a crucial role in transforming stress-related signals and in delaying neuronal cell death [19,24,33,39,59]. On the other hand, several investigators have reported that p38 was activated in microglia under various neuronal diseases [5,43,58]. Watson et al. reported that phosphorylated p38 IR was observed in the microglia in regions adjacent to, but not within, the dying CA1 neurons of the hippocampus following ischemia, and they suggested that p38 played a role in the microglial response to brain lesions [58]. Furthermore, the morphology of the microglia detected by phosphorylated p38 IR demonstrated activated microglia in the medial part of the substantia gelatinosa, Lissauer’s tract and the dorsal root entry zone of the avulsed side, which are likely to suffer from the direct impact of avulsion [13]. In contrast, in the axotomy models, the morphology demonstrated mainly ramified microglia. These phenomena implied that activated microglia detected by phosphorylated p38 IR play a role in phagocytizing the debris and foreign substances after root avulsion, since avulsed

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stress is accompanied by degeneration in dorsal parts of the spinal cord, whereas axotomized stress is not. In conclusion, we demonstrated changes in the expression of Hsp27, etc., in response to acute or subacute molecular episodes of astrocytes and pre-synaptic terminals on the sensory neurons in the avulsed dorsal horn, and we compared these episodes with findings following peripheral nerve axotomy. Hsp27 is a most useful marker for demonstrating stress-related sites following avulsion injury. Recently, Ramer et al. demonstrated the functional regeneration of the rhizotomized sensory axons into the spinal cord of the rat treated with several neurotrophic factors [44]. We hope that the expression of Hsp27 will be available as a stress-related marker for clinical studies in the avulsion model.

Acknowledgements The work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (Grant No. 12680733 awarded to T.I.) We thank Dr. Jian Wen He, Prof. M. Kawabuchi of the Department of Anatomy and Cell Biology, Graduate School of Medical Science, Kyushu University, and Ms. K. Hatanaka for their excellent technical support. The English used in this manuscript was revised by Miss K. Miller (Royal English Language Centre, Fukuoka, Japan).

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