ARTICLE |
Correspondence to: Tetsuro Takamatsu, Dept. of Pathology and Cell Regulation, Kyoto Prefectural U. of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan.
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Summary |
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We immunohistochemically and morphometrically examined the expression of gap junction protein connexin (Cx) in normal and crush-injured rat sciatic nerves using confocal laser scanning microscopy. Cx26 was localized in the perineurium and Cx43 was present in the perineurium and the epineurium, whereas Cx32 was confined to the paranodal regions of the nodes of Ranvier. Double labeling for connexins and laminin revealed that Cx43 was localized in multiple layers of the perineurium, whereas Cx26 was confined to the innermost layer. Double labeling for connexins and a tight junction protein, occludin, showed that occludin frequently coexisted with Cx43 but existed separately from Cx26 in the perineurium. After crush injury, the pattern of normal Cx32 expression was initially lost but recovered, whereas Cx43 rapidly appeared in the endoneurium and its expression was subsequently attenuated. Although crush injury produced no apparent alteration in Cx43 and occludin in the perineurium, a rapid increase and a subsequent decrease in the frequency of Cx26-positive spots during nerve regeneration were shown by morphometric analysis. These results indicate that Cx26, Cx32, and Cx43 are expressed differently in various types of cells in peripheral nerves and that their expressions are differentially regulated after injury. The expression of connexins and occludin in the perineurium suggests that perineurial cells are not uniform in type and that the regulation of gap junctions and tight junctions is closely related in the perineurium. (J Histochem Cytochem 47:937948, 1999)
Key Words: connexin, rat sciatic nerve, perineurial cell, occludin
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Introduction |
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Gap junctions are the transmembrane channels that directly link neighboring cells and mediate reciprocal exchange of low molecular weight (less than 1000 Da) metabolites and ions, including second messengers such as cAMP, inositol trisphosphate, and Ca2+, between cells in contact. Gap junctional intercellular communication is considered to play a crucial role in the maintenance of homeostasis and morphogenesis, cell differentiation, and growth control in multicellular organisms (
The peripheral nervous system consists of three different compartments, i.e., the endoneurium, perineurium, and epineurium (from inside to outside). The endoneurial compartment contains axons and their surrounding Schwann cells, collagen fibers, fibroblasts, capillaries, and a few mast cells. The perineurial compartment that encloses the endoneurial compartment is made of concentric layers of flattened fibroblast-like cells called perineurial cells (
Gap junctional intercellular communication constitutes an important part of cellcell communication in the peripheral nervous system. It has been shown that Cx32 is localized at the paranodal regions of the nodes of Ranvier and SchmidtLantermann incisures in the peripheral nervous system (
In this study we performed immunofluorescence studies in intact rat sciatic nerves using antibodies against Cx26-, Cx32-, and Cx43-specific peptides, in combination with confocal laser scanning microscopy, to elucidate which connexins are expressed in the peripheral nervous system. Double labeling was also performed by combining connexin immunolabeling with antibodies to laminin (a component of basal lamina) or occludin, an integral tight junction protein (
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Materials and Methods |
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Animals
Adult Wistar rats weighing 250350 g were sacrificed under deep diethyl ether anesthesia and sciatic nerves were exposed and removed. The specimens were immediately frozen in liquid nitrogen with Tissue-Tek OCT compound (Miles; Elkhart, IN) and stored at -80C.
Nerve Crush Injury
Fifteen adult Wistar rats were anesthetized by IP injection of sodium pentobarbital (4 mg/100 g body weight). Anesthesia was monitored and confirmed by the absence of pain reflexes. One side of the sciatic nerve was exposed and crushed for 10 sec at a sciatic notch with fine forceps. The muscles were then reattached at the suture line and the wound was closed. After various intervals (4, 7, 14, 21, or 28 days after crush injury, three rats/group), animals were sacrificed with diethyl ether and the crushed sciatic nerves were removed for analyses. The sciatic nerve from the contralateral side was collected as a control.
Immunohistochemistry
The applied primary antibodies, species, dilutions, and sources are given in Table 1. Serial 1020-µm frozen sections were prepared, air-dried, and fixed in absolute ethanol at -20C for 15 min. After incubation in PBS, pH 7.4, supplemented with 5% skim milk for 15 min at room temperature to block nonspecific labeling, the primary antibodies (see Table 1) were applied overnight at 4C. After three 5-min washes with PBS, incubation with fluorescein isothiocyanate (FITC)-labeled anti-rabbit or anti-mouse antibodies (1:300 dilution; DAKO, Copenhagen, Denmark) was performed for 1 hr at 37C. After final rinses in PBS, the sections were mounted in Vectorshield (Vector Laboratories; Burlingame, CA). All antibody dilutions were made in PBS containing 1% bovine serum albumin (Sigma; St Louis, MO) and 1% sodium azide with 0.1% Triton X-100. As negative controls, specimens were incubated in the absence of the primary antibodies.
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A rabbit polyclonal anti-protein gene product 9.5 (PGP9.5) antibody (1:500 dilution; UltraClone, Isle of Wight, UK) was used for serial sections from crush-injured nerves to assess the presence of regenerating axonal fibers (
Some sections were double labeled for Cx26 and Cx43 by utilizing the rabbit anti-Cx26 (
Confocal Laser Scanning Microscopy
The sections were viewed with a confocal laser scanning microscope (Olympus FLUOVIEW system; Tokyo, Japan) coupled to an inverted microscope (IX70; Olympus) equipped with an oil immersion objective (PlanApo x60, NA = 1.4; Olympus). An argonkrypton laser generated excitation bands at 488 nm for FITC, 568 nm for Texas Red, and monochromatic light for differential interference contrast (DIC) images. Fluorescence images were collected with emission filters for 510550 nm for FITC and 585610 nm for Texas Red. Simultaneous images (800 x 600 pixels, 12 bits each) of FITC and Texas Red labels were obtained and stored. In some cases, DIC images were obtained in parallel with fluorescence images and three-channel images were constructed with FLUOVIEW imaging software. Digital images were transferred to a Macintosh computer (Apple Computer) equipped with Photoshop software (Adobe System) and printed on an ink-jet printer (PM-750C; Epson, Tokyo, Japan).
Morphometric Analysis
For morphometric assessment of connexin-immunolabeled spots, digital images were collected by adjusting the sensitivity of the photomultiplier so that the spectrum of label intensities in each section spanned within the 4096-level scale. Section planes were obtained at 1-µm steps along the z-axis using the objective x60 and a zoom setting of 2 or 3. A single optical plane was then selected and the image was converted to 8 bits (256 level), and transferred to the NIH image program (Wayne Rasband; NIH, Bethesda, MD) in a Macintosh computer. A binary overlay was created and spots were morphometrically analyzed by counting their number (frequency) and measuring their area (size). The extent of co-localization of occludin (green signals) with connexins (red signals) was evaluated as the percentage of yellow area in the green or red area. In the crush-injury model, each image was obtained 510 mm proximal or distal to the site of the crush injury, and gap junctional spots were counted and measured in the same way as above. As a control, data were obtained from untreated sciatic nerves. Data on each group were collected from more than six images and were expressed as the mean ± SD.
Statistical Analysis
Statistical analysis was performed using StatView software (Abacus Concepts). Data on the size and frequency of immunolabeled connexin spots in normal sciatic nerve were examined by the KruskalWallis test or the MannWhitney U-test. Data in the crush model were examined by two-factor factorial ANOVA and Scheffé test. Significant differences were defined by p<0.05.
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Results |
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Normal Rat Sciatic Nerves
Indirect immunohistochemistry on transverse sections of normal rat sciatic nerves demonstrated the expression of Cx26, Cx32, and Cx43. The polyclonal rabbit antibody against Cx26-specific peptides (
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In transverse sections, Cx32 immunolabeling was detected in some neural fibers of the endoneurium but not in the perineurium or the epineurium (Figure 1C and Figure 1D). Labeling was localized to the paranodal regions of the nodes of Ranvier in the longitudinal sections (Figure 6A and Figure 6B). Identical results were obtained with both mouse and rabbit antibodies against Cx32 (not shown).
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With the polyclonal rabbit antibody against Cx43, abundant punctate Cx43 immunolabeling was detectable in multiple layers of the perineurium, and a small number of unevenly distributed punctate spots were also detected in the epineurium (Figure 1E and Figure 1F). However, no Cx43 immunoreactivity was found in the endoneurium except in endoneurial vessels (not shown). Identical results were obtained by use of the monoclonal mouse antibody against Cx43 (Figure 2B, Figure 3, and Figure 4B). The immunohistochemical localization of connexins and other proteins in this study is summarized in Table 2.
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The anti-Cx26 and anti-Cx32 antibodies demonstrated the expected punctate patterns of gap junctional plaques in rat livers, as did the anti-Cx43 antibodies in rat hearts (not shown). In the absence of the primary antibodies, nonspecific fluorescence was not noted in the sciatic nerve with FITC-labeled anti-rabbit or anti-mouse antibodies (Figure 1G1J). Table 3 summarizes morphometric data on the size and frequency of immunolabeled connexin spots in the normal rat sciatic nerve. There was no significant difference among the different antibodies for each connexin.
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Double Labeling
To elucidate the localization of Cx26 and Cx43 in the perineurium in more detail, we performed double labeling for Cx26 or Cx43 and laminin, a component of the basal lamina of perineurial cells. The rabbit polyclonal anti-laminin antibody revealed linear labeling in multiple layers of the perineurium and around neural fibers (Figure 2), in accordance with previously reported findings (
To assess whether both connexins are co-localized at the same gap junction plaque, double labeling for Cx26 and Cx43 was performed using a rabbit anti-Cx26 antibody (
Immunolabeling for occludin, an integral tight junction protein, was utilized to investigate the localization relationship between connexins and tight junctions in the perineurium. Occludin-positive punctate spots formed short lines in multiple layers of the perineurium (Figure 4A and Figure 4B) and in endoneurial vessels (not shown). Double labeling for Cx26 and occludin showed different localization of the proteins. Cx26 was present in the innermost layer of the perineurium, whereas the expression of occludin was distributed in multiple layers but not across layers (Figure 4A). In contrast, double labeling for Cx43 and occludin revealed that punctate spots of both proteins combined and formed a continuous linear structure, and that both proteins frequently coexisted in the same linear structure (Figure 4B). At the resolution of confocal microscopy, Cx43-positive spots appeared to be located between occludin-positive spots, and some yellow spots indicating co-localization of Cx43 (red) and occludin (green) were observed. Morphometric analysis of uninjured sciatic nerves showed that the ratio of co-localization (yellow) area to the occludin (green) and to the Cx43 (red) areas was 13.1 ± 2.2% and 36.4 ± 6.1% (mean ± SD), respectively. In contrast, little yellow area was found in double labeling for Cx26 (red) and occludin (green), and the ratio of co-localization (yellow) area to the occludin (green) and Cx26 (red) areas was 0.1 ± 0.2% and 1.2 ± 2.2% (mean ± SD), respectively.
Crush-injured Sciatic Nerves
To examine alterations in the expression of connexins after peripheral nerve crush injury, immunolabeling followed by morphometric analysis for Cx26, Cx32, and Cx43 was performed at various time points after an experimentally induced nerve crush. In the longitudinal sections of uninjured rat sciatic nerves, intact axons were revealed as continuous fibers with anti-PGP9.5 antibody. Four days after injury, the continuous fibers disappeared at the crushed and distal sites. Fourteen days after injury, a few axonal fibers were observed at the crushed site but not at the distal site. At 21 days after injury, many regenerating fibers appeared at the distal site (not shown).
In the longitudinal sections of uninjured sciatic nerves, punctate Cx26 immunoreactivity was localized to the innermost layer of the perineurium (Figure 5A and Figure 5B), consistent with the results in transverse sections (Figure 1B). The Cx26 localization in the perineurium did not change after crush injury (Figure 5C and Figure 5D). However, morphometric analysis at the distal site of the perineurium after injury revealed that Cx26 frequency increased at 4 days after injury and then decreased, the number of the Cx26-positive signals at 728 days after injury being significantly smaller than at 4 days after injury (Figure 8A). The sizes of Cx26-positive spots at 21 and 28 days after injury showed a tendency to become smaller than those of uninjured nerves, although there was no significant difference (p=0.57 for control vs 21 days distal and p=0.16 for control vs 28 days distal) (Figure 8B). No obvious change in the frequency and size of Cx26-immunoreactive spots was found in the perineurium at the proximal site of the injury (Figure 8A and Figure 8B). No clear immunolabeling for Cx26 was detected in the endoneurium and the epineurium of either uninjured or injured nerves.
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Cx32 immunoreactivity was restricted to the paranodal regions of the nodes of Ranvier in the endoneurium in the longitudinal sections of uninjured nerves and showed a symmetrical punctate pattern (Figure 6A and Figure 6B). Four days after injury, the normal symmetrical pattern of Cx32 was lost and Cx32-positive spots appeared fragmented at the distal site (Figure 6C). Fourteen days after injury, the crushed and distal regions of injury showed a complete reduction in Cx32 immunoreactivity (Figure 6D). Twenty-one days after injury, small spotty and irregular labeling appeared at the distal site of the endoneurium (Figure 6E). Twenty-eight days after injury, the symmetrical configuration of Cx32 immunoreactivity recovered in the distal segments (Figure 6F). No obvious change in Cx32 was detected at the proximal site of the injury (not shown).
In the longitudinal sections of uninjured sciatic nerves, immunolabeling for Cx43 demonstrated many spots in multiple layers of the perineurium and a small number of spots in the epineurium, but no immunoreactivity in the endoneurium (Figure 7A and Figure 7B), similar to that in the transverse sections (Figure 1F). No alterations in Cx43 immunolabeling (Figure 7C7G) or in the frequency and size of Cx43-positive signals (Figure 8C and Figure 8D) were detected in the perineurium during the entire period after injury. However, Cx43- positive spots appeared in the endoneurium after crush. Four days after injury, some Cx43-positive spots were detected in the endoneurium not only at the proximal site adjacent to the injury but also at distal sites (Figure 7C). Fourteen days after injury, the number of Cx43-positive spots in the endoneurium gradually attenuated (Figure 7D). DIC images of Cx43 immunolabeling and adjacent sections stained with hematoxylineosin indicated that Cx43-positive spots in the endoneurium were localized in/around axonal and myelin debris (not shown). Twenty-one days after injury, a few Cx43-positive spots were detected in the endoneurium, some of which were localized in/around the debris and others of which were located in fibrous structures in the DIC image (Figure 7E and Figure 7F). At this stage, regenerating axonal fibers were first found in these fibrous structures at the distal site of the injury by immunohistochemical labeling for PGP9.5 (not shown). Twenty-eight days after injury, the number of Cx43-positive spots in the endoneurium further decreased, but the spots existed outside neural fibers (Figure 7G).
Morphometric analysis demonstrated that the frequency of Cx43-positive spots at the distal site of the endoneurium markedly increased at 4 days after injury and then significantly decreased at 21 or 28 days after injury compared with that at 4 days after injury (Figure 8E). The endoneurial Cx43 that appeared at the proximal and distal sites after crush injury showed no significant change in size after injury (Figure 8F).
Double labeling for connexins and occludin was also performed to determine whether the relationship among these proteins is changed after crush injury. No obvious changes in the immunolabeling for occludin (Figure 5E and Figure 7H) and laminin (not shown) were observed in the perineurium at either the proximal or the distal sites after crush injury. Double labeling for Cx26 and occludin showed that the relationship between them did not change after injury, i.e., they were rarely co-localized in the perineurium in crushed nerves (Figure 5E and Figure 5F). In contrast, double labeling for Cx43 and occludin revealed that Cx43 frequently coexisted with occludin and that they were sometimes co-localized in multiple layers of the perineurium even after injury (Figure 7H and Figure 7I). Morphometric data confirmed no significant change in the extent of co-localization of Cx43 with occludin at either the proximal or the distal sites after injury (Figure 9).
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Discussion |
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In this study we demonstrated immunohistochemically that Cx26, Cx32, and Cx43 exhibited different expression patterns in intact peripheral nerves and that their expression was differentially modulated after crush injury. In intact nerves, Cx26-positive spots were unevenly distributed in the innermost layer of the perineurium, whereas abundant punctate Cx43 immunolabeling was detectable in multiple layers of the perineurium and a small number of punctate spots were also detected in the epineurium. Cx32 was confined to the paranodal regions of the nodes of Ranvier. Double labeling for Cx43 and occludin revealed that both proteins frequently existed in the same linear structures and were sometimes co-localized. During peripheral nerve regeneration after crush injury, the expression of Cx32 was altered and Cx43-positive spots appeared in the endoneurium. Morphometric data on Cx26 in the perineurium after injury showed a rapid increase and a subsequent decrease in frequency. There were no obvious changes in the expression of occludin and Cx43 in the perineurium.
We have good grounds for believing that our immunohistochemical data shown in this study exhibit credibility. For each connexin, Cx26, Cx32, and Cx43, identical results were obtained with different antibodies, including mouse monoclonal and rabbit polyclonal antibodies. Furthermore, confocal laser scanning microscopy enabled us to acquire simultaneous high-resolution images of two different connexins, or one connexin with occludin or laminin, as well as DIC images.
As far as we are aware, this is the first report on Cx26 localization in the perineurium of intact peripheral nerves. Before this study, although
For Cx43, our present study showed abundant punctate Cx43 immunolabeling in multiple layers of the perineurium and a few punctate spots in the epineurium, but no Cx43 immunoreactivity in the endoneurium except in endoneurial vessels. These results are consistent with previous reports showing that Cx43 immunoreactivity is localized to perineurial cell layers, the epineurium, and blood vessels of the endoneurium (
Our results of double labeling for Cx43 and occludin demonstrated that punctate spots of both proteins combined and formed continuous linear structures, with some co-localization in multiple layers of the perineurium, suggesting a close relationship between gap junctions and tight junctions among perineurial cells in each perineurial layer. The early freeze-fracture study by
Our results of the localization of Cx26, Cx43, and occludin in the perineurium also indicate that perineurial cells are not uniform in type through the multiple layers and may be made up of different populations. This is because some perineurial cells in the innermost layer possess Cx26, whereas outer perineurial cells in multiple layers express Cx43 and occludin but not Cx26. The difference in the properties of perineurial cells may be reflected in our present data on the expression of connexins after crush injury, showing that Cx26 in the innermost layer of the perineurium was changed during axonal regeneration, whereas Cx43 in the multiple layers did not alter. In addition, perineurial cells in the innermost layer may have different functions in addition to the diffusion barrier via tight junctions. For example, these innermost cells might receive signals from the endoneurial environment and regulate functions in the outer perineurial cells.
Cx32 is the first gap junction protein discovered in peripheral nerves (
In our crush injury study, changes in the expression of Cx32 and appearance of Cx43 spots were observed in the endoneurium at the distal site of the injury. After the loss of PGP9.5 expression in degenerated nerve fibers, the expression of Cx32 was reduced. Cx32 expression was recovered in the distal segments after the neural fibers regenerated, a process that appears to correspond to remyelination after nerve crush, as previous reports have indicated (
In contrast, in the perineurium, crush injuries caused no apparent alterations in the expression of occludin and Cx43 at the distal site, whereas the frequency and size of Cx26-positive spots in the innermost layer were modulated during axonal regeneration. These findings indicate that the endoneurial environment may influence the innermost layer of the perineurium but not the outer layers. The maintenance of a close relationship between occludin and Cx43 in the outer perineurial cells may be relevant to barrier functions against stimuli from the endoneurium.
In conclusion, our present study demonstrates that Cx26, Cx32, and Cx43 are expressed differently in various types of cells in intact peripheral nerves, and that these expressions are differentially modulated during axonal regeneration after crush injury. Because upregulation of Cx46 has been reported after nerve injury (
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Acknowledgments |
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Supported in part by a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Science and Culture.
We gratefully acknowledge the generous gift of the polyclonal anti-connexin26 antibody from Dr Y. Shibata (Kyushu University School of Medicine; Fukuoka, Japan) and that of the monoclonal anti-connexin32 (6-3G11) antibody from Dr A. Takeda (Department of Medical Biology, Ehime University; Ehime, Japan). We thank Mr D. Mrozek for advice on English usage.
Received for publication November 3, 1998; accepted March 9, 1999.
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